Epigenetic Regulation Of Hippo Pathway In Clear Cell Renal Cell Carcinoma

BackgroundClear cell renal cell carcinoma(ccRCC)is the most common type of renal carcinoma affecting many people worldwide.Although the 5-year survival rate for localized lesions is 65%,But the survival rate after metastasis is less than 10.Epigenetics regulation plays an important role in the progress of ccRCC.In this study,we found that miR-335 and RNA methyltransferases METTL14 regulate Hippo signaling pathways through different epigenetic pathways and thus affect cell proliferation,invasion and migration.On the one hand,Our study confirms H3K9me1/2 histone demethylase is KDM3A a new miR-335 regulatory target gene,KDM3A high in ccRCC,its upregulation is involved in ccRCC progress.We also found that the core molecules of the Hippo pathway YAP 1 a direct downstream target of the KDM3A,CHIP-qPCR confirmed KDM3A enrichment on the YAP1 promoter.On the other hand,We found that RNA methyltransferase was METTL14 low expression in ccRCC,It is significantly related to poor prognosis.Through a series of IP experiments,it is confirmed that METTL14 regulate LATS1 posttranscriptional levels in a m6A dependent manner,affect the activity of Hippo signaling pathways,and then participate in the regulation of ccRCC cell proliferation,invasion and migration.Anyway,Our study found that miR-335 and METTL14 regulate Hippo signaling pathways through different epigenetic pathways that affect the progression of ccRCC,For the progress of ccRCC research,it provides new ideas and potential new therapeutic targets.Methods and results1.The results showed that miR-335 expression was low in ccRCC,and the CCK8、transwell and scratch test showed that overexpression inhibited the proliferation,invasion and migration of 786-O cells.2.KDM3A is a miR-335 target gene,which can inhibit the expression of KDM3A at the RNA and protein levels.The results of cell function experiments show that the proliferation,invasion and migration ability of 786-O cells can be enhanced when overexpression is KDM3A,and the tumor-promoting function can be inhibited when overexpression is miR-335.3.The results of qPCR、western blot and CHIP-qPCR experiments suggest that KDM3A can promote YAP1 expression by binding to the promoter region of the YAP1 gene,while overexpression can inhibit the promotion of YAP 1 gene.The effects of overexpression miR-335/KDM3A/YAP1 signal axis on the proliferation,invasion and migration invasion and migration of 786-O cells were proved by CCK-8、transwell and scratch experiments;4.m6A quantitative results showed that the m6A level of ccRCC decreased significantly,the low expression of METTL14 was found by Shengxin analysis combined with clinical sample verification,and the cell function experiment showed that the low expression increased the proliferation,invasion and migration ability of tumor cells.5.qPCR、WB、immunofluorescence and nucleoplasm separation experiments showed that the RNA and protein levels of LATS1 decreased,YAP1 phosphorylation decreased,nuclear entry increased and downstream oncogene expression was initiated.6.RNA half-life experiments,MeRIP-qPCR、RIP-qPCR and RNA-pulldown experiments found that YTHDF2 can identify the m6A modification sites of the LATS1mRNA and maintain its stability.7.PDX model found that the expression of ki67 in clinical samples was decreased after treatment with depressor.Conclusions1.miR-335 low expression in ccRCC,cell experiments confirmed that low expression miR-335 can promote the proliferation and migration of tumor cells.2.KDM3A is a miR-335 target gene that promotes YAP1 transcription through binding to the promoter region.3.METTL14 low expression in ccRCC,cell experiments confirmed that low expression METTL14 can promote the proliferation and migration of tumor cells.4.YTHDF2 can recognize the m6A modification sites of METTL14 to the LATS1mRNA,maintain its stability,promote the increase of protein level,and thus enhance the expression of YAP 1 oncogenes.5.miR-335 and METTL14 regulate Hippo signaling pathways through different epigenetic pathways and then affect ccRCC progression.miR-335-mimics and de-inhibitors are potential targeted therapeutic agents.