| ObjectiveRenal Cell Carcinoma(RCC)is one of the most common malignancies.The incidence of RCC has been increasing in recent years.The onset of RCC is insidious,there are no apparent symptoms in the early stage.RCC is characterized by a high malignant degree and high metastasis rate.Metastasis is one of the most common causes of death in patients with RCC.Partial nephrectomy or radical nephrectomy is the main treatment for early-stage localized tumors,but about 30%of patients will have local recurrence or distant metastasis after surgery,which threatens the health of people.The treatment of metastatic RCC is limited,the sensitivity to traditional chemotherapy and radiotherapy is poor,and the five-year survival rate is only 10%.With the use of molecular targeted drugs such as tyrosine-kinase inhibitor,Sunitinib,Sorafenib and Pazopanib,the survival benefit of patients with metastatic RCC has been significantly improved.Due to the heterogeneity of RCC,the response of patients with the same clinical stage to the same treatment regimen may vary greatly.Meanwhile,primary or acquired resistance to molecular targeted drugs or immune checkpoint inhibitors makes clinical management challenging.At the same time,the molecular mechanism of metastatic RCC is not clear,and there is no effective target for early detection and treatment of metastatic RCC.Therefore,to explore the biological mechanism regulating metastasis of RCC,and to use new targets for early intervention and treatment is an urgent scientific problem to be solved.Epithelial-mesenchymal transition(EMT)is a process in which cells change from epithelioid to Mesenchymal,often accompanied by a variety of cellular features,such as cell morphological changes,loss of polarity,increased invasiveness,resistant to anoikis,and secretion of extracellular matrix.EMT influences tumor metastasis.It can not only improve the motility ability and invasion of tumor cells,promote local invasion and infiltration,but also make tumor cells acquire stem characteristics,and promote cell survival and colonization.EMT is a dynamic,plastic process,and research has shown that epigenetics can modulate tumor cell EMT and influence tumor metastasis.With the development of high-throughput sequencing technology in recent years,RNA modification,especially the N6-methyladenosine(m~6A)modification,has been paid more and more attention.M~6A modification plays a complex role in the development of many types of tumors.The aim of this study is to establish a EMT model of renal carcinoma cells and to explore the role and mechanism of m~6A modification in EMT and metastasis of RCC,so as to provide new ideas and theoretical basis for basic research of metastatic RCC,in order to find a new target for early prediction and treatment of metastatic RCC.Methods1.The 786-O and OSRC2 RCC cell lines were treated with Transforming growth factor Beta 1(TGF-β1,10 ng/m L)in vitro,and the cell morphology was observed,and the expression of the characteristic markers of EMT in RCC cell lines was detected by Western Blot and q PCR,it was proved that renal carcinoma cells developed EMT.2.After establishing EMT model of RCC cell lines,the migration and invasion ability were compared by the scratch wound healing assay and transwell invasion assay.3.After EMT,the migration and invasion of RCC cell lines were significantly enhanced.The m~6A-modified RNA immunoprecipitation sequencing(me RIP-seq)was performed,and the bioinformatics analysis was performed to detect the difference of m~6A modification patterns between the two groups.At the same time,the m RNA sequencing was performed.4.Western Blot and q PCR were used to detect the expression of m~6A modification-related genes in EMT and control RCC cell lines.5.YTHDF1 was upregulated significantly in RCC cell lines undergoing EMT processes.To explore the mechanism of TGF-β1-mediated up-regulation of YTHDF1 in RCC cell lines,Smad2 and Smad3,the downstream genes of TGF-β1 signaling pathway were specifically knocked down by small interfering RNA(si RNA),Western Blot and q PCR were used to detect the expression of YTHDF1.6.The promoter sequence of YTHDF1 was cloned into PGL3 plasmid and transfected into RCC cell lines.The dual-luciferase reporter experiments were performed,and the effects of Smad2 and Smad3 on the activity of TGF-β1-mediated YTHDF1 promoter were investigated.7.It was found that the expression of Smad2 could significantly affect the activity of YTHDF1 promoter mediated by TGF-β1.After designing specific primers,TGF-β1 was used to treat RCC cell lines,and Ch IP-q PCR was performed to verify the binding of Smad2 protein to YTHDF1 promoter.8.RCC cell line with stable YTHDF1 overexpression and knockdown were established.The expression of EMT related markers in RCC cell lines was detected by Western Blot.At the same time,the effects of YTHDF1 expression on migration and invasion of RCC cell lines were examined by the scratch wound healing assay and transwell invasion assay.Luc reporter was used to label the RCC cell lines,then the stable RCC cell lines were selected and injected into nude mice via tail vein to construct the model of lung metastasis.The effect of YTHDF1 expression on lung metastasis of RCC was evaluated.9.The expression of proteins in YTHDF1-knockdown RCC cell lines was detected by mass spectrometry.After combining with me RIP-seq data and literature review,we showed that Rap1a might be the potential target gene of YTHDF1 in EMT of RCC cell lines.10.The level of m~6A modification of Rap1a was detected by me RIP-q PCR with m~6A antibody in RCC cell lines treated with TGF-β1.In addition,the binding ratio of YTHDF1protein to Rap1a m RNA was detected by RIP-q PCR with YTHDF1 antibody.11.The effect of TGF-β1 on the expression of Rap1a protein was explored by Western Blot after treatment with TGF-β1 or overexpressing YTHDF1.YTHDF1 was knocked down in TGF-β1-treated RCC cells to observe the effect on the expression of Rap1a.12.It was found that the level of m~6A modification of Rap1a was increased and the binding ratio of YTHDF1 to Rap1a was increased in TGF-β1-mediated EMT process.The effect of TGF-β1 on the protein translation of Rap1a was detected by RNC-q PCR.13.The effect of knock-down of Rap1a on the migration and invasion of YTHDF1-overexpressed RCC cell lines was observed.The expression of EMT markers was detected.Results1.EMT models of 786-O and OSRC2 cell lines were successfully constructed and confirmed.The morphological changes and protein levels were consistent with the characteristics of EMT.2.Compared with control group,TGF-β1-induced EMT cells exhibited increased migration and invasion abilities.3.The combination of m RNA sequencing and me RIP sequencing showed that EMT-related pathways were significantly enriched in RCC cell lines undergoing EMT.4.The protein and m RNA expression of YTHDF1 increased significantly in RCC cell lines undergoing EMT.5.Smad2 knock-down could significantly inhibit the expression of YTHDF1 in RCC cell line mediated by TGF-β1.6.The dual luciferase reporter gene experiment showed that TGF-β1 enhanced the activity of YTHDF1 promoter in RCC cell line,and Smad2 knock-down inhibited TGF-β1-induced luciferase reporter gene activity in YTHDF1 promoter region.7.The results of Ch IP-q PCR showed that Smad2 protein could bind to YTHDF1promoter region.8.YTHDF1 knock-down inhibited EMT process and migration and invasion of RCC cell lines.The overexpression of YTHDF1 could promote EMT and enhance migration and invasion of RCC cell lines.The lung metastasis model showed that YTHDF1 knock-down could inhibit the lung metastasis of RCC.9.The combination of proteomics and me RIP-seq revealed that Rap1a may be a potential downstream target of YTHDF1 in TGF-β1-mediated EMT process in RCC cell line.10.In the process of TGF-β1-indeced EMT,the m~6A modification level of Rap1a was significantly increased,and the binding ratio of Rap1a m RNA to YTHDF1 protein was increased.11.Overexpression of YTHDF1 or treatment of TGF-β1 enhanced the protein level of Rap1a.TGF-β1 could not increase the level of Rap1a when YTHDF1 was knocked down.12.TGF-β1 promoted the translation efficiency of Rap1a m RNA.Overexpression of YTHDF1 could significantly enhance the protein translation of Rap1a.13.Knock-down of Rap1a inhibited YTHDF1-indeced EMT and invasion of RCC cell line.ConclusionWe constructed EMT model of RCC cell lines and found that the malignant potential of RCC cell lines was increased and the m~6A modification pattern was changed during EMT,in which the expression of YTHDF1 was significantly increased.Expression of YTHDF1 affected EMT and invasion and metastasis of RCC cell lines.It was found that TGF-β1/Smad2 signal pathway could enhance the expression of YTHDF1 by regulating the transcription of YTHDF1.YTHDF1 could bind to the m~6A-modified Rap1a m RNA and promote the protein translation of Rap1a,thus regulating EMT process and migration and invasion of RCC cell lines. |
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