The Study On Human Renal Cancer Cell Line786-O By Lentivirus-Mediated Clusterin Gene Silencing

Background and Objection:The incidence of renal cell carcinoma(RCC) ranks the2nd in the urinary tumor. Many patients don’t received therapy in time because the early symptoms are not obvious. Majority of the RCC are not sensitive to chemotherapy and radiotherapy. So it is necessary to search for the new method. RNAi is the breaking of a double-stranded RNA (dsRNA) matching a specific gene sequence into short pieces called short interfering RNA(siRNA), which triggers the degradation of mRN A that matches its sequence. As one of the noval gene blocking technologies, it is expected to become new antitumor therapy.CLU has been taken much attention in the role of antiapoptosis, and it is likely to become a new target for renal cancer gene therapy. In this experiment, on the basis of detect the CLU gene expression significantly in human renal cell carcinoma786-O and ACHN cells, we design and synthesis siRNA that match of the CLU gene, construct a lentivirus-mediated vector for RNA interference (RNAi) of clusterin(CLU) and testify the silence efficiency of a lentivirus-mediated vector for RNA interference such as proliferation and apoptosis in human renal cancer cell line 786-0and ACHN. Gene expression profiling analysis was used by gene expression array.Methods and materials:Different short hairpin RNAs targeting CLU gene-specific sequence were designed to link with pGCSIL-GFP vector. Lentiviral vectors for different short hairpin RNAs targeting the coding region of human CLU mRNA (CLU-RNAi-LV) and another control vector containing a nonsense sequence were constructed. The recombinant lentiviral vectors were harvested from293T cells and were used to transfect human renal cancer cell lines786-0and ACHN. Expressions of CLU mRNA and protein in the cells were detected by real time PCR and Western blot, respectively. After that, the proliferation inhibitory rate (IR) of786-0cells was detected by MST-1.Wound healing assay, flow cytometry were applied to examine the effect of CLU silence on the proliferation and apoptosis in786-0cells. Differentially expressed genes screening before or after clusterin silence in human renal cancer cell line786-0were detected by Axon GenePix4000B microarray scanner.Results:The lentiviral vectors CLU-RNAi-LV were constructed successfully and confirmed by DNA sequencing. CLU gene targeting via RNAi mediated by lentivirus vector had obviously inhibitory effects on expressions of CLU mRNA and protein in the human renal cancer cell line786-0and ACHN compared with the control vector. The mRNA and protein expressions of CLUg gene in the siRNA group were significantly inhibited by the CLU-RNAi-LV. The growth, proliferation and migration of786-0cell line were significantly suppressed and apoptosis of786-0cell line was promoted. There were1853differentially expressed genes.Among of them,up-regulation genes were1270,down-regulation genes were583.GO analysis showed that biological process,cellular component and molecular function were the top three counts of the significant enrichment terms.Pathway analysis showed that the top four up-regulation enrichment pathways were cell adhesion molecules,allograft rejection,phagosome and staphylococcus,whereas the top five down-regulation pathways were mTOR signaling pathway,MARK signaling,non-small cell lung cancer,pathways in cancer.Conclusion:The CLU gene and protein were significantly expressed in renal clear cell carcinoma786-0. A recombinant lentiviral vector expressing shRNAs targeting CLU gene and a human renal cancer cell line786-0with CLU gene stable interference we reestablished successfully. Lentivirus-mediated CLU RNAi can specifically inhibit endogenous CLU expression in human renal carcinoma cell line786-0and ACHN with CLU gene stable interference. Silencing CLU gene by the RNAi technique can not only decrease effectively the expressions of CLU gene and protein. Several efficient short hairpin RNA(shRNA) sequences were selected. CLU-RNAi-LV can also effectively inhibit proliferation and promote apoptosis in human renal carcinoma cell line786-O.There are1853differentially expressed genes.Among of them,up-regulation genes are1270,down-regulation genes are583.GO analysis shows that biological process,cellular component and molecular function are the top three counts of the significant enrichment terms.Pathway analysis shows that the top four up-regulation enrichment pathways are cell adhesion molecules,allograft rejection,phagosome and staphylococcus,whereas the top five down-regulation pathways are mTOR signaling pathway,MARK signaling,non-small cell lung cancer,pathways in cancer.Differentially expressed genes screening on gene expression array before or after clusterin silence in renal cancer cell line786-0shows that only small amount of genes are interfered among the human total genome.But there are complex pathways associated with down-regulated or up-related genes.All datus suggested that the CLU gene play an important role in the renal cancer’s occurrence and development,CLU gene may be a target of gene therapy in renal cell cancer.