Inhibitory Mechanism Of Mir-630 On The Expression Of Organic Cation Transporter OCT2 In Renal Cell Carcinoma

As the top 3 malignancies in urogenital system,R enal cell carcinoma(RCC)caused around 140,000 casualty each year,seriously imperiling human health.Although there are plenty of drug transporters expressed in kidney,the expression change in cancer pathology of those transporters turns out to result in the multidrug resistance of cancer chemotherapy,which leads to the inefficient chemotherapy(10%-15%)and high mortality.Therefore,chemotherapy can only be the complementary clinical treat of partial nephrectomy.Epigenetic factors play important role in the regulation ofgene misexpression in the progress of cancer.Previous studies have shown the transcriptional repression of OCT2 is regulated by DNA methylation and histone acylation.There is high methylation modification in CpG island of OCT2 promoter,which causes the loss of c-Myc binding in promoter E-box site and its recruit of MLL1,a transcriptional factor responsible for catalyzing the active methylation of Lsy4 of H3(H3K4me3)at the OCT2 promoter region.Meantime results showed the exceptional high expression of histone acetylate HDAC7 and HDAC9,which impairs histone acylation and finally leads to transcriptional repression of OCT2.Our research focused on other epigenetic regulation mechanism causing loss of OCT2 in RCC.microRNA(miRNA)is a small non-coding RNA molecule,participating in the regulation of most biological process.In order to inquire into the role miRNAs paly in the loss of OCT2 in RCC,we cross-matched several miRNA databases and bioinformatical softwares to predict potential miRNAs tending to bind OCT2 3'-UTR region.We found hsa-miR-630,155,181,431,488 as five miRNAs targets for further expression analyzing in RCC tissues and paired normal tissues.Results showed miR-630 and miR-155 were up regulated in RCC tissues.For onwards investigation,we used dual-luciferase reportergene assay to assess its binding ability to OCT2 3'-UTR.miR-630 displayed significant binding to OCT2 3'-UTR and inhibiting OCT2 expression while miR-155 displayed no binding sign.After mutation the binding site of miR-630 in OCT23'-UTR,the inhibition disappeared.We take RCC cell lines 786-O and 769-P as our models.When miR-630 was overexpressed in cells,the mRNA level of OCT2 was significantly repressed and its uptake to classic fluorescence substrate ASP+was reduced.Vice versa,cells were treated with miR-630 inhibitor with concentrationgradient,OCT2 mRNA was up regulated and cells showed higher ability to uptake OCT2 classic substrate MPP+.However the up regulation was unable to reach the level that DNMT inhibitor could induce.Then we construct miR-630 and OCT2 co-expressed cell model to further investigation.MTT result of co-expressed cells indicated cellsgot higher resistance ability than OCT2 overexpressed cells when treated with anti-cancer drug oxaliplatin and ICP-MS analysis of intra-cellular Pt+showed miR-630 could suppress OCT2 uptake of oxaliplatin.Afterwards,we constructed 786-O-OCT2-miR-630,786-O-OCT2,786-O xenograft models to reveal in vivo impact of miR-630.After ad ministrated with oxaliplatin,we found thegrowth rate of tumors in 786-O-OCT2-miR-630group displayed no significant difference to 786-O,indicating miR-630 could raise drug resistance in RCC via suppressing OCT2 function.To elucidate the mechanism of miR-630 high-expression,we analyzed the expression of c-Myc in RCC tissues and paired normal tissues.Results showed high expression of c-Myc in RCC tissue both mRNA and protein levels.dual-luciferase reportergene assay also showed c-Myc could bind to miR-630 encodinggene promoter and activate its transcription.To reverse its activation,two binding site must be mutated synchronously.Chromatin Immunoprecipitation(ChIP)of tissues demonstrated c-Myc which was more likely to bind to miR-630 encodinggene in RCC tissues was the regulatory factor in miR-630 upregulation.Collectively,c-Myc can activate miR-630 transcription and high expression of MIR-630 will repress OCT2 transcription and then lower the cytotoxicity of anticancer drugs like oxaliplatin.This study provides new target for drug development and new coad minstration strategy for clinical chemotherapy.