bims-rimeca 2022-07-31 papers

bims-rimeca

Biomed News

on RNA methylation in cancer

Issue of 2022–07–31
twelve papers selected by
Sk Ramiz Islam, Saha Institute of Nuclear Physics

The m6A RNA Modification Quantity and the Prognostic Effect of Reader YTHDC2 in Colorectal Cancer.
m6A Regulator-Based Methylation Modification Patterns Characterized by Distinct Tumor Microenvironment Immune Profiles in Rectal Cancer.
N6-Methyladenosine RNA-Binding Protein YTHDF1 in Gastrointestinal Cancers: Function, Molecular Mechanism and Clinical Implication.
Methyltransferase-like 3 facilitates lung cancer progression by accelerating m6A methylation-mediated primary miR-663 processing and impeding SOCS6 expression.
WWP2 overexpression inhibits the antitumor effects of doxorubicin in hepatocellular carcinoma.
N6 -methyladenosine RNA demethylase FTO regulates extracellular matrix-related genes and promotes pancreatic cancer cell migration and invasion.
The function of Wtap in N6-adenosine methylation of mRNAs controls T cell receptor signaling and survival of T cells.
m6A methyltransferase METTL3-induced lncRNA SNHG17 promotes lung adenocarcinoma gefitinib resistance by epigenetically repressing LATS2 expression.
N6-Methyladenosine-modified lncRNA LINREP promotes Glioblastoma progression by recruiting the PTBP1/HuR complex.
SQSTM1/p62 regulate breast cancer progression and metastasis by inducing cell cycle arrest and regulating immune cell infiltration.
GOT2 Silencing Promotes Reprogramming of Glutamine Metabolism and Sensitizes Hepatocellular Carcinoma to Glutaminase Inhibitors.
N6-Methyladenosine-induced miR-143-3p promotes intervertebral disc degeneration by regulating SOX5.

Clin Med Insights Oncol. 2022 ;16 11795549221104441

The m6A RNA Modification Quantity and the Prognostic Effect of Reader YTHDC2 in Colorectal Cancer.

Tianyu Liu, Wentao Tang, Yijiao Chen, Yu Liu, Donghao Xu, Yudong Jiang, Shizhao Zhou, Xiaorui Qin, Li Ren, Wenju Chang, Jianmin Xu.

   Background: N6-methyladenosine (m6A) modification plays crucial roles in cancers. However, its alteration in colorectal cancer (CRC) is still poorly described. The purpose of this study is to explore the change of m6A modification and the function of m6A binding protein YTHDC2 in CRC.
Methods: The global level of m6A modification was detected by mass spectrometry and dot blotting assay. The expression of YTHDC2 was investigated using The Cancer Genome Atlas and using real-time polymerase chain reaction (RT-qPCR), western blotting, and immunohistochemistry based on CRC tissues. Kaplan-Meier analysis and Cox proportional hazards regression were performed to analyze the prognostic value of YTHDC2. RNA immunoprecipitation (RIP)-seq and m6A immunoprecipitation (MeRIP)-seq were used to explore the direct targets of YTHDC2. Gene oncology (GO) and Gene Set Enrichment Analysis (GSEA) were used to explore the pathways that could be influenced by YTHDC2.
Results: No significant difference was observed in the global level of m6A modification on total RNA or mRNA between CRC and adjacent nontumor tissues. We further found a significant decreasing of YTHDC2 in CRC tissues. Kaplan-Meier analysis indicated that lower expression of YTHDC2 was related to the worse disease-free survival and overall survival. In addition, lower expression of YTHDC2 was an independent worse prognostic factor in univariate and multivariate Cox regression analysis. Using YTHDC2-RIP-seq and MeRIP-seq, we identified that YTHDC2 could participate in several important biological signal pathways.
Conclusions: In summary, this study suggested that the global level of m6A did not change in CRC and identified that lower YTHDC2 as a prognostic marker for worse survival of CRC.

Keywords:  Colorectal cancer; N6-methyladenosine; RNA modification; YTHDC2; prognostic biomarker

DOI:  https://doi.org/10.1177/11795549221104441
Front Oncol. 2022 ;12 879405

m6A Regulator-Based Methylation Modification Patterns Characterized by Distinct Tumor Microenvironment Immune Profiles in Rectal Cancer.

Kaili Liao, Jialing Hu, Yu Huang, Siji Yu, Qijun Yang, Fan Sun, Chengfeng Wu, Yunqi Cheng, Wenyige Zhang, Xue Zhang, Hongyu Li, Xiaozhong Wang.

   Background: Previous studies reported the related role of RNA n6-methyladenosine (m6A) modification in tumorigenesis and development. However, it is not clear whether m6A modification also plays a potential role in the immune regulation of rectal cancer (RC) and the formation of tumor microenvironment.
Methods: In this study, we screened 23 m6A regulatory factors from 369 rectal cancer specimens, further determined the modification patterns of m6A in RC, and systematically linked these modification patterns with the characteristics of TME cell infiltration. The principal component analysis (PCA) algorithm was used to evaluate the m6A modification pattern of a single tumor related to immune response.
Results: Three different m6A modification patterns were found in the measurement results, which are related to different clinical results and biological pathways. TME identification results show that the identified m6A pattern is closely related to immune characteristics. According to the m6Ascore extracted from m6A-related signature genes, RC patients were divided into high and low score subgroups combined with tumor mutation burden. Patients with high tumor mutation burden and higher m6Ascore have a significant survival advantage and enhanced immune infiltration. Further analysis showed that patients with higher m6Ascore had higher PD-L1 expression levels and showed better immune response and lasting clinical benefits.
Conclusions: M6A modification plays a crucial role in the formation of TME diversity and complexity. The evaluation of the m6A modification mode will help us to enhance our understanding of the characteristics of TME infiltration and provide new insights for more effective immunotherapy strategies.

Keywords:  M6A modification; immune profiles; immunotherapy; rectal cancer; tumor microenvironment

DOI:  https://doi.org/10.3389/fonc.2022.879405
Cancers (Basel). 2022 Jul 18. pii: 3489. [Epub ahead of print]14(14):

N6-Methyladenosine RNA-Binding Protein YTHDF1 in Gastrointestinal Cancers: Function, Molecular Mechanism and Clinical Implication.

Danyu Chen, Henley Cheung, Harry Cheuk-Hay Lau, Jun Yu, Chi Chun Wong.

  N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic cell mRNA, and this modification plays a key role in regulating mRNA translation, splicing, and stability. Emerging evidence implicates aberrant m6A as a crucial player in the occurrence and development of diseases, especially GI cancers. Among m6A regulators, YTHDF1 is the most abundant m6A reader that functionally connects m6A-modified mRNA to its eventual fate, mostly notably protein translation. Here, we summarized the function, molecular mechanisms, and clinical implications of YTHDF1 in GI cancers. YTHDF1 is largely upregulated in multiple GI cancer and its high expression predicts poor patient survival. In vitro and in vivo experimental evidence largely supports the role of YTDHF1 in promoting cancer initiation, progression, and metastasis, which suggests the oncogenic function of YTHDF1 in GI cancers. Besides, YTHDF1 overexpression is associated with changes in the tumor microenvironment that are favorable to tumorigenesis. Mechanistically, YTHDF1 regulates the expression of target genes by promoting translation, thereby participating in cancer-related signaling pathways. Targeting YTHDF1 holds therapeutic potential, as the overexpression of YTHDF1 is associated with tumor resistance to chemotherapy and immunotherapy. In summary, YTHDF1-mediated regulation of m6A modified mRNA is an actionable target and a prognostic factor for GI cancers.

Keywords:  N6-methyladenosine; YTHDF1; colorectal cancer; drug resistance; gastric cancer; gastrointestinal cancer; immunotherapy; liver cancer; oncogenic signaling

DOI:  https://doi.org/10.3390/cancers14143489
J Cancer Res Clin Oncol. 2022 Jul 30.

Methyltransferase-like 3 facilitates lung cancer progression by accelerating m6A methylation-mediated primary miR-663 processing and impeding SOCS6 expression.

Shengshu Li, Xiaoxin Lu, Dongyang Zheng, Weizong Chen, Yuzhu Li, Fang Li.

   OBJECTIVE: Lung cancer (LC) remains a threatening health issue worldwide. Methyltransferase-like protein 3 (METTL3) is imperative in carcinogenesis via m6A modification of microRNAs (miRNAs). This study estimated the effect of METTL3 in LC by regulating m6A methylation-mediated pri-miR-663 processing.
METHODS: miR-663 expression in 4 LC cell lines and normal HBE cells was determined using RT-qPCR. A549 and PC9 LC cells selected for in vitro studies were transfected with miR-663 mimics or inhibitor. Cell viability, migration, invasion, proliferation, and apoptosis were detected by CCK-8, Transwell, EdU, and flow cytometry assays. The downstream target genes and binding sites of miR-663 were predicted via Starbase database and validated by dual-luciferase assay. LC cells were delivered with oe-METTL3/sh-METTL3. Crosslinking between METTL3 and DGCR8 was verified by co-immunoprecipitation. Levels of m6A, miR-663, and pri-miR-663 were measured by m6A dot blot assay and RT-qPCR. m6A modification of pri-miR-663 was verified by Me-RIP assay. Finally, the effects of METTL3 in vivo were ascertained by tumor xenograft in nude mice.
RESULTS: miR-663 was upregulated in LC cells, and miR-663 overexpression promoted cell proliferation, migration, invasion, and inhibited apoptosis, but miR-663 knockdown exerted the opposite effects. miR-663 repressed SOCS6 expression. SOCS6 overexpression annulled the promotion of miR-663 on LC cell growth. METTL3 bound to DGCR8, and METTL3 silencing elevated the levels of pri-miR-663 and m6A methylation-modified pri-miR-663, and suppressed miR-663 maturation and miR-663 expression. METTL3 facilitated tumor growth in mice through the miR-663/SOCS6 axis.
CONCLUSION: METTL3 promotes LC progression by accelerating m6A methylation-mediated pri-miR-663 processing and repressing SOCS6.

Keywords:  Lung cancer; Methyltransferase-like 3; Migration and invasion; Primary miR-663; SOCS6; Tumor formation assay; m6A methylation

DOI:  https://doi.org/10.1007/s00432-022-04128-5
Cell Biol Int. 2022 Jul 26.

WWP2 overexpression inhibits the antitumor effects of doxorubicin in hepatocellular carcinoma.

Yong Qin, Chao-Jun Wang, Hai-Lin Ye, Guan-Xiong Ye, Shi Wang, De-Biao Pan, Jun Wang, He-Juan Shen, Sheng-Qian Xu.

  Hepatocellular carcinoma (HCC) is a common liver cancer that accounts for 90% of cases. Doxorubicin exhibits a broad spectrum of antitumor activity and is one of the most active agents in HCC. WW domain-containing protein 2 (WWP2) is highly expressed in HCC tissues and activates protein kinase B (AKT) signaling pathway to enhance tumor metastasis. However, the role of WWP2 in the glycolysis and antitumor effects of doxorubicin and the epigenetic alterations of WWP2 in HCC remain to be elucidated. The levels of WWP2 and N6-methyladenosine methyltransferase-like 3 (METTL3) in clinical samples and cells were investigated. WWP2 were silenced or overexpressed to study the role of WWP2 in regulating cell proliferation, colony formation, and glycolysis. RNA immunoprecipitation was performed to test m6 A levels. Quantitative reverse-transcription polymerase chain reaction (RT-PCR) and Western blot were used to measure mRNA and protein, respectively. WWP2 silencing inhibits cell proliferation, colony formation, and glycolysis, while WWP2 overexpression has the inverse effects via the AKT signaling pathway. Silencing WWP2 enhances doxorubicin's antitumor effect, while WWP2 overexpression suppresses doxorubicin's antitumor effect. Data also support that METTL3 mediates WWP2 m6A modification, and m6A reader, IGF2BP2, binds to the methylated WWP2 to promote the stability of WWP2, leading to upregulation of WWP2. METTL3 mediates WWP2 m6A modification, which can be recognized and bound by IGF2BP2 to increase the stability of WWP2, leading to WWP2 overexpression which inhibits the antitumor effects of doxorubicin through METTL3/WWP2/AKT/glycolysis axis.

Keywords:  AKT signaling; doxorubicin; glycolysis; hepatocellular carcinoma; m6A RNA methylation

DOI:  https://doi.org/10.1002/cbin.11856
Cancer Med. 2022 Jul 25.

N6 -methyladenosine RNA demethylase FTO regulates extracellular matrix-related genes and promotes pancreatic cancer cell migration and invasion.

Wei Wang, Ying He, Lun Wu, Lu-Lu Zhai, Long-Jiang Chen, Li-Chao Yao, Kai-Huan Yu, Zhi-Gang Tang.

  Pancreatic cancer (PC) is a deadly disease, and its post-transcriptional gene regulation mechanism remains unclear. The abundant extracellular matrix (ECM) in PC plays an important role in tumor progression. This study is the first to focus on the role of N6 -methyladenosine (m6 A) RNA methylation, an emerging post-transcriptional regulatory mechanism, in the regulation of the ECM in PC. Here, we found that ADAMTS2, COL12A1, and THBS2 were associated with the prognosis of PC by comprehensive analysis of differentially expressed genes from two independent GEO expression profile datasets and m6 A-related genes in RMVar database (PAAD). GO and KEGG enrichment analysis found that these m6 A-related targets are chiefly functionally concentrated in the ECM region and participate in ECM signal transduction. Correlation analysis revealed that these genes can be regulated by the demethylase FTO. Cell biology function assays showed that knockdown of FTO-inhibited PC cell abilities to migrate and invade in vitro. qRT-PCR and MeRIP experiments showed that after knockdown of FTO, the mRNA levels of ADAMTS2, COL12A1, and THBS2 and their m6 A modification levels were significantly reduced. These results indicate that m6 A RNA demethylation is associated with the regulation of ECM in PC. In conclusion, m6 A RNA demethylase FTO regulates ECM-related genes and promotes PC cell abilities to migrate and invade, our work provides a new perspective on the molecular mechanism of PC progression.

Keywords:  RNA methylation; extracellular matrix; m6A; pancreatic cancer

DOI:  https://doi.org/10.1002/cam4.5054
Nat Immunol. 2022 Jul 25.

The function of Wtap in N6-adenosine methylation of mRNAs controls T cell receptor signaling and survival of T cells.

Taku Ito-Kureha, Cristina Leoni, Kayla Borland, Giulia Cantini, Marian Bataclan, Rebecca N Metzger, Gregor Ammann, Anne B Krug, Annalisa Marsico, Stefanie Kaiser, Stefan Canzar, Stefan Feske, Silvia Monticelli, Julian König, Vigo Heissmeyer.

T cell antigen-receptor (TCR) signaling controls the development, activation and survival of T cells by involving several layers and numerous mechanisms of gene regulation. N6-methyladenosine (m6A) is the most prevalent messenger RNA modification affecting splicing, translation and stability of transcripts. In the present study, we describe the Wtap protein as essential for m6A methyltransferase complex function and reveal its crucial role in TCR signaling in mouse T cells. Wtap and m6A methyltransferase functions were required for the differentiation of thymocytes, control of activation-induced death of peripheral T cells and prevention of colitis by enabling gut RORγt+ regulatory T cell function. Transcriptome and epitranscriptomic analyses reveal that m6A modification destabilizes Orai1 and Ripk1 mRNAs. Lack of post-transcriptional repression of the encoded proteins correlated with increased store-operated calcium entry activity and diminished survival of T cells with conditional genetic inactivation of Wtap. These findings uncover how m6A modification impacts on TCR signal transduction and determines activation and survival of T cells.

DOI: https://doi.org/10.1038/s41590-022-01268-1
Cell Death Dis. 2022 Jul 28. 13(7): 657

m6A methyltransferase METTL3-induced lncRNA SNHG17 promotes lung adenocarcinoma gefitinib resistance by epigenetically repressing LATS2 expression.

Heng Zhang, Shao-Qiang Wang, Li Wang, Hang Lin, Jie-Bo Zhu, Ri Chen, Lin-Feng Li, Yuan-Da Cheng, Chao-Jun Duan, Chun-Fang Zhang.

Gefitinib has been widely applied for the treatment of lung adenocarcinoma (LUAD). However, the long-term application of gefitinib usually leads to acquired drug resistance in tumour patients, resulting in clinical treatment failure. Small nucleolar host gene 17 (SNHG17) has been shown to play a regulatory role in LUAD progression. Nevertheless, the role of SNHG17 in LUAD gefitinib resistance remains elusive. The expression pattern of SNHG17 was examined in tissues and cell lines of gefitinib-sensitive and gefitinib-resistant LUAD, respectively. Gain- and loss-of-function experiments were employed to assess the biological functions of SNHG17 in cell proliferation and apoptosis, as well as aggressive phenotypes of LUAD cells. MeRIP-qPCR and colorimetric quantificational analysis were performed to detect m6A modifications and contents. Fluorescence in situ hybridisation (FISH) and subcellular fractionation analysis were used to reveal the distribution of SNHG17. RIP and ChIP assays were performed to further validate the SNHG17/EZH2/LATS2 regulatory axis. A xenograft tumour growth assay was conducted to evaluate the role of SNHG17 in LUAD gefitinib resistance in vivo. SNHG17 was upregulated in gefitinib-resistant LUAD tissues and cell lines. Functional assays showed that SNHG17 aggravated the malignant phenotypes of gefitinib-resistant LUAD cells. In addition, METTL3-mediated N6-methyladenosine modification could induce the upregulation of SNHG17by stabilising its RNA transcript. Mechanistically, SNHG17 epigenetically repressed the expression of LATS2 by recruiting EZH2 to the promoter region of LATS2. The regulatory role of the SNHG17/EZH2/LATS2 axis in LUAD gefitinib resistance was further supported in vivo. Collectively, our findings suggested that SNHG17 induced by METTL3 could promote LUAD gefitinib resistance by epigenetically repressing LATS2 expression.

DOI: https://doi.org/10.1038/s41419-022-05050-x
Cell Death Differ. 2022 Jul 23.

N6-Methyladenosine-modified lncRNA LINREP promotes Glioblastoma progression by recruiting the PTBP1/HuR complex.

Xiaoshuai Ji, Zihao Liu, Jiajia Gao, Xin Bing, Dong He, Wenqing Liu, Yunda Wang, Yanbang Wei, Xianyong Yin, Fenglin Zhang, Min Han, Xiangdong Lu, Zixiao Wang, Qian Liu, Tao Xin.

Glioblastoma multiforme (GBM) is acknowledged as the most aggressive primary brain tumor in adults. It is typically characterized by the high heterogeneity which corresponds to extensive genetic mutations and complex alternative splicing (AS) profiles. Known as a major repressive splicing factor in AS, polypyrimidine tract-binding protein 1 (PTBP1) is involved in the exon skipping events of multiple precursor mRNAs (pre-mRNAs) in GBM. However, precise mechanisms that modulate the expression and activity of PTBP1 remain to be elucidated. In present study, we provided evidences for the role of a long intergenic noncoding RNA (LINREP) implicated in the regulation of PTBP1-induced AS. LINREP interacted with PTBP1 and human antigen R (HuR, ELAVL1) protein complex and protected PTBP1 from the ubiquitin-proteasome degradation. Consequently, a broad spectrum of PTBP1-induced spliced variants was generated by exon skipping, especially for the skipping of reticulon 4 (RTN4) exon 3. Interestingly, LINREP also promoted the dissociation of nuclear UPF1 from PTBP1, which increased the binding of PTBP1 to RTN4 transcripts, thus enhancing the skipping of RTN4 exon 3 to some extent. Besides, HuR recruitment was essential for the stabilization of LINREP via a manner dependent on N6-methyladenosine (m6A) formation and identification. Taken together, our results demonstrated the functional significance of LINREP in human GBM for its dual regulation of PTBP1-induced AS and its m6A modification modality, implicating that HuR/LINREP/PTBP1 axis might serve as a potential therapeutic target for GBM.

DOI: https://doi.org/10.1038/s41418-022-01045-5
Genes Dis. 2022 Sep;9(5): 1332-1344

SQSTM1/p62 regulate breast cancer progression and metastasis by inducing cell cycle arrest and regulating immune cell infiltration.

Jia-Long Qi, Jin-Rong He, Cun-Bao Liu, Shu-Mei Jin, Xu Yang, Hong-Mei Bai, Yan-Bing Ma.

  The autophagy adaptor protein SQSTM1/p62 is overexpressed in breast cancer and has been identified as a metastasis-related protein. However, the mechanism by which SQSTM1/p62 contributes to breast cancer progression and tumor microenvironment remains unclear. This study revealed that silencing SQSTM1/p62 expression suppressed breast cancer progression via regulating cell proliferation and reshaping the tumor microenvironment (TME). Here, we found that SQSTM1/p62 was overexpressed in multiple human cancer tissue types and that was correlated with poor patient overall survival (OS) and disease-free survival (DFS). Moreover, we found that short-hairpin RNA (shRNA)-mediated knockdown of p62 expression significantly inhibited cell proliferation, migration, and invasion, and promoted cell death in vitro, as well as suppressed breast cancer growth and lung metastasis in vivo. In addition, flow cytometry analysis of splenocytes and tumor infiltrating lymphocytes (TILs) indicated that the numbers of CD8α+ interferon (IFN)-γ+ cells (CTLs) and CD4+IFN-γ+ (Th1) cells were increased while those of CD4+IL-4+ (Th2) cells, tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) were decreased. RT-PCR analyses showed that the gene expression of Th1/Th2 cytokines changed in the tumor microenvironment. Silencing SQSTM1/p62 suppressed tumor cell lung metastasis. Together, our results provide strong evidence that silencing tumor cell SQSTM1/p62 inhibited tumor growth and metastasis through cell cycle arrest and TME regulation. This finding provides a novel molecular therapeutic strategy for breast cancer progression and metastasis treatment.

Keywords:  Breast cancer; Cell cycle; Metastasis; SQSTM1/p62; Tumor microenvironment

DOI:  https://doi.org/10.1016/j.gendis.2021.03.008
Cancer Res. 2022 Jul 27. pii: CAN-22-0042. [Epub ahead of print]

GOT2 Silencing Promotes Reprogramming of Glutamine Metabolism and Sensitizes Hepatocellular Carcinoma to Glutaminase Inhibitors.

Yunzheng Li, Binghua Li, Yanchao Xu, Liyuan Qian, Tiancheng Xu, Gang Meng, Huan Li, Ye Wang, Laizhu Zhang, Xiang Jiang, Qi Liu, Yuanyuan Xie, Chunxiao Cheng, Beicheng Sun, Decai Yu.

Hepatocellular carcinoma (HCC) is one of the primary liver malignancies with a poor prognosis. Glutamic-oxaloacetic transaminase 2 (GOT2) is a highly tissue-specific gene in the liver, but the roles GOT2 plays in the progression of HCC remain unclear. Here, we report that GOT2 is downregulated in HCC tumor tissues and that low expression of GOT2 is associated with advanced progression and poor prognosis. In HCC cells, knockdown of GOT2 promoted proliferation, migration, and invasion. In mouse models of HCC, loss of GOT2 promoted tumor growth as well as hematogenous and intrahepatic metastasis. Mechanistically, silencing of GOT2 enhanced glutaminolysis, nucleotide synthesis, and GSH synthesis by reprogramming glutamine metabolism to support the cellular antioxidant system, which activated the PI3K/AKT/mTOR pathway to contribute to HCC progression. Furthermore, HCC with low expression of GOT2 was highly dependent on glutamine metabolism and sensitive to the glutaminase inhibitor CB-839 in vitro and in vivo. Overall, GOT2 is involved in glutamine metabolic reprogramming to promote HCC progression and may serve as a therapeutic and diagnostic target for HCC.

DOI: https://doi.org/10.1158/0008-5472.CAN-22-0042
Bone. 2022 Jul 22. pii: S8756-3282(22)00180-6. [Epub ahead of print] 116503

N6-Methyladenosine-induced miR-143-3p promotes intervertebral disc degeneration by regulating SOX5.

Daokuan Gao, Bo Hu, Baiyang Ding, Quanlai Zhao, Yu Zhang, Liang Xiao.

  Intervertebral disc degeneration is the basic cause of lumbocrural pain, which not only causes pain and but also serious economic burdens on patients. Increasingly more evidence has shown that tumor necrosis factor-α (TNF-α) is involved in the pathological process of intervertebral disc degeneration, but the specific molecular mechanism is still unclear. This study investigated the potential mechanism and function of methyltransferase-like 3 (METTL3)/miR-143-3p/SOX5 regulatory axis in nucleus pulposus cells under the action of TNF-α. Human nucleus pulposus cells were treated with TNF-α to construct an in vitro model of intervertebral disc degeneration. Flow cytometry, quantitative reverse-transcription PCR (RT-qPCR), Western blot (WB) and luciferase assays were used to identify the mechanism of action of miR-143-3p in the course of intervertebral disc degeneration in vitro and the downstream targeted regulatory molecules. The role of miR-143-3p in intervertebral disc degeneration was also validated by in vivo. RT-qPCR, WB, coimmunoprecipitation (Co-IP) and flow cytometry were used to verify the regulatory effect of METTL3 on miR-143-3p maturation. RT-qPCR and WB were adopted to detect differences in METTL3, miR-143-3p and SOX5 expression in human nucleus pulposus tissue. miR-143-3p in nucleus pulposus cells was involved in the regulation of extracellular matrix metabolism and apoptosis after TNF-α stimulation, and intervertebral disc degeneration was relieved by effectively regulating miR-143-3p expression. Subsequent experiments showed that the downstream direct target gene of miR-143-3p was SOX5 and that miR-143-3p negatively regulated the expression of SOX5. In addition, METTL3 promoted miR-143-3p maturation, and METTL3 and miR-143-3p were significantly upregulated in degenerative nucleus pulposus, an effect that was significantly negatively correlated with low SOX5 expression. In conclusion, TNF-α upregulates METTL3, METTL3 promotes miR-143-3p maturation, and miR-143-3p inhibits the transcriptional activity of SOX5 through targeted binding, thereby inducing intervertebral disc degeneration. The inhibition of METTL3 or miR-143-3p expression may be an effective way to treat intervertebral disc degeneration.

Keywords:  METTL3; Nucleus pulposus cells; SOX5; TNF-α; miRNA

DOI:  https://doi.org/10.1016/j.bone.2022.116503