The Knockdown Of Long Non-coding RNA PVT1 Suppressed The Proliferation Of Clear Cell Renal Cell Carcinoma

Objective:Long non-coding RNA PVT1 may act as oncogene and play an important role in clear cell Renal Cell Carcinoma,moreover,was co-overexpressed in clear cell Renal Cell Carcinoma with MYC protein.This study aims to investigate the role of PVT1 in clear cell Renal Cell Carcinoma,to clarify whether the expression of MYC and biological functions of clear cell Renal Cell Carcinoma will vary after the knockdown of PVT1.Methods:Total RNA was extracted from clear cell Renal Cell Carcinoma cell lines 786-0,ACHN and Renal Tubular Epithelial cell line HK-2.Quantitative real-time PCR was performed after reverse transcription to investigate whther the expression of RNA of1 PVT1 and MYC in 786-0 and ACHN cell lines differ from that in HK-2 cell line.Then 786-0 and ACHN cell lines were stably transfected using lentiviral vectors carrying shPVTli and shNCi,cells successfully transfected were screened using puromycin,and green fluorescent cells were observed under fluorescence microscopy.Afterwards,total RNA was extracted from cells successfully transfected with shPVTli and shNCi respectively.Quantitative real-time PCR was performed after reverse transcription to identify the expression of PVT1 and MYC RNAs after successful transfection.Afterwards,cells were harvested and the total protein was extracted.The expression of MYC protein was detected by western-blot.Subsequent cell proliferation was detected by CCK8 method in the experimental group(786-0 shPVTli)and the control group(786-0 shNCi).Additional Colony Formation Assay was performed as well.Cell cycles were detected by flow cytometry.Tumor Formation Assay was performed on nude mice using ACHN cell line successfully transfected with shPVTli and shNCi.Result:Quantitative real-time PCR results of 786-0,ACHN and HK-2 cell lines showed that the expression of PVT1 and MYC RNAs were significantly higher in 786-0 and ACHN cell lines than that in HK-2 cell line.The expression of PVT1 RNA in 786-0 and ACHN cell lines transfected by shPVTli was significantly lower than that of cells transfected by shNCi while the expression of MYC mRNA did not differ.Western blot results found that the knockdown of PVT1 by shPVTli could inhibit the expression level of MYC protein in 786-0 and ACHN cell lines.The proliferation of 786-0 cells transfected by shPVTli was significantly inhibited(P<0.05).The colony formation rate of the experimental group were 14.5%,15.6%,14.6%,lower than that in the control group which were showed to be 22.1%,21.7%and 23.4%respectively.G0/G1 phase cell cycle arrest was observed in 786-0 cell line after the knockdown of PVT 1 expression.Tumor Formation Assay in nude mice presented that the tumorigenic ability of ACHN cell line which was transfected by shPVTli was obviously decreased.Conclusions:The expression levels of PVT1 and MYC RNA in clear cell Renal Cell Carcinoma cell lines 786-0 and ACHN were higher than those in renal tubular epithelial cell line HK-2.The inhibition of the expression of PVT1 leads to the down-regualtion of MYC protein but not MYC mRNA in 786-0 and ACHN cell lines.In addition,PVT1 knockdown can inhibit the cell proliferation ability and cell clone formation rate,moreover,block the cell cycle and reduce its tumorigenesis ability.It is speculated that PVT1 knockdown can arrest cell cycle and decrease the expression level of MYC protein and proliferation of clear cell Renal Cell Carcinoma.