bims-rimeca 2022-11-13 papers

bims-rimeca

Biomed News

on RNA methylation in cancer

Issue of 2022–11–13
thirty-six papers selected by
Sk Ramiz Islam, Saha Institute of Nuclear Physics

FTO Inhibits Epithelial Ovarian Cancer Progression by Destabilising SNAI1 mRNA through IGF2BP2.
m6A-Related Genes Contribute to Poor Prognosis of Hepatocellular Carcinoma.
METTL3 promotes colorectal cancer metastasis by promoting the maturation of pri-microRNA-196b.
Mechanism of METTL14-mediated m6A modification in non-small cell lung cancer cell resistance to cisplatin.
METTL3-mediated m6A modification of lnc RNA RHPN1-AS1 enhances cisplatin resistance in ovarian cancer by activating PI3K/AKT pathway.
Involvement of METTL3 in arsenite-induced skin lesions by targeting the SOCS3/STAT3/Krt signaling pathway.
Prognostic and clinicopathological value of m6A regulators in human cancers: a meta-analysis.
The Mechanism and Role of N6-Methyladenosine (m6A) Modification in Atherosclerosis and Atherosclerotic Diseases.
Identifying Prognostic Biomarkers Related to m6A Modification and Immune Infiltration in Renal Cell Carcinoma.
Aberrant elevation of FTO levels promotes liver steatosis by decreasing the m6A methylation and increasing the stability of SREBF1 and ChREBP mRNAs.
m6A demethylase FTO renders radioresistance of nasopharyngeal carcinoma via promoting OTUB1-mediated anti-ferroptosis.
Mettl14-mediated m6A modification enhances the function of Foxp3+ regulatory T cells and promotes allograft acceptance.
FMR1 promotes the progression of colorectal cancer cell by stabilizing EGFR mRNA in an m6A-dependent manner.
IGF2BP2 Promotes Epithelial to Mesenchymal Transition and Metastasis through Stabilizing HMGA1 mRNA in Gastric Cancer.
Deciphering the map of METTL14-mediated lncRNA m6A modification at the transcriptome-wide level in breast cancer.
Pt(IV)-Deferasirox Prodrug Combats DNA Damage Repair by Regulating RNA N6-Methyladenosine Methylation.
ALKBH5-mediated m6A modification of lincRNA LINC02551 enhances the stability of DDX24 to promote hepatocellular carcinoma growth and metastasis.
Polychlorinated biphenyls alter hepatic m6A mRNA methylation in a mouse model of environmental liver disease.
Comprehensive Analysis Revealed the Potential Roles of N6-Methyladenosine (m6A) Mediating E. coli F18 Susceptibility in IPEC-J2 Cells.
m6A readers, writers, erasers, and the m6A epitranscriptome in breast cancer.
N6 -methyladenosine-modified lncRNA ARHGAP5-AS1 stabilises CSDE1 and coordinates oncogenic RNA regulons in hepatocellular carcinoma.
Comprehensive pan-cancer analysis of N7-methylguanosine regulators: Expression features and potential implications in prognosis and immunotherapy.
Maslinic Acid Suppresses High Glucose-induced Inflammation by Epigenetically Inhibiting TXNIP Expression.
Translational Regulation by eIFs and RNA Modifications in Cancer.
Construction of m6A-based prognosis signature and prediction for immune and anti-angiogenic response.
Methylation-driven gene DLL3 is a potential prognostic biomarker in ocular melanoma correlating with metastasis.
Detection of m6A from direct RNA sequencing using a multiple instance learning framework.
Knockdown of METTL5 inhibits the Myc pathway to downregulate PD-L1 expression and inhibits immune escape of hepatocellular carcinoma cells.
Aberrant DNA and RNA Methylation Occur in Spinal Cord and Skeletal Muscle of Human SOD1 Mouse Models of ALS and in Human ALS: Targeting DNA Methylation Is Therapeutic.
Guanine nucleotide-binding protein 2, GNBP2, accelerates the progression of clear cell renal cell carcinoma via regulation of STAT3 signaling transduction pathway.
Low expression of PEBP1P2 promotes metastasis of clear cell renal cell carcinoma by post-transcriptional regulation of PEBP1 and KLF13 mRNA.
Role of N6‑methyladenosine in the pathogenesis, diagnosis and treatment of pancreatic cancer (Review).
METTL14 is required for exercise-induced cardiac hypertrophy and protects against myocardial ischemia-reperfusion injury.
m6A and miRNA jointly regulate the development of breast muscles in duck embryonic stages.
SUMO1-modified DNA methyltransferase 1 induces DNA hypermethylation of VWC2 in the development of colorectal cancer.
YTHDF1 regulates endoplasmic reticulum stress, NF-κB, MAPK and PI3K-AKT signaling pathways in inflammatory osteoclastogenesis.

Cancers (Basel). 2022 Oct 25. pii: 5218. [Epub ahead of print]14(21):

FTO Inhibits Epithelial Ovarian Cancer Progression by Destabilising SNAI1 mRNA through IGF2BP2.

Meige Sun, Xiaocui Zhang, Fangfang Bi, Dandan Wang, Xin Zhou, Xiao Li, Qing Yang.

  Fat mass and obesity-associated protein (FTO) regulates critical pathways in various diseases, including malignant tumours. However, the functional link between FTO and its target genes in epithelial ovarian cancer (EOC) development remains to be elucidated. In this study, the biological functions of FTO were verified in vitro and in vivo. The m6A modification and the binding sites of SNAI1 mRNA were confirmed by m6A RNA immunoprecipitation (MeRIP) and RIP experiments. The actinomycin D assay was used to test the stability of RNA. We found that FTO was downregulated with increased m6A levels in EOC. Reduced expression of FTO was associated with a higher FIGO stage in patients with EOC. Mechanistically, FTO decreased the m6A level and stability of SNAI1 mRNA, causing downregulation of SNAI1 and inhibiting epithelial-mesenchymal transition (EMT). Furthermore, FTO-mediated downregulation of SNAI1 expression depended on IGF2BP2, which acted as an m6A reader binding to the 3' UTR region of SNAI1 mRNA to promote its stability. In conclusion, FTO inhibits SNAI1 expression to attenuate the growth and metastasis of EOC cells in an m6A-IGF2BP2-dependent manner. Our findings suggest that the FTO-IGF2BP2-SNAI1 axis is a potential therapeutic target in EOC.

Keywords:  EMT; FTO; IGF2BP2; SNAI1; m6A; metastasis; ovarian cancer

DOI:  https://doi.org/10.3390/cancers14215218
Comput Math Methods Med. 2022 ;2022 2427987

m6A-Related Genes Contribute to Poor Prognosis of Hepatocellular Carcinoma.

Yan Zou, Guichun Jiang, Yanmin Xie, Hui Li.

Background: Hepatocellular carcinoma (HCC) is one of the most common and lethal digestive system cancers worldwide. N6-methyladenosine (m6A) modification plays an essential role in diverse critical biological processes and may participate in the development and progression of HCC.
Methods: We downloaded transcriptome data and clinical data from TCGA as the training set. COX and LASSO screened prognostic m6A genes. ROC and Kaplan-Meier curve analysis evaluated the effectiveness of the model. ICGC and our center data were used as verification sets.
Results: We include the "writer (METTL3, METTL14, WTAP, KIAA1429, RBM15, ZC3H13)," the "reader (YTHDC1, YTHDC2, YTHDF1, YTHDF2, HNRNPC)," and the "eraser (FTO, ALKBH5)" in the study. We obtained YTHDF2, YTHDF1, METTL3, and KIAA1429 through differential analysis, survival analysis, and LASSO regression analysis. The prediction model was established based on the expression of these 4 molecules. HCC patients were divided into "high-risk" and "low-risk" groups to compare survival differences. The model suggested a poor prognosis in the validation sets.
Conclusion: The four-m6A-related-gene combination model was an independent prognostic factor of HCC and could improve the prediction of the prognosis of HCC.

DOI: https://doi.org/10.1155/2022/2427987
J Cancer Res Clin Oncol. 2022 Nov 08.

METTL3 promotes colorectal cancer metastasis by promoting the maturation of pri-microRNA-196b.

Lanlan Huang, Danlu Liang, Yu Zhang, Xiaoting Chen, Junxiong Chen, Chuangyu Wen, Huanliang Liu, Xiaorong Yang, Xiangling Yang, Shaoqiang Lin.

   PURPOSE: Methyltransferase-like 3 (METTL3), a key member of the m6A methyltransferase complex, is upregulated in multiple human malignancies and plays a role in regulating tumor migration. This study aimed to reveal the underlying mechanism by which METTL3 in regulates the metastasis of colorectal cancer (CRC).
METHODS: We compared METTL3 expression levels in CRC tumor tissues and adjacent nontumor tissues by immunohistochemistry (IHC). The functional roles of METTL3 in CRC were assessed by real-time cell migration assays, wound-healing assays and Transwell assays. miRNA sequencing (miRNA-seq), RNA-binding protein immunoprecipitation (RIP) assays and N6-methyladenosine immunoprecipitation (MeRIP) assays were performed to confirm the molecular mechanism underlying the involvement of METTL3 in CRC cell metastasis.
RESULTS: We found that METTL3 was overexpressed in CRC tissues. METTL3 knockdown significantly inhibited CRC cell migration and invasion, while METTL3 overexpression had the opposite effects. Furthermore, we demonstrated that METTL3 regulates miR-196b expression via an N6-methyladenosine (m6A)-pri-miR-196b-dependent mechanism and thereby promotes CRC metastasis.
CONCLUSION: This study shows the important role of METTL3 in CRC metastasis and provides novel insight into m6A modification in CRC metastasis.

Keywords:  Cancer metastasis; Colorectal cancer; METTL3; m6A; miR-196b

DOI:  https://doi.org/10.1007/s00432-022-04429-9
J Mol Med (Berl). 2022 Nov 09.

Mechanism of METTL14-mediated m6A modification in non-small cell lung cancer cell resistance to cisplatin.

Shulei Gong, Shiyang Wang, Mingrui Shao.

  Methyltransferase-like 14 (METTL14) mediates N6-methyladenosine (m6A) modification to influence cancer progression. This study aims to determine the mechanism of METTL14-mediated m6A in non-small cell lung cancer (NSCLC) cell resistance to cisplatin (DDP). METTL14, miR-19a-5p, RBM24, and AXIN1 expressions in NSCLC tissues/cells were determined. DDP-resistant cell line was obtained, followed by the interference of METTL14 expression. NSCLC cells were treated with DDP to establish a drug-resistant cell line, and METTL14 expression in cells was intervened. The IC50 of NSCLC cells to DDP was measured by CCK-8 assay. NSCLC cell proliferation and apoptosis were observed by clone formation assay and flow cytometry. The content of m6A in total RNA in tissues and cells of NSCLC patients was detected using m6A Methylation Quantification Kit. The expressions of DGCR8-bound pri-miR-19a and m6A-modified pri-miR-19a were detected. The binding relationships between miR-19a-5p and RBM24 and RBM24 and AXIN1 were validated using dual-luciferase assay and RNA immunoprecipitation. Finally, mouse xenograft tumor model was established to verify the role of METTL14 in vivo. METTL14 was highly expressed in NSCLC. METTL14 silencing diminished IC50 to DDP, repressed NSCLC cell proliferation, and enhanced apoptosis. METTL14-mediated m6A induced recognition and processing of pri-miR-19a by DGCR8, thus promoting the transition of pri-miR-19a to miR-19a-5p, repressing RBM24 expression, reducing the binding of RBM24 and AXIN1, and suppressing AXIN1 transcription. miR-19a-5p overexpression or RBM24/AXIN1 silencing abolished the effect of METTL14 silencing on NSCLC cell resistance to DDP. METTL14 silencing in vivo enhanced the suppressive role of DDP to tumor growth. Collectively, METTL14-mediated m6A modification facilitated NSCLC cell resistance to DDP via miR-19a-5p/RBM24/AXIN1 axis. KEY MESSAGES: • METTL14 is highly expressed NSCLC and further increased in DDP-resistant cells. • METTL14 silencing attenuates DDP resistance of NSCLC cells. • METTL14 promotes the nature of pri-miR-19a by upregulating pri-miR-19a m6A level. • miR-19a-5p targets RBM24, thus reducing the binding of RBM24 and AXIN1 and inhibiting AXIN1 transcription. • METTL14 silencing in vivo enhances the suppressive role of DDP to tumor growth.

Keywords:  Cisplatin resistance; METTL14; Non-small cell lung cancer; Pri-miR-19a; RBM24; miR-19a-5p

DOI:  https://doi.org/10.1007/s00109-022-02268-2
J Clin Lab Anal. 2022 Nov 06. e24761

METTL3-mediated m6A modification of lnc RNA RHPN1-AS1 enhances cisplatin resistance in ovarian cancer by activating PI3K/AKT pathway.

Shoubin Cui.

   BACKGROUND: Cisplatin resistance is a big challenge for ovarian cancer (OC) therapy. The abnormal expression of long noncoding RNAs (lncRNAs) regulated by N6-methyladenosine (m6A) modification has been confirmed to play the crucial roles in OC. The aim of this study is to explore the regulatory mechanism of lncRNA RHPN1-AS1 on OC with cisplatin resistance.
METHODS: The real-time reverse transcription-polymerase chain reaction was carried out to confirm the expression of RHPN1-AS1 and methyltransferase-like 3 (METTL3) in OC. The effects of RHPN1-AS1 on cisplatin-resistant OC cells were identified by cell functional experiments and animal experiment. Western blotting was performed to detect the effect of RHPN1-AS1 on PI3K/AKT pathway. Moreover, methylated RNA immunoprecipitation and RNA stability assays confirmed the interaction between RHPN1-AS1 and METTL3.
RESULTS: RHPN1-AS1 and METTL3 were confirmed to be overexpressed in OC. After transfecting RHPN1-AS1 overexpression or RHPN1-AS1 knockdown vectors into cisplatin-resistant OC cells, it was found that upregulating RHPN1-AS1 contributed to cell viability, migration, invasion, and tumor growth in vivo. In addition, RHPN1-AS1 could enhance the protein levels of PI3K and phosphorylated AKT in cisplatin-resistant OC cells, and METTL3 could enhance the stability of RHPN1-AS1 by the m6A modification.
CONCLUSION: Overall, this study revealed that METTL3-mediated m6A modification of RHPN1-AS1 accelerates cisplatin resistance in OC by activating PI3K/AKT pathway.

Keywords:  METTL3; PI3K/AKT; RHPN1-AS1; cisplatin; ovarian cancer

DOI:  https://doi.org/10.1002/jcla.24761
Environ Pollut. 2022 Nov 08. pii: S0269-7491(22)01848-6. [Epub ahead of print] 120634

Involvement of METTL3 in arsenite-induced skin lesions by targeting the SOCS3/STAT3/Krt signaling pathway.

Fan Yang, Aihua Zhang.

  Arsenic is a common environmental pollutant, typically affecting the skin most severely. Recent studies have shown that arsenic's toxicity may be linked to N6-methyladenosine (m6A), an abundant and dynamic epigenetic RNA modification. However, it is not completely understood how m6A contributes to arsenite-induced skin lesions. Herein, it is shown that methyltransferase-like 3 (METTL3) plays a crucial role in the involvement of arsenite-induced skin lesions in an m6A-dependent manner. Using bioinformatic analysis and experimental approaches, we demonstrate that arsenite induces METTL3 upregulation, represses suppressors of cytokine signaling 3 (SOCS3) expression in an m6A- YTH m6A RNA binding protein 2 (YTHDF2)-dependent manner, and leads to the aberrant activation of the Janus kinase (JAK)2/signal transducer and activator of transcription 3(STAT3) signaling pathway. We further found that the activated transcription factor STAT3 binds to the promoter regions of Krt1 and Krt10, promoting their transcription, which ultimately leads to arsenite-induced skin lesions. In conclusion, our study reveals the role of m6A in arsenite-induced skin lesions through the activation of the JAK2/STAT3/Krt signaling axis. The findings provide new insight into the potential molecular mechanisms underlying arsenic toxicity regulation through m6A modification.

Keywords:  Arsenite; METTL3; N(6)-methyladenosine; STAT3/Krt signaling pathway; Skin lesions

DOI:  https://doi.org/10.1016/j.envpol.2022.120634
Aging (Albany NY). 2022 Nov 07. 14

Prognostic and clinicopathological value of m6A regulators in human cancers: a meta-analysis.

Zhangci Su, Leyao Xu, Xinning Dai, Mengyao Zhu, Xiaodan Chen, Yuanyuan Li, Jie Li, Ruihan Ge, Bin Cheng, Yun Wang.

 BACKGROUND: N6-methyladenosine (m6A) is the most abundant epigenetic modification. Although the dysregulation of m6A regulators has been associated with cancer progression in several studies, its relationship with cancer prognosis and clinicopathology is still controversial. Therefore, we evaluated the prognostic and clinicopathological value of m6A regulators in cancers by performing a comprehensive meta-analysis.
METHODS: The PubMed, Cochrane Library, Web of Science, and Embase databases were searched up to April 2022. Hazard ratios were used to analyze the association between m6A with prognosis. We also analyze the relationship between m6A and clinicopathology using odds ratios.
RESULTS: METTL3 overexpression predicted poor overall survival and disease-free survival in cancer patients (p < 0.001) such as gastric cancer (p < 0.001), esophageal squamous cell carcinoma (p < 0.001), oral squamous cell carcinoma (p = 0.002) and so on. Additionally, METTL3 overexpression was associated with poor pT stage (p < 0.001), pN stage (p < 0.001), TNM stage (p < 0.001), tumor size >5 cm (p < 0.001) and vascular invasion (p = 0.024). Conversely, METTL14 overexpression was positively associated with better OS (p < 0.001), negatively with poor pT stage (p = 0.001), pM stage (p = 0.002), pN stage (p = 0.011) and TNM stage (p < 0.001). Moreover, KIAA1429 overexpression was associated with poor OS (p = 0.001). YTHDF1 overexpression was also associated with advanced pM stage (p < 0.001) and tumor size >5 cm (p < 0.001). However, ALKBH5 overexpression was negatively associated with vascular invasion (p = 0.032).
CONCLUSIONS: High expression of METTL3 predicted poor outcome. In contrast, high expression of METTL14 was associated with better outcome. Thus, we suggest that among all the m6A regulators, METTL3 and METTL14 could be potential prognostic markers in cancers.

Keywords: cancers; clinicopathology; m6A regulators; meta-analysis; prognosis

DOI: https://doi.org/10.18632/aging.204371
J Cardiovasc Dev Dis. 2022 Oct 25. pii: 367. [Epub ahead of print]9(11):

The Mechanism and Role of N6-Methyladenosine (m6A) Modification in Atherosclerosis and Atherosclerotic Diseases.

Quandan Tan, Song He, Xinyi Leng, Danni Zheng, Fengkai Mao, Junli Hao, Kejie Chen, Haisong Jiang, Yapeng Lin, Jie Yang.

  N6-methyladenosine (m6A) modification is a newly discovered regulatory mechanism in eukaryotes. As one of the most common epigenetic mechanisms, m6A's role in the development of atherosclerosis (AS) and atherosclerotic diseases (AD) has also received increasing attention. Herein, we elucidate the effect of m6A on major risk factors for AS, including lipid metabolism disorders, hypertension, and hyperglycemia. We also describe how m6A methylation contributes to endothelial cell injury, macrophage response, inflammation, and smooth muscle cell response in AS and AD. Subsequently, we illustrate the m6A-mediated aberrant biological role in the pathogenesis of AS and AD, and analyze the levels of m6A methylation in peripheral blood or local tissues of AS and AD, which helps to further discuss the diagnostic and therapeutic potential of m6A regulation for AS and AD. In summary, studies on m6A methylation provide new insights into the pathophysiologic mechanisms of AS and AD, and m6A methylation could be a novel diagnostic biomarker and therapeutic target for AS and AD.

Keywords:  N6-methyladenosine (m6A); atherosclerosis; atherosclerotic diseases; diagnostic biomarkers; targeted therapeutics

DOI:  https://doi.org/10.3390/jcdd9110367
Genes (Basel). 2022 Nov 07. pii: 2059. [Epub ahead of print]13(11):

Identifying Prognostic Biomarkers Related to m6A Modification and Immune Infiltration in Renal Cell Carcinoma.

Junjie Ye, Peng Li, Huijiang Zhang, Qi Wu, Dongrong Yang.

   BACKGROUND: Renal cell carcinoma (RCC) is the largest category of kidney tumors and usually does not have a good prognosis. N6-methyladenosine(m6A) and immune infiltration have received increased attention because of their great influence on the clinical outcome and prognosis of cancer patients.
METHODS: We identified hub genes through multi-dimensional screening, including DEGs, PPI analysis, LASSO regression, and random forest. Meanwhile, GO/KEGG enrichment, cMAP analysis, prognostic analysis, m6A prediction, and immune infiltration analysis were performed to understand the potential mechanism and screen therapeutic drugs.
RESULTS: We screened 275 downregulated and 185 upregulated genes using three GEO datasets and the TCGA dataset. In total, 82 candidate hub genes were selected using STRING and Cytoscape. Enrichment analysis illustrated that the top 3 biological process terms and top 1 KEGG term were related to immunity. cMAP analysis showed some antagonistic molecules can be candidate drugs for the treatment of RCC. Then, six hub genes (ERBB2, CASR, P2RY8, CAT, PLAUR, and TIMP1) with strong predictive values for prognosis and clinicopathological features were selected. Meanwhile, P2RY8, ERBB2, CAT, and TIMP1 may obtain m6A modification by binding METTL3 or METTL14. On the other hand, differential expression of CAT, ERBB2, P2RY8, PLAUR, and TIMP1 affects the infiltration of the majority of immune cells.
CONCLUSIONS: We identified six hub genes through multi-dimensional screening. They all possess strong predictive value for prognosis and clinicopathological features. Meanwhile, hub genes may regulate the progression of RCC via an m6A- and immunity-dependent mechanism.

Keywords:  biomarker; immune infiltration; m6A modification; renal cell carcinoma

DOI:  https://doi.org/10.3390/genes13112059
J Mol Cell Biol. 2022 Nov 10. pii: mjac061. [Epub ahead of print]

Aberrant elevation of FTO levels promotes liver steatosis by decreasing the m6A methylation and increasing the stability of SREBF1 and ChREBP mRNAs.

Zhili Tang, Chao Sun, Ying Yan, Zhoumin Niu, Yuying Li, Xi Xu, Jing Zhang, Yuting Wu, Yan Li, Li Wang, Cheng Hu, Zhuoyang Li, Jingjing Jiang, Hao Ying.

  Previous studies have indicated an association of fat mass and obesity-associated (FTO) with nonalcoholic fatty liver disease (NAFLD), the most common chronic liver disease worldwide. This study aimed to decipher the complex role of FTO in hepatic lipid metabolism. We found that a decrease in N6-methyladenosine (m6A) RNA methylation in the liver of mice fed with high-fat diet (HFD) was accompanied by an increase in FTO expression. Overexpression of FTO in the liver promoted triglyceride accumulation by upregulating the expression of lipogenic genes. Mechanistical studies revealed that FTO could stabilize the mRNAs of sterol regulatory element binding transcription factor 1 (SREBF1) and carbohydrate responsive element binding protein (ChREBP), two master lipogenic transcription factors, by demethylating m6A sites. Knockdown of either SREBF1 or ChREBP attenuated the lipogenic effect of FTO, suggesting that they are bona fide effectors for FTO in regulating lipogenesis. Insulin could stimulate FTO transcription through a mechanism involving the action of intranuclear insulin receptor beta, while knockdown of FTO abrogated the lipogenic effect of insulin. Inhibition of FTO by entacapone decreased the expression of SREBF1, ChREBP, and downstream lipogenic genes, ameliorating liver steatosis in HFD-fed mice. Thus, our study established a critical role of FTO in both the insulin-regulated hepatic lipogenesis and the pathogenesis of NAFLD and provided a potential strategy for treating NAFLD.

Keywords:  ChREBP; FTO; NAFLD; SREBF1; lipogenesis; m6A

DOI:  https://doi.org/10.1093/jmcb/mjac061
Transl Oncol. 2022 Nov 04. pii: S1936-5233(22)00235-2. [Epub ahead of print]27 101576

m6A demethylase FTO renders radioresistance of nasopharyngeal carcinoma via promoting OTUB1-mediated anti-ferroptosis.

Wei-Mei Huang, Zhi-Xun Li, Ying-Hui Wu, Zhi-Ling Shi, Jing-Lin Mi, Kai Hu, Ren-Sheng Wang.

  Radiotherapy is a valid treatment for nasopharyngeal carcinoma (NPC), and radioresistance is the main cause of local NPC treatment failure. However, the underlying mechanisms and valuable markers of radioresistance for NPC remain have not been established. In this study, we observed that the m6A mRNA demethylase fat mass and obesity-associated protein (FTO) was significantly upregulated in radioresistant NPC tissues and cells relative to parental radiosensitive NPC tissues and cells. FTO enhances radioresistance by repressing radiation-induced ferroptosis in NPC. Mechanistically, FTO acts as an m6A demethylase to erase the m6A modification of the OTUB1 transcript and promote the expression of OTUB1, thereby inhibiting the ferroptosis of cells induced by radiation and finally triggering the radiotherapy resistance of NPC. Furthermore, our in vivo experiment results showed that the FTO inhibitor, FB23-2, and the ferroptosis activator, erastin, altered tumor responsiveness to radiotherapy in NPC cell lines and patient-derived xenografts. Our findings reveal, for the first time, that FTO enhances NPC radiotherapy resistance by withstanding radiation-induced ferroptosis, suggesting that FTO may serve as a potential therapeutic target and valuable prognostic biomarker in patients with NPC.

Keywords:  Nasopharyngeal carcinoma (NPC), FTO, Ferroptosis; OTUB1; m6A

DOI:  https://doi.org/10.1016/j.tranon.2022.101576
Front Immunol. 2022 ;13 1022015

Mettl14-mediated m6A modification enhances the function of Foxp3+ regulatory T cells and promotes allograft acceptance.

Yanzhuo Liu, Yinglin Yuan, Zili Zhou, Yuanyuan Cui, Yan Teng, Hao Huang, Hao Yuan, Yanling Zhang, Lu Yang, Gaoping Zhao.

 N6-methyladenosine (m6A), the most prevalent form of internal mRNA modification, is extensively involved in Treg cells differentiation and function. However, the involvement of m6A in functional Treg cells for transplantation tolerance remains to be elucidated. By using an experimental transplantation mouse model, we found that m6A levels in Treg cells were altered during the induction of transplant tolerance by performing a dot blotting assay. Subsequently, we used the heterogenic Treg-specific Mettl14 knockout mice (Foxp3-Mettl14f/+ cKO) to reduce METTL14 expression and performed islets allograft transplantation. Our result revealed that reduced expression of METTL14 prevented Treg cells expansion and promoted the infiltration of CD4+ and CD8+ T cells around the allograft, which led to rapid allograft rejection in Foxp3-Mettl14 f/+ cKO mice. The expression of regulatory cytokines including IL-10 and TGF-β was significantly decreased in Foxp3-Mettl14 f/+ cKO mice, and the suppressive function of Treg cells was also abrogated. In addition, an analysis of RNA-seq data revealed that the SOCS family (SOCS1, SOCS2 and SOCS3) is the subsequent signaling pathway affected by the METTL14 mediated m6A modification in Treg cells to modulate the suppressive function after transplantation. Taken together, our study showed for the first time that the METTL14-mediated m6A modification is essential for the suppressive function of Treg cells in transplantation and may serve as a regulatory element of Treg cell-based therapy in transplant medicine.

Keywords: Mettl14; N6-methyladenosine; Treg function; allograft acceptance; transplantation

DOI: https://doi.org/10.3389/fimmu.2022.1022015
Cell Death Dis. 2022 Nov 08. 13(11): 941

FMR1 promotes the progression of colorectal cancer cell by stabilizing EGFR mRNA in an m6A-dependent manner.

Yuhan Hu, Qingzu Gao, Shuai Ma, Pei Yu, Shuang Ding, Xiaofei Yao, Zheying Zhang, Shuya Lu, Manman Lu, Jinghang Zhang, Yanling Wang, Xinlai Qian, Jiateng Zhong.

FMR1, a new m6A reader, is known to be involved in the regulation of cancer progression. However, its role, regulatory mechanism, and clinical significance in colorectal cancer (CRC) are elusive. Here, we showed that FMR1 was upregulated in CRC, and it promoted proliferation and metastasis of CRC cells in vitro and in vivo. Mechanically, FMR1 recognized the m6A-modification site in EGFR mRNA, a key molecule in cancer occurrence and targeted therapy, sustained its stability and maintained its expression in an m6A-dependent manner, thereby promoting the tumorigenesis and metastasis of CRC. And the effect of FMR1 knockdown in CRC cells could be abolished by METTL3. Furthermore, FMR1 shRNA plasmid carried by attenuated Salmonella has an effective anti-tumor effect in vivo. Collectively, we identified the METTL3/FMR1/EGFR axis in the progression of CRC. This novel mechanism indicated that the METTL3/FMR1/EGFR axis is a potential target for early therapeutic intervention in CRC progression.

DOI: https://doi.org/10.1038/s41419-022-05391-7
Cancers (Basel). 2022 Oct 31. pii: 5381. [Epub ahead of print]14(21):

IGF2BP2 Promotes Epithelial to Mesenchymal Transition and Metastasis through Stabilizing HMGA1 mRNA in Gastric Cancer.

Jun Ouyang, Junqing Li, Dongwei Li, Jianlong Jiang, Tengfei Hao, Yujian Xia, Xiaofang Lu, Changhua Zhang, Yulong He.

  As an RNA-binding protein, insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) is involved in enhancing the progression of a few malignant tumors by recognizing N6-methyladenosine on targeted RNA. However, the specific effects of IGF2BP2 on gastric cancer (GC) and the underlying mechanisms remain unclear. In this study, the expression level of IGF2BP2 was evaluated by analyzing data from a public database and performing immunohistochemical staining with GC specimens. The effect of IGF2BP2 on GC cell metastasis was investigated by Transwell assays and animal studies. RNA immunoprecipitation (RIP) was performed to identify potential mRNA bound to IGF2BP2. The levels of these identified RNAs were measured by RT-PCR, while corresponding proteins were quantified via Western blot. It was revealed that IGF2BP2 expression in GC tissues was significantly upregulated, and its overexpression was significantly associated with worse survival in GC patients. The aberrant expression of IGF2BP2 was demonstrated to promote the invasion and metastasis of GC cells by both in vivo and in vitro experiments. In subsequent experiments, it was then verified that by directly interacting with HMGA1 mRNA, IGF2BP2 augmented its stability and thus increased its expression. The knocking down of IGF2BP2 could significantly decrease the migration and invasion of GC cells, which could be reversed by increasing HMGA1 expression. Additionally, both in vitro and in vivo epithelial-mesenchymal transition (EMT) of GC cells were enhanced by IGF2BP2/HMGA1 axis. In conclusion, it was proven in our study that the IGF2BP2/HMGA1/EMT axis contributed to GC metastasis, suggesting its potential as a novel predictive and therapeutic biomarker for GC.

Keywords:  EMT; HMGA1; IGF2BP2; gastric cancer; metastasis

DOI:  https://doi.org/10.3390/cancers14215381
J Clin Lab Anal. 2022 Nov 06. e24754

Deciphering the map of METTL14-mediated lncRNA m6A modification at the transcriptome-wide level in breast cancer.

Dandan Yi, Fazhan Xu, Ru Wang, Chaoyu Jiang, Jiabo Qin, YiHsuan Lee, Xianbiao Shi, Jianfeng Sang.

   BACKGROUND: Emerging studies have demonstrated the critical role of RNA m6A methylation in tumor progression, whereas lncRNA m6A modification profiles in breast cancer remain largely unknown. Our previous study has shown that METTL14 accelerates breast cancer migration and invasion in an m6A-dependent manner, making it critical to analyze METTL14-mediated m6A modification at a transcriptome-wide scale in breast cancer.
METHODS: Here, we performed MeRIP-seq analysis in METTL14 overexpressed and control MDA-MB-231 cells. Conjoint analysis of MeRIP-seq and RNA-seq data was used to select lncRNAs with m6A methylation and differential expression. Finally, the screened lncRNA was verified by MeRIP-PCR and its function was studied via transwell assay.
RESULTS: Our results determined that high expression of METLL14 results in 3996 hypermethylation peaks from 3107 transcripts, and 4100 hypomethylation peaks from 2918 transcripts. Furthermore, conjoint analysis of MeRIP-seq and RNA-seq data identified 25 lncRNAs with discrepant methylation and simultaneously discrepant expression, among which the top 10 differentially expressed LncRNAs were AC026401.3, CYTOR, LINC01943, AC084125.2, FLJ20021, LINC00472, and NORAD, MALAT1, AL161431.1, and LINC01764. Moreover, over-expressed METTL14 stimulated the m6A modification of AC084125.2, while decreasing its expression. Compared to adjacent tissues, AC084125.2 was lowly expressed in tumors and could be used as a biomarker in the diagnosis of breast cancer. Meanwhile, AC084125.2 inhibited the migration and invasion of cancer cells.
CONCLUSION: In conclusion, METTL14-mediated m6A modification of lncRNAs, which might provide reference for future intervention in tumor progression.

Keywords:  MeRIP-seq; N6-methyladenosine; RNA-seq; breast cancer; long non-coding RNA

DOI:  https://doi.org/10.1002/jcla.24754
J Med Chem. 2022 Nov 10. 65(21): 14692-14700

Pt(IV)-Deferasirox Prodrug Combats DNA Damage Repair by Regulating RNA N6-Methyladenosine Methylation.

Zheng-Yin Pan, Yu-Yi Ling, Hang Zhang, Liang Hao, Cai-Ping Tan, Zong-Wan Mao.

DNA damage repair is considered to be an important mechanism of cisplatin resistance, and the roles of iron homeostasis in action mechanisms of cisplatin have not been studied yet. Herein, a Pt(IV) prodrug (DFX-Pt) integrating cisplatin and the clinical oral iron-chelating agent deferasirox (DFX) is found to be highly active toward cisplatin-insensitive triple-negative breast cancer cells both in vitro and in vivo. RNA-sequencing shows that DFX-Pt can downregulate genes related to the double-strand break (DSB) damage pathway significantly. DFX-Pt can reduce cellular free iron, regulate the expression of the RNA demethylase, and elevate the levels of RNA N6-methyladenosine (m6A), which degrades the DSB-related genes in an m6A-dependent manner. In all, we first reveal the roles of RNA modification in mechanisms of combating DNA damage repair and show that the combination of iron homeostasis intervention may bring new treatment regimens for cisplatin resistance.

DOI: https://doi.org/10.1021/acs.jmedchem.2c01224
Cell Death Dis. 2022 Nov 05. 13(11): 926

ALKBH5-mediated m6A modification of lincRNA LINC02551 enhances the stability of DDX24 to promote hepatocellular carcinoma growth and metastasis.

Hongwei Zhang, Yachong Liu, Wei Wang, Furong Liu, Weijian Wang, Chen Su, He Zhu, Zhibin Liao, Bixiang Zhang, Xiaoping Chen.

As the most important RNA epigenetic regulation in eukaryotic cells, N6-metheyladenosine (m6A) modification has been demonstrated to play significant roles in cancer progression. However, this modification in long intergenic non-coding RNAs (lincRNAs) and the corresponding functions remain elusive. Here, we showed a lincRNA LINC02551 was downregulated by AlkB Homolog 5 (ALKBH5) overexpression in a m6A-dependent manner in hepatocellular carcinoma (HCC). Functionally, LINC02551 was required for the growth and metastasis of HCC. Mechanistically, LINC02551, a bona fide m6A target of ALKBH5, acted as a molecular adaptor that blocked the combination between DDX24 and a E3 ligase TRIM27 to decrease the ubiquitination and subsequent degradation of DDX24, ultimately facilitating HCC growth and metastasis. Thus, ALKBH5-mediated LINC02551 m6A methylation was required for HCC growth and metastasis.

DOI: https://doi.org/10.1038/s41419-022-05386-4
Environ Res. 2022 Oct 28. pii: S0013-9351(22)02013-8. [Epub ahead of print]216(Pt 3): 114686

Polychlorinated biphenyls alter hepatic m6A mRNA methylation in a mouse model of environmental liver disease.

Belinda J Petri, Kellianne M Piell, Banrida Wahlang, Kimberly Z Head, Kalina Andreeva, Eric C Rouchka, Matthew C Cave, Carolyn M Klinge.

  Exposure to polychlorinated biphenyls (PCBs) has been associated with liver injury in human cohorts and with nonalcoholic steatohepatitis (NASH) in mice fed a high fat diet (HFD). N (6)-methyladenosine (m6A) modification of mRNA regulates transcript fate, but the contribution of m6A modification on the regulation of transcripts in PCB-induced steatosis and fibrosis is unknown. This study tested the hypothesis that PCB and HFD exposure alters the levels of m6A modification in transcripts that play a role in NASH in vivo. Male C57Bl6/J mice were fed a HFD (12 wks) and administered a single oral dose of Aroclor1260, PCB126, or Aroclor1260 + PCB126. Genome-wide identification of m6A peaks was accomplished by m6A mRNA immunoprecipitation sequencing (m6A-RIP) and the mRNA transcriptome identified by RNA-seq. Exposure of HFD-fed mice to Aroclor1260 decreased the number of m6A peaks and m6A-containing genes relative to PCB vehicle control whereas PCB126 or the combination of Aroclor1260 + PCB126 increased m6A modification frequency. ∼41% of genes had one m6A peak and ∼49% had 2-4 m6A peaks. 117 m6A peaks were common in the four experimental groups. The Aroclor1260 + PCB126 exposure group showed the highest number (52) of m6A-peaks. qRT-PCR confirmed enrichment of m6A-containing fragments of the Apob transcript with PCB exposure. A1cf transcript abundance, m6A peak count, and protein abundance was increased with Aroclor1260 + PCB126 co-exposure. Irrespective of the PCB type, all PCB groups exhibited enriched pathways related to lipid/lipoprotein metabolism and inflammation through the m6A modification. Integrated analysis of m6A-RIP-seq and mRNA-seq identified 242 differentially expressed genes (DEGs) with increased or reduced number of m6A peaks. These data show that PCB exposure in HFD-fed mice alters the m6A landscape offering an additional layer of regulation of gene expression affecting a subset of gene responses in NASH.

Keywords:  Epitranscriptome; High fat diet; Liver; PCBs; Readers; Writers; m6A

DOI:  https://doi.org/10.1016/j.envres.2022.114686
Int J Mol Sci. 2022 Nov 06. pii: 13602. [Epub ahead of print]23(21):

Comprehensive Analysis Revealed the Potential Roles of N6-Methyladenosine (m6A) Mediating E. coli F18 Susceptibility in IPEC-J2 Cells.

Zhengchang Wu, Yifu Wang, Tong Li, Li Yang, Jian Jin, Shenglong Wu, Wenbin Bao.

  Post-weaning diarrhea caused by enterotoxigenic Escherichia coli F18 (E. coli F18) causes significant economic losses for pig producers. N6-methyladenosine (m6A) is a highly abundant epitranscriptomic marker that has been found to be involved in regulating the resistance of host cells to pathogenic infection, but its potential role in E. coli F18-exposed intestinal porcine epithelial cells (IPEC-J2) remains undetermined. Here, we demonstrated that m6A and its regulators modulate E. coli F18 susceptibility. Briefly, we revealed that the Wilms' tumor 1-associating protein (WTAP) expressions were markedly elevated in IPEC-J2 cells upon E. coli F18 exposure. WTAP are required for the regulation of E. coli F18 adhesion in IPEC-J2 cells. Additionally, WTAP knockdown significantly suppressed m6A level at N-acetyllactosaminide beta-1,6-N-acetylglucosaminyl-transferase (GCNT2) 3'UTR, resulting in the enhancement of TH N6-methyladenosine RNA binding protein 2 (YTHDF2)-mediated GCNT2 mRNA stability. Subsequently, the altered GCNT2 expressions could inhibit the glycosphingolipid biosynthesis, thus improving resistance to E. coli F18 infection in IPEC-J2. Collectively, our analyses highlighted the mechanism behind the m6A-mediated management of E. coli F18 susceptibility, which will aid in the development of novel approaches that protect against bacterial diarrhea in piglets.

Keywords:  E. coli F18; IPEC-J2; N6-methyladenosine (m6A); susceptibility

DOI:  https://doi.org/10.3390/ijms232113602
J Mol Endocrinol. 2022 Nov 01. pii: JME-22-0110. [Epub ahead of print]

m6A readers, writers, erasers, and the m6A epitranscriptome in breast cancer.

Belinda J Petri, Carolyn M Klinge.

Epitranscriptomic modification of RNA regulates human development, health, and disease. The true diversity of the transcriptome in breast cancer including chemical modification of transcribed RNA (epitranscriptomics) is not well understood due to limitations of technology and bioinformatic analysis. N-6-methyladenosine (m6A) is the most abundant epitranscriptomic modification of mRNA and regulates splicing, stability, translation, and intracellular localization of transcripts depending on m6A association with reader RNA binding proteins. m6A methylation is catalyzed by the METTL3 complex and removed by the specific m6A demethylase ALKBH5, with FTO's role as an 'eraser' uncertain. In this review, we provide and overview of epitranscriptomics related to mRNA and focus on m6A in mRNA and its detection. We summarize current knowledge on altered levels of writers, readers, and erasers of m6A and their roles in breast cancer and association with prognosis. We summarize studies identifying m6A peaks and sties in genes in breast cancer cells.

DOI: https://doi.org/10.1530/JME-22-0110
Clin Transl Med. 2022 Nov;12(11): e1107

N6 -methyladenosine-modified lncRNA ARHGAP5-AS1 stabilises CSDE1 and coordinates oncogenic RNA regulons in hepatocellular carcinoma.

Jiandong Liu, Nasha Zhang, Jiajia Zeng, Teng Wang, Yue Shen, Chi Ma, Ming Yang.

   BACKGROUND: Hepatocellular carcinoma (HCC) ranks fourth among the malignancies leading to cancer-related deaths all around the world. It is increasingly evident that long non-coding RNAs (lncRNAs) are a key mode of hepatocarcinogenesis. As the most prevalent mRNA modification form, N6 -methyladenosine (m6 A) regulates gene expression by impacting multiple aspects of mRNA metabolism. However, there are still no reports on genome-wide screening and functional annotation of m6 A-methylated lncRNAs in HCC.
METHODS: The m6 A modification and biologic functions of ARHGAP5-AS1 in HCC were investigated through a series of biochemical assays. Clinical implications of ARHGAP5-AS1 were examined in tissues from HCC patients.
RESULTS: After systematically analysing the m6 A-seq data of HCC cells, we identified 22 candidate lncRNAs with evidently dysregulated m6 A levels. Among these lncRNAs, we found that ARHGAP5-AS1 is the lncRNA with the highest levels of m6 A modification and significantly increased expression in HCC specimens. METTL14 acts as the m6 A writer of ARHGAP5-AS1 and IGF2BP2 stabilises the lncRNA as its m6 A reader. ARHGAP5-AS1 remarkably promotes malignant behaviours of HCC cells ex vivo and in vivo. We identified oncoprotein CSDE1 working as the interacting protein of the lncRNA and TRIM28 as the E3 ligase of CSDE1 in HCC. Interestingly, ARHGAP5-AS1 could attenuate interactions between CSDE1 and TRIM28, which prevents the degradation of CSDE1 via the ubiquitin-proteasome pathway. Elevated levels of CSDE1 coordinate oncogenic RNA regulons, promote translation of VIM and RAC1 and activate the ERK pathway, which contributes to HCC prognosis.
CONCLUSIONS: Our study reveals a new paradigm in m6 A-modified lncRNAs controlling CSDE1-mediated oncogenic RNA regulons and highlights lncRNAs as potential targets for future therapeutics against HCC.

Keywords:  ARHGAP5-AS1; CSDE1; N6-methyladenosine; hepatocellular carcinoma; lncRNA

DOI:  https://doi.org/10.1002/ctm2.1107
Front Genet. 2022 ;13 1016797

Comprehensive pan-cancer analysis of N7-methylguanosine regulators: Expression features and potential implications in prognosis and immunotherapy.

Wei Wei, Chao Liu, Caihong Wang, Meng Wang, Wei Jiang, Yaqian Zhou, Shuqun Zhang.

  Although immunotherapy has made great strides in cancer therapy, its effectiveness varies widely among individual patients as well as tumor types, and there is an urgent need to develop biomarkers for effectively assessing immunotherapy response. In recent years, RNA methylation regulators have demonstrated to be novel potential biomarkers for prognosis as well as immunotherapy of cancers, such as N6-methyladenine (m6A) and 5-methylcytosine (m5C). N7-methylguanosine (m7G) is a prevalent RNA modification in eukaryotes, but the relationship between m7G regulators and prognosis as well as tumor immune microenvironment is still unclear. In this study, a pan-cancer analysis of 26 m7G regulators across 17 cancer types was conducted based on the bioinformatics approach. On the one hand, a comprehensive analysis of expression features, genetic variations and epigenetic regulation of m7G regulators was carried out, and we found that the expression tendency of m7G regulators were different among tumors and their aberrant expression in cancers could be affected by single nucleotide variation (SNV), copy number variation (CNV), DNA methylation and microRNA (miRNA) separately or simultaneously. On the other hand, the m7Gscore was modeled based on single sample gene set enrichment analysis (ssGSEA) for evaluating the relationships between m7G regulators and cancer clinical features, hallmark pathways, tumor immune microenvironment, immunotherapy response as well as pharmacotherapy sensitivity, and we illustrated that the m7Gscore exhibited tight correlations with prognosis, several immune features, immunotherapy response and drug sensitivity in most cancers. In conclusion, our pan-cancer analysis revealed that m7G regulators may exert critical roles in the tumor progression and immune microenvironment, and have the potential as biomarkers for predicting prognosis, immunotherapy response as well as candidate drug compounds for cancer patients.

Keywords:  N7-methylguanosine; immunotherapy; pan-cancer analysis; prognosis; tumor immune microenvironment

DOI:  https://doi.org/10.3389/fgene.2022.1016797
Curr Med Sci. 2022 Nov 09.

Maslinic Acid Suppresses High Glucose-induced Inflammation by Epigenetically Inhibiting TXNIP Expression.

Lin Wang, Yi-Fu Fan, Bing-Rui Li, Wen-Hui Sun, Li-Na Wang.

   OBJECTIVE: Hyperglycemia-induced inflammation and subsequent endothelial injuries ultimately lead to the pathogenesis of cardiovascular diseases associated with high mortality, such as atherosclerosis. Maslinic acid (MA) is a phytochemical with anti-inflammatory activity. However, it remains unknown whether it can inhibit diabetes-associated cardiovascular inflammation. The present study aimed to determine the effect of MA on high glucose-induced endothelial inflammation and apoptosis in human umbilical vein endothelial cells (HUVECs) and to explore the underlying mechanism.
METHODS: HUVECs were treated with high glucose to induce inflammation and apoptosis. Apoptosis was determined by flow cytometry. CCK-8 assay was used to examine cell viability. Production levels of cytokines were detected by quantitative realtime PCR (qPCR) and ELISA. Protein expression levels and signaling pathways activation were detected by Western blotting. RNA immunoprecipitation and qPCR were used to determine the N6-methyladenosine (m6A) levels of target mRNAs.
RESULTS: MA promoted the recruitment of RNA demethylase ALKBH5 to TXNIP mRNA, and subsequently enhanced its m6A demethylation. By this means, MA decreased the stability of TXNIP mRNA and downregulated its expression level. Subsequently, reactive oxygen species (ROS) and production of pro-inflammatory cytokines, including TNF-α, IL-6 and IL-1β, were inhibited. And high glucose-induced apoptosis in HUVECs was inhibited by MA.
CONCLUSION: MA ameliorates high glucose-induced endothelial inflammation and injury, serving as a new potential therapeutic application for protecting against diabetes-associated atherosclerosis and other inflammatory diseases.

Keywords:  N 6-methyladenosine; RNA demethylase ALKBH5; inflammation; maslinic acid

DOI:  https://doi.org/10.1007/s11596-022-2657-6
Genes (Basel). 2022 Nov 06. pii: 2050. [Epub ahead of print]13(11):

Translational Regulation by eIFs and RNA Modifications in Cancer.

Linzhu Zhang, Yaguang Zhang, Su Zhang, Lei Qiu, Yang Zhang, Ying Zhou, Junhong Han, Jiang Xie.

  Translation is a fundamental process in all living organisms that involves the decoding of genetic information in mRNA by ribosomes and translation factors. The dysregulation of mRNA translation is a common feature of tumorigenesis. Protein expression reflects the total outcome of multiple regulatory mechanisms that change the metabolism of mRNA pathways from synthesis to degradation. Accumulated evidence has clarified the role of an increasing amount of mRNA modifications at each phase of the pathway, resulting in translational output. Translation machinery is directly affected by mRNA modifications, influencing translation initiation, elongation, and termination or altering mRNA abundance and subcellular localization. In this review, we focus on the translation initiation factors associated with cancer as well as several important RNA modifications, for which we describe their association with cancer.

Keywords:  2′-O-dimethyladenosine (m6Am); 5-methylcytosine (m5C); N4-acetylcytidine (ac4C); N6; N6-methyladenosine (m6A); RNA modification; pseudouridine (Ψ); translation initiation factor; tumor

DOI:  https://doi.org/10.3390/genes13112050
Front Mol Biosci. 2022 ;9 1034928

Construction of m6A-based prognosis signature and prediction for immune and anti-angiogenic response.

Xiang-Xu Wang, Li-Hong Wu, Qiong-Yi Dou, Liping Ai, Yajie Lu, Shi-Zhou Deng, Qing-Qing Liu, Hongchen Ji, Hong-Mei Zhang.

  Background: Increasing evidence illustrated that m6A regulator-mediated modification plays a crucial role in regulating tumor immune and angiogenesis microenvironment. And the combination of immune checkpoint inhibitor and anti-angiogenic therapy has been approved as new first-line therapy for advanced HCC. This study constructed a novel prognosis signature base on m6A-mediated modification and explored the related mechanism in predicting immune and anti-angiogenic responses. Methods: Gene expression profiles and clinical information were collected from TCGA and GEO. The ssGSEA, MCPCOUNT, and TIMER 2.0 algorithm was used to Estimation of immune cell infiltration. The IC50 of anti-angiogenic drugs in GDSC was calculated by the "pRRophetic" package. IMvigor210 cohort and Liu et al. cohort were used to validate the capability of immunotherapy response. Hepatocellular carcinoma single immune cells sequencing datasets GSE140228 were collected to present the expression landscapes of 5 hub genes in different sites and immune cell subpopulations of HCC patients. Results: Three m6A clusters with distinct immune and angiogenesis microenvironments were identified by consistent cluster analysis based on the expression of m6A regulators. We further constructed a 5-gene prognosis signature (termed as m6Asig-Score) which could predict both immune and anti-angiogenic responses. We illustrated that high m6Asig-Score is associated with poor prognosis, advanced TNM stage, and high TP53 mutation frequency. Besides, the m6Asig-Score was negatively associated with immune checkpoint inhibitors and anti-angiogenic drug response. We further found that two of the five m6Asig-Score inner genes, B2M and SMOX, were associated with immune cell infiltration, immune response, and the sensitivity to sorafenib, which were validated in two independent immunotherapy cohorts and the Genomics of Drug Sensitivity in Cancer (GDSC) database. Conclusion: We constructed a novel prognosis signature and identified B2M and SMOX for predicting immune and anti-angiogenic efficacy in HCC, which may guide the combined treatment strategies of immunotherapy and anti-angiogenic therapy in HCC.

Keywords:  hepatocellular carcinoma; immunotherapy; m6A modification; prognostic signature; tumor microenvironment

DOI:  https://doi.org/10.3389/fmolb.2022.1034928
Front Oncol. 2022 ;12 964902

Methylation-driven gene DLL3 is a potential prognostic biomarker in ocular melanoma correlating with metastasis.

Ludi Yang, Gaoming Wang, Hanhan Shi, Shichong Jia, Jing Ruan, Ran Cui, Shengfang Ge.

   Background: Ocular melanoma is an aggressive malignancy with a high rate of metastasis and poor prognosis. Increasing evidence indicated that DNA methylation plays an important role in the occurrence and development of ocular melanoma. Hence, exploring new diagnostic and prognostic biomarkers at the genetic level may be beneficial to the prognosis of patients with ocular melanoma.
Methods: We collected DNA methylation and gene expression profiles of human UM (uveal melanoma) and CM (conjunctival melanoma) samples from various datasets. We conducted differential methylation and expression analyses to screen the potential biomarkers. Correlation analysis was performed to investigate the relationships between the expression level of DLL3 (delta-like protein 3) and the methylation level of its corresponding CpGs. We explored the prognostic and diagnostic value of DLL3 in UM and CM. Functional annotation and GSEA (gene set enrichment analysis) were applied to get insight into the possible biological roles of DLL3. A cohort of 60 ocular melanoma patients as well as UM and CM cell lines were used to validate our findings in bioinformatic analyses.
Results: We found that DLL3 was a methylation-driven gene correlating with UM metastasis. The CpGs of DLL3 are mainly located in the gene body and their methylation level positively correlated to DLL3 expression. Multivariate Cox regression analysis revealed that DLL3 was an independent protective factor for UM patients. High DLL3 expression significantly prolonged the overall survival and disease-free survival of UM patients. DLL3 also showed a promising power to distinguish CM from normal tissues. Functional annotation exhibited that DLL3 may suppress UM progression through modulating immune activities and down-regulating various signaling pathways. External datasets, biospecimens, and cell lines further validated the aberrant expression and prognostic role of DLL3 in ocular melanoma.
Conclusion: Methylation-driven gene DLL3 could serve as a new potential diagnostic and prognostic biomarker in ocular melanoma. Our findings may contribute to improving the clinical outcomes of patients with UM or CM.

Keywords:  DLL3; metastasis; methylation; ocular melanoma; prognosis

DOI:  https://doi.org/10.3389/fonc.2022.964902
Nat Methods. 2022 Nov 10.

Detection of m6A from direct RNA sequencing using a multiple instance learning framework.

Christopher Hendra, Ploy N Pratanwanich, Yuk Kei Wan, W S Sho Goh, Alexandre Thiery, Jonathan Göke.

RNA modifications such as m6A methylation form an additional layer of complexity in the transcriptome. Nanopore direct RNA sequencing can capture this information in the raw current signal for each RNA molecule, enabling the detection of RNA modifications using supervised machine learning. However, experimental approaches provide only site-level training data, whereas the modification status for each single RNA molecule is missing. Here we present m6Anet, a neural-network-based method that leverages the multiple instance learning framework to specifically handle missing read-level modification labels in site-level training data. m6Anet outperforms existing computational methods, shows similar accuracy as experimental approaches, and generalizes with high accuracy to different cell lines and species without retraining model parameters. In addition, we demonstrate that m6Anet captures the underlying read-level stoichiometry, which can be used to approximate differences in modification rates. Overall, m6Anet offers a tool to capture the transcriptome-wide identification and quantification of m6A from a single run of direct RNA sequencing.

DOI: https://doi.org/10.1038/s41592-022-01666-1
J Chemother. 2022 Nov 11. 1-10

Knockdown of METTL5 inhibits the Myc pathway to downregulate PD-L1 expression and inhibits immune escape of hepatocellular carcinoma cells.

Wenjie Xu, Shuyi Liu, Genshan Zhang, Jin Liu, Gang Cao.

  The incidence of hepatocellular carcinoma (HCC) is raised annually, which causes a great harm to people's health. This research aimed to investigate the influence and mechanism of methyltransferase-like 5 (METTL5) in HCC. According to The Cancer Genome Atlas (TCGA) database, METTL5 levels and prognosis was analyzed in HCC. Next, in HCC tissues and cells, METTL5 expression was examined via quantitative real-time polymerase chain reaction (qRT-PCR). Biological behaviors of HCC cells were assessed by Cell Counting Kit-8 (CCK-8), colony formation, transwell and flow cytometry assays. Gene Set Enrichment Analysis (GSEA) was applied to predict METTL5 related pathway. The possible binding sites of programmed cell death 1 ligand 1 (PD-L1) and myelocytomatosis viral oncogene (Myc) was predicted by JASPAR database. Western blot was utilized to test the change of PD-L1 and Myc pathway related proteins [cellular (c)-Myc, chaperonin containing TCP1 subunit 2 (CCT2) and chromobox protein homolog 3 (CBX3)]. In HCC tissues and cells, METTL5 expression was increased. High METTL5 expression was associated with poor prognosis. Knockdown of METTL5 inhibited HCC cell proliferation and invasion, induced cell apoptosis and reduced the expression of PD-L1, c-Myc, CCT2 and CBX3. The bind between PD-L1 and the Myc promoter in HCC cells was confirmed using Chip and luciferase reporter assays. Moreover, the influences of knockdown of METTL5 on PD-L1 expression and HCC cell biological behaviors were reversed by overexpression of Myc. Knockdown of METTL5 inhibited PD-L1 expression and malignant cell behavior of HCC through inhibiting the Myc pathway.

Keywords:  Hepatocellular carcinoma; METTL5; Myc pathway; cell apoptosis; cell invasion; cell proliferation

DOI:  https://doi.org/10.1080/1120009X.2022.2143614
Cells. 2022 Oct 31. pii: 3448. [Epub ahead of print]11(21):

Aberrant DNA and RNA Methylation Occur in Spinal Cord and Skeletal Muscle of Human SOD1 Mouse Models of ALS and in Human ALS: Targeting DNA Methylation Is Therapeutic.

Lee J Martin, Danya A Adams, Mark V Niedzwiecki, Margaret Wong.

  Amyotrophic lateral sclerosis (ALS) is a fatal disease. Skeletal muscles and motor neurons (MNs) degenerate. ALS is a complex disease involving many genes in multiple tissues, the environment, cellular metabolism, and lifestyles. We hypothesized that epigenetic anomalies in DNA and RNA occur in ALS and examined this idea in: (1) mouse models of ALS, (2) human ALS, and (3) mouse ALS with therapeutic targeting of DNA methylation. Human superoxide dismutase-1 (hSOD1) transgenic (tg) mice were used. They expressed nonconditionally wildtype (WT) and the G93A and G37R mutant variants or skeletal muscle-restricted WT and G93A and G37R mutated forms. Age-matched non-tg mice were controls. hSOD1 mutant mice had increased DNA methyltransferase enzyme activity in spinal cord and skeletal muscle and increased 5-methylcytosine (5mC) levels. Genome-wide promoter CpG DNA methylation profiling in skeletal muscle of ALS mice identified hypermethylation notably in cytoskeletal genes. 5mC accumulated in spinal cord MNs and skeletal muscle satellite cells in mice. Significant increases in DNA methyltransferase-1 (DNMT1) and DNA methyltransferase-3A (DNMT3A) levels occurred in spinal cord nuclear and chromatin bound extracts of the different hSOD1 mouse lines. Mutant hSOD1 interacted with DNMT3A in skeletal muscle. 6-methyladenosine (6mA) RNA methylation was markedly increased or decreased in mouse spinal cord depending on hSOD1-G93A model, while fat mass and obesity associated protein was depleted and methyltransferase-like protein 3 was increased in spinal cord and skeletal muscle. Human ALS spinal cord had increased numbers of MNs and interneurons with nuclear 5mC, motor cortex had increased 5mC-positive neurons, while 6mA was severely depleted. Treatment of hSOD1-G93A mice with DNMT inhibitor improved motor function and extended lifespan by 25%. We conclude that DNA and RNA epigenetic anomalies are prominent in mouse and human ALS and are potentially targetable for disease-modifying therapeutics.

Keywords:  6-methyladenosine; CpG island; Dnmt3A; FTO; cytosine methylation; motor neuron

DOI:  https://doi.org/10.3390/cells11213448
Genes Genomics. 2022 Nov 08.

Guanine nucleotide-binding protein 2, GNBP2, accelerates the progression of clear cell renal cell carcinoma via regulation of STAT3 signaling transduction pathway.

Qingfei Cao, Jiaji Ma, Weitao Li, Peng Hong, Tong Shen, Ming Tong.

   BACKGROUND: Guanine nucleotide-binding protein 2 (GNBP2) is a GTPase that has critical roles in host immunity and some types of cancer, but its function in clear cell renal cell carcinoma (ccRCC) is not fully understood.
OBJECTIVE: This work explored the role of GNBP2 in ccRCC progression and the underlying molecular mechanism.
METHODS: Two public human cancer databases TNMplot and TISIDB were employed to analyze the expression pattern of GNBP2 during ccRCC progression and the correlation between GNBP2 expression and clinical features of ccRCC patients. GNBP2 functions in ccRCC cells were determined by EdU staining, flow cytometry, scratch wound assay, transwell assay, and xenograft model. Gene expression was evaluated using qPCR, Western blot, immunofluorescence staining, and immunohistochemical staining.
RESULTS: GNBP2 expression was significantly elevated in ccRCC tissues and increased gradually with the increasing tumor grades. Patients with higher GNBP2 expression had shorter overall survival times. Knockdown of GNBP2 suppressed tumor cell proliferation and cell cycle progression and reduced the capability of migration and invasion, while GNBP2 overexpression exhibited protumor effects. GNBP2 silencing by RNA interference significantly inhibited the tumor growth of tumor-bearing nude mice and decreased the proliferation marker Ki67. Mechanistically, GNBP2 downregulation suppressed the STAT3 signaling transduction, as it reduced the phosphorylation of STAT3 and modulated the expression of the target genes, including c-Myc, MMP2, N-cadherin, and E-cadherin.
CONCLUSION: These findings reveal that GNBP2 promotes ccRCC progression by regulating STAT3 signaling transduction, indicating that GNBP2 might be a promising molecular target for ccRCC therapy.

Keywords:  Cell signaling; Clear cell renal cell carcinoma; Disease progression; Guanine nucleotide-binding protein 2; Signal transducer and transcription activator 3

DOI:  https://doi.org/10.1007/s13258-022-01334-w
Exp Hematol Oncol. 2022 Nov 08. 11(1): 87

Low expression of PEBP1P2 promotes metastasis of clear cell renal cell carcinoma by post-transcriptional regulation of PEBP1 and KLF13 mRNA.

Lei Yang, Haoli Yin, Yi Chen, Chun Pan, Hexing Hang, Yanwen Lu, Wenliang Ma, Xin Li, Weidong Gan, Hongqian Guo, Dongmei Li.

   BACKGROUND: Pseudogenes play an essential role in tumor occurrence and progression. However, the functions and mechanisms of pseudogenes in clear cell renal cell carcinoma (ccRCC) remain largely elusive.
METHODS: We quantified PEBP1P2 expression in ccRCC tissues and cells using fluorescence in situ hybridization and real-time PCR. Besides, we evaluated the role of PEBP1P2 in ccRCC using a lung metastasis model and a transwell assay. Finally, we documented the interactions between PEBP1P2, PEBP1, and KLF13 by performing luciferase, RNA immunoprecipitation, RNA pulldown, and targeted RNA demethylation assays.
RESULTS: Low PEBP1P2 expression correlates significantly with advanced stages and poor prognosis in ccRCC patients. Besides, PEBP1P2 overexpression inhibits ccRCC metastasis formation in vivo and in vitro. Interestingly, PEBP1P2 directly interacted with 5-methylcytosine (m5C)-containing PEBP1 mRNA and recruited the YBX1/ELAVL1 complex, stabilizing PEBP1 mRNA. In addition, PEBP1P2 increased KLF13 mRNA levels by acting as a sponge for miR-296, miR-616, and miR-3194.
CONCLUSIONS: PEBP1P2 inhibits ccRCC metastasis formation and regulates both PEBP1 and KLF13. Therefore, molecular therapies targeting PEBP1P2 might be an effective treatment strategy against ccRCC and other cancers with low PEBP1P2 levels.

Keywords:  Clear cell renal cell carcinoma; KLF13; PEBP1; PEBP1P2; m5C modification

DOI:  https://doi.org/10.1186/s40164-022-00346-2
Int J Oncol. 2023 Jan;pii: 4. [Epub ahead of print]62(1):

Role of N6‑methyladenosine in the pathogenesis, diagnosis and treatment of pancreatic cancer (Review).

Tong Ye, Jiaxin Wang, Haiying Zhao, Guiping Zhao, Peng Li.

  Pancreatic cancer (PC) ranks as the seventh leading cause of cancer‑associated mortality, and is predicted to become the third leading cause of cancer‑associated mortality by the year 2025. Although advanced modalities of diagnosis and treatment have been continuously emerging, the mortality rate (466,003) approximated to that of the morbidity rate (495,773) in 2020. N6‑methyladenosine (m6A) has been shown to be methylated on the sixth N atom of adenine in RNA, which occurs co‑transcriptionally and serves to regulate gene expression post‑transcriptionally. The discovery of m6A has heralded a new era in the scientific investigation of PC. In the present review article, the classical conception of m6A and emerging hypotheses regarding its role are summarized, and the function of m6A in carcinogenesis and progression of PC is then discussed, followed by the potential roles of m6A in the diagnosis of PC and in therapeutic applications. However, this new era is only at the initial stages, and the extent to which m6A influences PC is still poorly understood. In view of this, the present review article also summarizes the developments at the frontier of the interaction between m6A and PC, and discusses strategies through which m6A may provide a promising avenue for anticancer therapy.

Keywords:  N6‑methyladenosine; diagnosis; pancreatic cancer; pathogenesis; treatment

DOI:  https://doi.org/10.3892/ijo.2022.5452
Nat Commun. 2022 Nov 09. 13(1): 6762

METTL14 is required for exercise-induced cardiac hypertrophy and protects against myocardial ischemia-reperfusion injury.

Lijun Wang, Jiaqi Wang, Pujiao Yu, Jingyi Feng, Gui-E Xu, Xuan Zhao, Tianhui Wang, H Immo Lehmann, Guoping Li, Joost P G Sluijter, Junjie Xiao.

RNA m6A modification is the most widely distributed RNA methylation and is closely related to various pathophysiological processes. Although the benefit of regular exercise on the heart has been well recognized, the role of RNA m6A in exercise training and exercise-induced physiological cardiac hypertrophy remains largely unknown. Here, we show that endurance exercise training leads to reduced cardiac mRNA m6A levels. METTL14 is downregulated by exercise, both at the level of RNA m6A and at the protein level. In vivo, wild-type METTL14 overexpression, but not MTase inactive mutant METTL14, blocks exercise-induced physiological cardiac hypertrophy. Cardiac-specific METTL14 knockdown attenuates acute ischemia-reperfusion injury as well as cardiac dysfunction in ischemia-reperfusion remodeling. Mechanistically, silencing METTL14 suppresses Phlpp2 mRNA m6A modifications and activates Akt-S473, in turn regulating cardiomyocyte growth and apoptosis. Our data indicates that METTL14 plays an important role in maintaining cardiac homeostasis. METTL14 downregulation represents a promising therapeutic strategy to attenuate cardiac remodeling.

DOI: https://doi.org/10.1038/s41467-022-34434-y
Front Vet Sci. 2022 ;9 933850

m6A and miRNA jointly regulate the development of breast muscles in duck embryonic stages.

Lihong Gu, Shunjin Zhang, Boling Li, Qicheng Jiang, Tieshan Xu, Yongzhen Huang, Dajie Lin, Manping Xing, Lili Huang, Xinli Zheng, Feng Wang, Zhe Chao, Weiping Sun.

  N6-methyladenosine (m6A) is an abundant internal mRNA modification and plays a crucial regulatory role in animal growth and development. In recent years, m6A modification has been found to play a key role in skeletal muscles. However, whether m6A modification contributes to embryonic breast muscle development of Pekin ducks has not been explored. To explore the role of m6A in embryonic breast muscle development of ducks, we performed m6A sequencing and miRNA sequencing for the breast muscle of duck embryos on the 19th (E19) and 27th (E27) days. A total of 12,717 m6A peaks were identified at E19, representing a total of 7,438 gene transcripts. A total of 14,703 m6A peaks were identified, which overlapped with the transcripts of 7,753 genes at E27. Comparing E19 and E27, we identified 2,347 differential m6A peaks, which overlapped with 1,605 m6A-modified genes (MMGs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that MMGs were enriched in multiple muscle- or fat-related pathways, which was also revealed from our analysis of differentially expressed genes (DEGs). Conjoint analysis of m6A-seq and RNA-seq data showed that pathways related to β-oxidation of fatty acids and skeletal muscle development were significantly enriched, suggesting that m6A modification is involved in the regulation of fat deposition and skeletal muscle development. There were 90 upregulated and 102 downregulated miRNAs identified between the E19 and E27 stages. Through overlapping analysis of genes shared by MMGs and DEGs and the targets of differentially expressed miRNAs (DEMs), we identified six m6A-mRNA-regulated miRNAs. Finally, we found that m6A modification can regulate fat deposition and skeletal muscle development. In conclusion, our results suggest that m6A modification is a key regulator for embryonic breast muscle development and fat deposition of ducks by affecting expressions of mRNAs and miRNAs. This is the first study to comprehensively characterize the m6A patterns in the duck transcriptome. These data provide a solid basis for future work aimed at determining the potential functional roles of m6A modification in adipose deposition and muscle growth.

Keywords:  breast muscles; ducks; embryo; m6A sequencing; miRNAs sequencing

DOI:  https://doi.org/10.3389/fvets.2022.933850
Neoplasma. 2022 Nov 11. pii: 220817N841. [Epub ahead of print]

SUMO1-modified DNA methyltransferase 1 induces DNA hypermethylation of VWC2 in the development of colorectal cancer.

Xiao-Hu Cheng, Tong-Tong Xu, Lian-Bang Zhou, Fang-Yuan Li, Song Wang, Huan-Ru Liang, Jing-Jing Tang, Chang-Qin Duan, Heng Jiang, Ying-Feng Zhang, Zhi-Ning Liu.

Aberrant DNA methylation of genes is closely linked to many aspects of tumor development. This study focuses on the effect of DNA hypermethylation of von Willebrand factor C domain containing 2 (VWC2) on colorectal cancer (CRC) progression and the underpinning mechanism. According to data in the bioinformatic systems, VWC2 had the highest degree of DNA methylation in colonic adenocarcinoma, and it showed DNA hypermethylation in rectal adenocarcinoma as well. CRC and the para-tumorous tissues were collected from 86 patients. VWC2 was expressed at low levels in CRC samples and inversely correlated with tumor stage and tumor biomarker expression. DNA hypermethylation and reduced expression of VWC2 were also detected in CRC HCT-116 and HT29 cells. VWC2 overexpression suppressed the malignant growth of cells in vitro and in vivo. Co-immunoprecipitation and western blot assays showed that small ubiquitin-like modifier 1 (SUMO1) mediated SUMOylation of DNA methyltransferase 1 (DNMT1) and strengthened its protein stability, which promoted DNA methylation and suppression of the VWC2 gene. In summary, this study demonstrates that SUMO1-mediated activation of DNMT1 induces DNA methylation and downregulation of VWC2 in CRC to augment cancer development.

DOI: https://doi.org/10.4149/neo_2022_220817N841
Arch Biochem Biophys. 2022 Nov 08. pii: S0003-9861(22)00349-6. [Epub ahead of print] 109464

YTHDF1 regulates endoplasmic reticulum stress, NF-κB, MAPK and PI3K-AKT signaling pathways in inflammatory osteoclastogenesis.

Mingli He, Di Li, Caihong Fang, Qiong Xu.

  Abnormal increases in osteoclast differentiation and activity contribute to excessive bone resorption in inflammatory bone diseases. The specific m6A-binding protein YT521-B homology domain family 1 (YTHDF1) participates in many physiopathological processes by regulating mRNA stability or translation. However, whether YTHDF1 is involved in the regulation of inflammatory osteoclastogenesis remains a mystery. This study revealed that YTHDF1 expression was upregulated during lipopolysaccharide (LPS)-stimulated osteoclast differentiation. Knockdown of Ythdf1 inhibited osteoclast formation, bone resorption and the expression of osteoclast-related genes (Tnfrsf11a, Traf6, Mmp9 and Acp5). Analysis of RNA sequencing data showed that the genes downregulated by Ythdf1 knockdown were closely associated with endoplasmic reticulum (ER) stress and osteoclast differentiation. Western blotting confirmed that Ythdf1 depletion suppressed activation of the ER stress-related PERK, IRE1α and ATF6 signaling pathways. The ER stress activator tunicamycin (Tm) partially rescued the decreased expression of Mmp9 and Acp5 caused by Ythdf1 deficiency. Meanwhile, Ythdf1 depletion inhibited the phosphorylation levels of key proteins in the NF-κB, MAPK and PI3K-AKT signaling pathways and decreased the mRNA stability of Tnfrsf11a, which is the major upstream signaling molecule that mediates the activation of these pathways during osteoclast differentiation. In conclusion, our findings suggest that Ythdf1 knockdown inhibits inflammatory osteoclast differentiation and function by suppressing ER stress signaling pathways. Ythdf1 knockdown also inactivates the signaling pathways involved in osteoclast differentiation by inhibiting Tnfrsf11a mRNA stability. These findings will help shed light on the molecular mechanisms of m6A-mediated epigenetic regulation in inflammatory osteoclastogenesis.

Keywords:  Endoplasmic reticulum stress; Lipopolysaccharide; Osteoclast differentiation; YTHDF1; mRNA stability

DOI:  https://doi.org/10.1016/j.abb.2022.109464