bims-rimeca Biomed News
on RNA methylation in cancer
Issue of 2021‒10‒03
twelve papers selected by
Sk Ramiz Islam
Saha Institute of Nuclear Physics
  1. Increased m6A-RNA methylation and FTO suppression is associated with myocardial inflammation and dysfunction during endotoxemia in mice.
  2. METTL3-dependent RNA m6A dysregulation contributes to neurodegeneration in Alzheimer's disease through aberrant cell cycle events.
  3. m6A methyltransferase METTL3-mediated lncRNA FOXD2-AS1 promotes the tumorigenesis of cervical cancer.
  4. N6-Methyladenosine-Related lncRNA Signature Predicts the Overall Survival of Colorectal Cancer Patients.
  5. Integrative Analysis of Biomarkers Through Machine Learning Identifies Stemness Features in Colorectal Cancer.
  6. METTL14-Mediated miR-30c-1-3p Maturation Represses the Progression of Lung Cancer via Regulation of MARCKSL1 Expression.
  7. FTO-stabilized lncRNA HOXC13-AS epigenetically upregulated FZD6 and activated Wnt/β-catenin signaling to drive cervical cancer proliferation, invasion, and EMT.
  8. METTL3-mediated mature miR-497-5p/195-5p inhibits trophoblast migration and invasion by targeting WWP1 in preeclampsia.
  9. METTL14 aggravates podocyte injury and glomerulopathy progression through N6-methyladenosine-dependent downregulating of Sirt1.
  10. A methyltransferase-like 14/miR-99a-5p/tribble 2 positive feedback circuit promotes cancer stem cell persistence and radioresistance via histone deacetylase 2-mediated epigenetic modulation in esophageal squamous cell carcinoma.
  11. MicroRNA-501-3p inhibits the proliferation of kidney cancer cells by targeting WTAP.
  12. m6A methylation promotes white-to-beige fat transition by facilitating Hif1a translation.
  1. Mol Cell Biochem. 2021 Sep 28.
    Increased m6A-RNA methylation and FTO suppression is associated with myocardial inflammation and dysfunction during endotoxemia in mice.
    Praveen K Dubey, Mallikarjun Patil, Sarojini Singh, Shubham Dubey, Paras Ahuja, Suresh Kumar Verma, Prasanna Krishnamurthy.
      Endotoxemia triggers life-threatening immune and cardiovascular response that leads to tissue damage, multi-organ failure, and death. The understanding of underlying molecular mechanisms is still evolving. N6-methyladenosine (m6A)-RNA modification plays key regulatory role in numerous biological processes. However, it remains unclear whether endotoxemia alters RNA methylation in the myocardium. In the current study, we investigated the effect of lipopolysaccharide (LPS)-induced endotoxemia on m6A-RNA methylation and its implications on myocardial inflammation and left ventricular (LV) function. Following LPS administration, mice showed increases in m6A-RNA methylation in the myocardium with a corresponding decrease in the expression of fat mass and obesity-associated protein (FTO, an m6A eraser/demethylase). The changes were associated with a significant increase in expression of myocardial inflammatory cytokine genes, such as IL-6, TNF-α, IL-1β, and reduced LV function. Moreover, rat cardiomyoblasts (H9c2) exposed to LPS showed similar changes (with increase in m6A-RNA methylation and inflammatory cytokine genes, whereas downregulation of FTO). Furthermore, methylated RNA immunoprecipitation assay showed hypermethylation and increase in the expression of IL-6 and TNF-α genes in LPS-treated H9c2 cells as compared to untreated cells. Interestingly, FTO knockdown in cardiomyocytes mimicked the above effects. Taken together, these data suggest that endotoxemia-induced m6A methylation might play a critical role in expression of cardiac proinflammatory cytokines, and modulation of m6A methylation might limit myocardial inflammation and dysfunction during endotoxemia.
    Keywords:  Cardiac dysfunction; Endotoxemia; FTO; RNA methylation; Sepsis
    DOI:  https://doi.org/10.1007/s11010-021-04267-2
  2. Mol Neurodegener. 2021 Sep 30. 16(1): 70
    METTL3-dependent RNA m6A dysregulation contributes to neurodegeneration in Alzheimer's disease through aberrant cell cycle events.
    Fanpeng Zhao, Ying Xu, Shichao Gao, Lixia Qin, Quillan Austria, Sandra L Siedlak, Kinga Pajdzik, Qing Dai, Chuan He, Wenzhang Wang, James M O'Donnell, Beisha Tang, Xiongwei Zhu.
      BACKGROUND: N6-methyladenosine (m6A) modification of RNA influences fundamental aspects of RNA metabolism and m6A dysregulation is implicated in various human diseases. In this study, we explored the potential role of RNA m6A modification in the pathogenesis of Alzheimer disease (AD).METHODS: We investigated the m6A modification and the expression of m6A regulators in the brain tissues of AD patients and determined the impact and underlying mechanism of manipulated expression of m6A levels on AD-related deficits both in vitro and in vivo.
    RESULTS: We found decreased neuronal m6A levels along with significantly reduced expression of m6A methyltransferase like 3 (METTL3) in AD brains. Interestingly, reduced neuronal m6A modification in the hippocampus caused by METTL3 knockdown led to significant memory deficits, accompanied by extensive synaptic loss and neuronal death along with multiple AD-related cellular alterations including oxidative stress and aberrant cell cycle events in vivo. Inhibition of oxidative stress or cell cycle alleviated shMettl3-induced apoptotic activation and neuronal damage in primary neurons. Restored m6A modification by inhibiting its demethylation in vitro rescued abnormal cell cycle events, neuronal deficits and death induced by METTL3 knockdown. Soluble Aβ oligomers caused reduced METTL3 expression and METTL3 knockdown exacerbated while METTL3 overexpression rescued Aβ-induced synaptic PSD95 loss in vitro. Importantly, METTL3 overexpression rescued Aβ-induced synaptic damage and cognitive impairment in vivo.
    CONCLUSIONS: Collectively, these data suggested that METTL3 reduction-mediated m6A dysregulation likely contributes to neurodegeneration in AD which may be a therapeutic target for AD.
    Keywords:  Aberrant cell cycle events; Alzheimer’s disease; Apoptosis; METTL3; RNA m6A modification; RNA methylation
    DOI:  https://doi.org/10.1186/s13024-021-00484-x
  3. Mol Ther Oncolytics. 2021 Sep 24. 22 574-581
    m6A methyltransferase METTL3-mediated lncRNA FOXD2-AS1 promotes the tumorigenesis of cervical cancer.
    Fei Ji, Yang Lu, Shaoyun Chen, Xiaoling Lin, Yan Yu, Yuanfang Zhu, Xin Luo.
      Recent studies have indicated that long noncoding RNA (lncRNA) and N6-methyladenosine (m6A) methylation modification play critical roles in human cancers; however, their regulation on cervical cancer is largely unclear. Here, our study tries to investigate the underlying mechanisms by which lncRNA FOXD2 adjacent opposite strand RNA 1 (FOXD2-AS1) modulates cervical cancer tumorigenesis. Results illuminated that FOXD2-AS1 expression was significantly upregulated in cervical cancer cells and tissue, which was closely correlated to the unfavorable prognosis. Functionally, gain and loss-of-function assays showed that FOXD2-AS1 promoted the migration and proliferation of cervical cancer cells. Besides, FOXD2-AS1 silencing repressed the tumor growth in vivo. Mechanistically, m6A methyltransferase methyltransferase-like 3 (METTL3) enhanced the stability of FOXD2-AS1 and maintained its expression. Moreover, FOXD2-AS1 recruited lysine-specific demethylase 1 (LSD1) to the promoter region of p21 to silence its transcription abundance. In conclusion, these findings support that METTL3/FOXD2-AS1 accelerates cervical cancer progression via a m6A-dependent modality, which may serve as a potential therapeutic target for cervical cancer.
    Keywords:  FOXD2-AS1; LSD1; METTL3; N6-methyladenosine; cervical cancer
    DOI:  https://doi.org/10.1016/j.omto.2021.07.004
  4. Genes (Basel). 2021 Aug 31. pii: 1375. [Epub ahead of print]12(9):
    N6-Methyladenosine-Related lncRNA Signature Predicts the Overall Survival of Colorectal Cancer Patients.
    Wei Song, Jun Ren, Wenzheng Yuan, Rensheng Xiang, Yuhang Ge, Tao Fu.
      BACKGROUND: The N6-methyladenosine (m6A) RNA modification can modify long non-coding RNAs (lncRNAs), thereby affecting the tumorigenesis and progression of tumors. However, the underlying role of m6A-modified lncRNAs in colorectal cancer (CRC) remains largely unknown. Therefore, our aim was to assess the prognostic value of m6A-modified lncRNAs in CRC patients.METHODS: The gene expression and clinicopathological data of CRC were extracted from The Cancer Genome Atlas (TCGA) database. Pearson correlation analysis was used to investigate the m6A-modified lncRNAs. Consensus clustering was conducted to identify molecular subtypes of CRC, and the clinical significance of molecular subtypes was identified. The least absolute shrinkage and selection operator analysis (LASSO) was applied to establish a risk signature. Finally, a prognostic nomogram with risk score and clinicopathological variables was established.
    RESULTS: In total, 29 m6A-modified lncRNAs were identified as prognostic lncRNAs. Two molecular clusters were identified and significant differences were found with respect to clinicopathological features and prognosis. Cluster1 is associated with poor overall survival (OS), down-regulation of Programmed cell death ligand-1 (PD-L1) expression, lower immune score, and less immune cell infiltration. Then, an m6A-modified lncRNA signature for predicting OS was constructed in the TCGA training cohort. The signature demonstrated favorable prediction performance in both training and validation sets. Compared with low-risk patients, patients with high risk showed worse clinical outcomes, lower immune scores, and downregulated PD-L1 expression. Further analysis indicated that the signature was an independent prognostic indicator, and then a prognostic nomogram based on risk score, tumor location, and tumor stage was established.
    CONCLUSIONS: Our study identified a seven m6A-modified lncRNA signature and established a prognostic nomogram that reliably predicts OS in CRC. These findings may improve the understanding of m6A modifications in CRC and provide insights into the prognosis and treatment strategy of CRC.
    Keywords:  N6-methyladenosine; colorectal cancer; long non-coding RNA; prognostic model
    DOI:  https://doi.org/10.3390/genes12091375
  5. Front Cell Dev Biol. 2021 ;9 724860
    Integrative Analysis of Biomarkers Through Machine Learning Identifies Stemness Features in Colorectal Cancer.
    Ran Wei, Jichuan Quan, Shuofeng Li, Hengchang Liu, Xu Guan, Zheng Jiang, Xishan Wang.
      Background: Cancer stem cells (CSCs), which are characterized by self-renewal and plasticity, are highly correlated with tumor metastasis and drug resistance. To fully understand the role of CSCs in colorectal cancer (CRC), we evaluated the stemness traits and prognostic value of stemness-related genes in CRC. Methods: In this study, the data from 616 CRC patients from The Cancer Genome Atlas (TCGA) were assessed and subtyped based on the mRNA expression-based stemness index (mRNAsi). The correlations of cancer stemness with the immune microenvironment, tumor mutational burden (TMB), and N6-methyladenosine (m6A) RNA methylation regulators were analyzed. Weighted gene co-expression network analysis (WGCNA) was performed to identify the crucial stemness-related genes and modules. Furthermore, a prognostic expression signature was constructed using the Lasso-penalized Cox regression analysis. The signature was validated via multiplex immunofluorescence staining of tissue samples in an independent cohort of 48 CRC patients. Results: This study suggests that high-mRNAsi scores are associated with poor overall survival in stage IV CRC patients. Moreover, the levels of TMB and m6A RNA methylation regulators were positively correlated with mRNAsi scores, and low-mRNAsi scores were characterized by increased immune activity in CRC. The analysis identified 34 key genes as candidate prognosis biomarkers. Finally, a three-gene prognostic signature (PARPBP, KNSTRN, and KIF2C) was explored together with specific clinical features to construct a nomogram, which was successfully validated in an external cohort. Conclusion: There is a unique correlation between CSCs and the prognosis of CRC patients, and the novel biomarkers related to cell stemness could accurately predict the clinical outcomes of these patients.
    Keywords:  N6-methyladenosine; cancer stem cell; colorectal cancer; machine learning; tumor immune microenvironment
    DOI:  https://doi.org/10.3389/fcell.2021.724860
  6. Mol Biotechnol. 2021 Sep 29.
    METTL14-Mediated miR-30c-1-3p Maturation Represses the Progression of Lung Cancer via Regulation of MARCKSL1 Expression.
    Fei Li, Jing Zhao, Lei Wang, Yantong Chi, Xiaori Huang, Wei Liu.
      Lung cancer (LC) is a pulmonary malignant tumor with extremely low 5-year survival rate. N6-methyladenosine (m6A) is confirmed to regulate diverse pathophysiological processes including cancers. Methyltransferase-like 14 (METTL14) is an important RNA methyltransferase in m6A modification. However, researches on the regulatory mechanism of METTL14 on LC progression are relatively rare. Tumor xenograft experiment was conducted to investigate the effect of METTL14 on LC in vivo. The relative expression of METTL14, miR-30c-1-3p, and myristoylated alanine-rich C kinase substrate-like protein-1 (MARCKSL1) in LC tissues and/or cell lines was determined using qRT-PCR. Western blot assay was used to measure the protein levels of METTL14 and MARCKSL1 in tumor xenograft model and/or LC cell lines. MTT, wound healing, and transwell assays were performed to detect LC cell viability and metastasis. RNA immunoprecipitation assay and qRT-PCR were used to verify the effects of METTL14 on pri-miR-30c-1-3p. The relationship between miR-30c-1-3p and MARCKSL1 was confirmed by the dual-luciferase reporter assay. METTL14 was remarkably downregulated in LC tissues and cell lines. METTL14 mediated the maturation of miR-30c-1-3p. The overexpressed METTL14 and overexpressed miR-30c-1-3p suppressed the cell viability and metastasis in LC. Meanwhile, the increased METTL14 also repressed the growth of tumor xenograft in vivo. In addition, MARCKSL1 was confirmed to be the target gene of miR-30c-1-3p. High expression of MARCKSL1 and low expression of miR-30c-1-3p reversed the suppressive effects of METTL14 overexpression on cell viability and metastasis. METTL14 promoted the maturation of miR-30c-1-3p and mediated MARCKSL1 expression to inhibit the progression of LC. This study may provide a new insight for the LC clinical therapy.
    Keywords:  Lung cancer; MARCKSL1; METTL14; N6-methyladenosine; miR-30c-1-3p
    DOI:  https://doi.org/10.1007/s12033-021-00406-8
  7. J BUON. 2021 Jul-Aug;26(4):26(4): 1279-1291
    FTO-stabilized lncRNA HOXC13-AS epigenetically upregulated FZD6 and activated Wnt/β-catenin signaling to drive cervical cancer proliferation, invasion, and EMT.
    Tongfei Wang, Weina Li, Biaofei Ye, Shuangyan Zhang, Xin Lei, Da Zhang.
      PURPOSE: Cervical cancer (CC) is the third most prevalent malignancy in women. Frizzled class receptor 6 (FZD6) is demonstrated to either activate or repress the activity of Wnt/β-catenin pathway, a crucial signaling involved in cancer development. However, the role of FZD6 in CC is unknown. The present study explored the function of FZD6 and its mechanism in CC.METHODS: The levels of FZD6, HOXC13-AS were detected in CC specimens and CC cell lines via qRT-PCR. Cell proliferation and invasion was explored via CCK-8 assay, colony formation assay and transwell assay. Luciferase reporter analysis, FISH, subcellular fractionation, chromatin immunoprecipitation and RNA immunoprecipitation were performed for investigating the molecular mechanism.
    RESULTS: FZD6 was up-regulated in CC. FZD6 silence retarded proliferation, invasion, and epithelial-to-mesenchymal transition (EMT), and inactivated Wnt/β-catenin. HOXC13 antisense RNA (HOXC13-AS) was up-regulated in CC and positively correlated with FZD6. Mechanistically, HOCX13-AS1 augmented FZD through cAMP-response element binding protein-binding protein (CBP)-modulated histone H3 on lysine 27 acetylation (H3K27ac). Additionally, fat mass and obesity-associated protein (FTO) reduced N6-methyladenosine (m6A) and stabilized HOXC13-AS in CC.
    CONCLUSIONS: In conclusion, this study firstly showed that FTO-stabilized HOXC13-AS epigenetically up-regulated FZD6 and activated Wnt/β-catenin signaling to drive CC proliferation, invasion, and EMT, suggesting HOXC13-AS as a potential target for CC treatment.
  8. Cell Cycle. 2021 Sep 30. 1-16
    METTL3-mediated mature miR-497-5p/195-5p inhibits trophoblast migration and invasion by targeting WWP1 in preeclampsia.
    Rui Li, Xia Qiu, Mei He, Jing Qiao, Jing He, Mei Zhong.
      Preeclampsia (PE) is a pregnancy-associated disorder caused by poor placentation. METTL3 as an RNA methyltransferase that plays an essential role in the regulation of the m6A modification. This work investigated the regulation of METTL3-mediated mature miR-497-5p/195-5p cluster in PE progression and identified the downstream mechanisms involved. Differentially expressed miRNAs in PE were obtained from the GSE96983 dataset. The miR-497-5p/195-5p levels in placental samples collected from 20 cases of PE patients and 18 cases of normal controls were measured by RT-qPCR. Effects of miR-497-5p/195-5p and WWP1 on trophoblast proliferation, migration, and invasion were analyzed by CCK8, EdU, wound healing and Transwell assays. Luciferase reporter and RIP experiments were conducted to verify the interaction of WWP1 with miR-497-5p/195-5p. Dot blot assay was performed to determine the m6A levels in PE. The m6A modification of pri-miR-497-5p/195-5p was determined by Me-RIP assay. Immunochemistry (IHC) and western blotting were used to examine the immunoreactivities and protein levels of METTL3 and WWP1 in placental samples from PE patients and normal controls. The miR-497-5p/195-5p levels were high in PE placenta. Functionally, overexpression of miR-497-5p/195-5p prevented trophoblast migration, invasion, and proliferation. WWP1 overexpression enhanced trophoblast migration, invasion, and proliferation. Mechanistically, WWP1 was verified to be targeted by miR-497-5p/195-5p. Moreover, METTL3 promoted the recognition of pri-miR-497-5p/195-5p by DGCR8 and enhanced the formation of mature miR-497-5p/195-5p in an m6A manner. We demonstrated that METTL3-mediated m6A modification promotes the transition of pri-miR-497-5p/195-5p to mature miRNAs, thereby upregulating miR-497-5p/195-5p to aggravate PE progression by targeting WWP1.
    Keywords:  METTL3; Preeclampsia; WWP1; m6A modification; miR-497-5p/195-5p
    DOI:  https://doi.org/10.1080/15384101.2021.1982527
  9. Cell Death Dis. 2021 Sep 27. 12(10): 881
    METTL14 aggravates podocyte injury and glomerulopathy progression through N6-methyladenosine-dependent downregulating of Sirt1.
    Zhihui Lu, Hong Liu, Nana Song, Yiran Liang, Jiaming Zhu, Jing Chen, Yichun Ning, Jiachang Hu, Yi Fang, Jie Teng, Jianzhou Zou, Yan Dai, Xiaoqiang Ding.
      Podocytes are known to play a determining role in the progression of proteinuric kidney disease. N6-methyladenosine (m6A), as the most abundant chemical modification in eukaryotic mRNA, has been reported to participate in various pathological processes. However, its role in podocyte injury remains unclear. In this study, we observed the elevated m6A RNA levels and the most upregulated METTL14 expression in kidneys of mice with adriamycin (ADR) and diabetic nephropathy. METTL14 was also evidently increased in renal biopsy samples from patients with focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy and in cultured human podocytes with ADR or advanced glycation end product (AGE) treatment in vitro. Functionally, we generated mice with podocyte-specific METTL14 deletion, and identified METTL14 knockout in podocytes improved glomerular function and alleviated podocyte injury, characterized by activation of autophagy and inhibition of apoptosis and inflammation, in mice with ADR nephropathy. Similar to the results in vivo, knockdown of METTL14 facilitated autophagy and alleviated apoptosis and inflammation in podocytes under ADR or AGE condition in vitro. Mechanically, we identified METTL14 knockdown upregulated the level of Sirt1, a well-known protective deacetylase in proteinuric kidney diseases, in podocytes with ADR or AGE treatment. The results of MeRIP-qPCR and dual-luciferase reporter assay indicated METTL14 promoted Sirt1 mRNA m6A modification and degradation in injured podocytes. Our findings suggest METTL14-dependent RNA m6A modification contributes to podocyte injury through posttranscriptional regulation of Sirt1 mRNA, which provide a potential approach for the diagnosis and treatment of podocytopathies.
    DOI:  https://doi.org/10.1038/s41419-021-04156-y
  10. Clin Transl Med. 2021 Sep;11(9): e545
    A methyltransferase-like 14/miR-99a-5p/tribble 2 positive feedback circuit promotes cancer stem cell persistence and radioresistance via histone deacetylase 2-mediated epigenetic modulation in esophageal squamous cell carcinoma.
    Zhenchuan Liu, Kaiqing Wu, Shaorui Gu, Wenli Wang, Shiliang Xie, Tiancheng Lu, Lei Li, Chenglai Dong, Xishi Wang, Yongxin Zhou.
      BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a highly aggressive and treatment-resistant tumor. The biological implications and molecular mechanism of cancer stem-like cells (CSCs) in ESCC, which contribute to therapeutic resistance such as radioresistance, remain elusive.METHODS: Quantitative real-time polymerase chain reaction, western blotting, immunohistochemistry, and in situ hybridization assays were used to detect methyltransferase-like 14 miR-99a-5p tribble 2 (METTL14/miR-99a-5p/TRIB2) expression in ESCC. The biological functions of METTL14/miR-99a-5p/TRIB2 were demonstrated in vitro and in vivo. Mass spectrum analysis was used to identify the downstream proteins regulated by TRIB2. Chromatin immunoprecipitation (IP), IP, N6 -methyladenosine (m6 A)-RNA IP, luciferase reporter, and ubiquitination assays were employed to explore the molecular mechanisms underlying this feedback circuit and its downstream pathways.
    RESULTS: We found that miR-99a-5p was significantly decreased in ESCC. miR-99a-5p inhibited CSCs persistence and the radioresistance of ESCC cells, and miR-99a-5p downregulation predicted an unfavorable prognosis of ESCC patients. Mechanically, we unveiled a METTL14-miR-99a-5p-TRIB2 positive feedback loop that enhances CSC properties and radioresistance of ESCC cells. METTL14, an m6 A RNA methyltransferase downregulated in ESCC, suppresses TRIB2 expression via miR-99a-5p-mediated degradation of TRIB2 mRNA by targeting its 3' untranslated region, whereas TRIB2 induces ubiquitin-mediated proteasomal degradation of METTL14 in a COP1-dependent manner. METTL14 upregulates miR-99a-5p by modulating m6 A-mediated, DiGeorge critical region 8-dependent pri-mir-99a processing. Hyperactivation of TRIB2 resulting from this positive circuit was closely correlated with radioresistance and CSC characteristics. Furthermore, TRIB2 activates HDAC2 and subsequently induces p21 epigenetic repression through Akt/mTOR/S6K1 signaling pathway activation. Pharmacologic inhibition of HDAC2 effectively attenuates the TRIB2-mediated effect both in vitro and in patient-derived xenograft models.
    CONCLUSION: Our data highlight the presence of the METTL14/miR-99a-5p/TRIB2 axis and show that it is positively associated with CSC characteristics and radioresistance of ESCC, suggesting potential therapeutic targets for ESCC treatment.
    Keywords:  cancer stem-like cells; esophageal squamous cell carcinoma; positive feedback loop; radioresistance
    DOI:  https://doi.org/10.1002/ctm2.545
  11. Cancer Med. 2021 Sep 30.
    MicroRNA-501-3p inhibits the proliferation of kidney cancer cells by targeting WTAP.
    Liujia He, Shiming Chen, Yufan Ying, Haiyun Xie, Jiangfeng Li, Xueyou Ma, Weiyu Wang, Haixiang Shen, Xiao Wang, Xiangyi Zheng, Liping Xie.
      BACKGROUND: Emerging evidence suggests that miR-501-3p plays an important role in the pathogenesis and progression of various carcinomas. However, its role and underlying mechanisms in renal cell carcinoma (RCC) remain to be elucidated.METHODS: Quantitative RT-PCR, western blot, and bioinformatics methods were used to evaluate the expression of miR-501-3p and Wilms' tumor 1-associating protein (WTAP) in RCC cell lines and clinical tissues. The effects of miR-501-3p on the proliferation of RCC cells were investigated using flow cytometric, colony formation, and CCK8 assays. The target gene of miR-501-3p was confirmed by western blotting, qRT-PCR, and dual-luciferase reporter assays. The levels of RNA methylation with N6-methyladenosine (m6 A) following miR-501-3p overexpression or knockdown of its target gene were quantified using a dot-blot assay.
    RESULTS: miR-501-3p expression was significantly downregulated in human RCC cell lines and tissues. In contrast, its overexpression markedly inhibited cancer cell proliferation in vitro by inducing G1 phase arrest. Moreover, WTAP was verified as a direct target gene of miR-501-3p. WTAP gene knockdown alone efficiently produced the same cancer-inhibiting effects as miR-501-3p overexpression, with the level of m6 A in RCC cells being decreased under both scenarios. The intermolecular interaction between miR-501-3p and WTAP was further substantiated by rescue experiments.
    CONCLUSION: RCC progression is regulated via the miR-501-3p/WTAP/CDK2 axis and is inhibited by the overexpression of miR-501-3p.
    Keywords:  WTAP; microRNA-501-3p; proliferation; renal cell carcinoma
    DOI:  https://doi.org/10.1002/cam4.4157
  12. EMBO Rep. 2021 Sep 27. e52348
    m6A methylation promotes white-to-beige fat transition by facilitating Hif1a translation.
    Ruifan Wu, Yushi Chen, Youhua Liu, Lenan Zhuang, Wei Chen, Botao Zeng, Xing Liao, Guanqun Guo, Yizhen Wang, Xinxia Wang.
      Obesity mainly results from a chronic energy imbalance. Promoting browning of white adipocytes is a promising strategy to enhance energy expenditure and combat obesity. N6-methyladenosine (m6A), the most abundant mRNA modification in eukaryotes, plays an important role in regulating adipogenesis. However, whether m6A regulates white adipocyte browning was unknown. Here, we report that adipose tissue-specific deletion of Fto, an m6A demethylase, predisposes mice to prevent high-fat diet (HFD)-induced obesity by enhancing energy expenditure. Additionally, deletion of FTO in vitro promotes thermogenesis and white-to-beige adipocyte transition. Mechanistically, FTO deficiency increases the m6A level of Hif1a mRNA, which is recognized by m6A-binding protein YTHDC2, facilitating mRNA translation and increasing HIF1A protein abundance. HIF1A activates the transcription of thermogenic genes, including Ppaggc1a, Prdm16, and Pparg, thereby promoting Ucp1 expression and the browning process. Collectively, these results unveil an epigenetic mechanism by which m6A-facilitated HIF1A expression controls browning of white adipocytes and thermogenesis, providing a potential target to counteract obesity and metabolic disease.
    Keywords:   Hif1a ; FTO; m6A; thermogenesis; translation
    DOI:  https://doi.org/10.15252/embr.202052348
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