LncRNA PVT1 Altecred 5-FU/DDP Sensitivity Of HCC Cells By Bounding P300 Induced P53 Transcription

Objective: Hepatocellular Carcinoma damage human health,and the exploration of its pathogenesis is still needs further study because of the low response to chemotherapy drugs in diagnosis and prognosis.Long noncoding RNA(lnc RNA)was found to be closely associated with the occurrence and development of liver cancer,but the specific molecular mechanism is still study deep enough.In this work,we research the regulation mechanism of longnon-coding RNA PVT1(lnc RNA PVT1)between p53,analysis the lnc RNA PVT1 function of regulating the multi-drug resistance related proteins MRP1 and the effect on drug sensitivity in liver cancer cell lines Hep G2.Methods: Bioinformatics and research software programs were used to predicted the possible protein targeted the lnc RNA PVT1;Quantitative real-time PCR was applied to examine the difference of MRP1,p21,Bax,MDM2,Cyclin D1 and acetylation p53 m RNA.Western blot tested the protein expression of p53,acetylation p53,MRP1,p21,Bax,MDM2 and Cyclin D1.MTT cell proliferation assay was performed to determine cell sensitive of 5-Fu and DDP;Cell proliferation was determined by(Edu)incorporation assay;Flow cytometry(FCM)analysis of apoptosis and cell cycle distribution;The RIP assay was conducted to examine putative direct binding of p300 to PVT1 RNA region(Upstate).Results: The results of bioinformatics software shown that lnc RNA PVT1 targeted p300 with the highest probability,97.2256.PVT1 is highly expressed in Hep G2 cells compared with normal liver cells of LO2 by real-time reverse transcription-polymerase chain reaction(q RT-PCR).And we selected the most efficient interference sequence of lnc RNA PVT1 by q RT-PCR,is si-PVT1-857.Moreover,the p53 m RNA expression has no significiant differences after konckdown lnc RNA PVT1(P > 0.05,n=3).And then,we detected the down-regulation expression of p53 downstream target gene of p21,Bax and MDM2,up-regulation m RNA expression of MRP1 in the si-PVT1 group compare with NC,but si-PVT1&C646 group has reverse effect compare with si-PVT1&DMSO group.The results of western blot shown that,compared with the control and NC group,Bcl-xl and cyclin D1 presented a low expression levels in Hep G2 cell after down-regulate lnc RNA PVT1,there are significant differences,but the expresion of p53 has no significant differences,si-PVT1&C646 group was partially restored the protein expression of Bcl-xl and Cyclin D1 compare with si-PVT1&DMSO group;Continue to detect the protein expression of acetylation p53,p21,Bax and MDM2,si-PVT1 group increased the expression of acetylation p53,p21,Bax and MDM2,and si-PVT1&C646 group was part of reducind those protein expression compare with si-PVT1&DMSO;With compare to Ctrl and NC,the expression of MRP1 was down-regulated in si-PVT1 group,si-PVT1&C646 tgroup was partially restored the protein expression of MRP1 compare with si-PVT1&DMSO.Compared with the contorl and NC group,si-PVT1 not only markedly suppressed Hep G2 cell growth and proliferation by MTT and Ed U assay in vitro cultured Hep G2 cells(P < 0.05),but also noticeably impaired cell cycle and promoted apoptosis of Hep G2 cell by Flow cytometry assay,and si-PVT1&C646 group has an opposite result compare with si-PVT1&DMSO.Under treatment of 5-Fu and DDP,down-expression PVT1 reduces cell survival rate,blocks cell cycle and induces apoptosis,but p300 inhibitor can restored these indicators.RNA immunoprecipitation(RIP)assay showed that P300 directly bound to PVT1 RNA,indicating that PVT1 interacting with P300 inhibits p53-mediated transcriptional activity.Conclusion:1.Lnc RNA PVT1 can targeted p300 protein;2.Lnc RNA PVT1 may regulation of p53 transcriptional activity;3.Interference lnc RNA PVT1 could enhance the chemotherapy drugs sensitivity;4.Interference lnc RNA PVT1 could enhance the chemotherapy drugs sensitivity,which could be associated with the mechanism of lnc RNA PVT1 increase p53 transcription activity by targeted p300.