The Effect Of Lentivirus-siRNA-GRP78 On Proliferation And Invasion Of Renal Cell Carcinoma

Renal cell carcinoma (renal cell carcinoma,RCC) referred as kidney cancer, is a kind of malignant tumor originated in the renal tubular epithelial cells of renal parenchyma. It is one of the most common malignant tumor in urinary system. Clear renal cell carcinoma (CRCC) is the most common pathological types of RCC. CRCC is given priority to treated with radical nephrectomy (radical nephrectomy,RN), because the early clinical symptom is not obvious so as too late for many patients in clinical. And part of patients had postoperative tumor recurrence or metastasis and multiple drug resistance which is not sensitive to chemotherapy treatment and poor curative effect. Immunotherapy due to long-term curative effect is not identified and the total remission rate is not high. Thus further study of the molecular mechanisms on RCC proliferation and invasion treatment response is critical to improve the early diagnosis and follow-up.GRP78 as a critial member of the family of GRP, has higher homology with heat shock protein which is belonging to one of heat shock protein 70 (HSP70) family members. GRP78 is highly expressive in tumor ceil and tumor tissues such as liver cancer, colon cancer and lung cancer, expression of glial cell tumor contour, and may be closely related with tumor apoptosis, drug resistance. But the role of GRP78 in RCC cancer cell proliferation and invasion is not clear yet.This topic early experiment has proved that GRP78 is highly expressed in the RCC. Preliminary discussed GRP78 gene may be related with the occurrence and development of RCC. Firstly this study will build a good small interference RNA (small interference RNA, siRNA) gene transfected into the expression of GRP78 in the kidney cells 786-0. Confirming siRNA-GRP78 in gene and protein level can effectively inhibit the expression of GRP78. Then research interference GRP78 gene on kidney cancer cell proliferation and cell after the influence of invasiveness. By adopting the method of RNA interference study of kidney cancer 786-0 after GRP78 gene proliferation of cells and the influence of the invasiveness, to provide certain theoretical and experimental basis for RCC gene therapy.Part 1The construction and identification of RNA interferencing GRP78 sequences lenti virusObjective To investigate the construction and identification of RNA interferencing GRP78 sequences lentivirus.Methods Designing and synthesizing the siRNA which can inhibit the GRP78 expression and transfecting with renal cancer cell 786-O.Checking the expression level of GRP78 mRNA and protein by Realtime-PCR and Western blot.We wanted to select a most efficient siRNA sequences for further study.Results Realtime-PCR results compared with blank control (1.85±0.0 8) and scrambled siRNA (1.79±0.15), siRNA-GRP78 significantly inhibite d the relative expression of GRP78 mRNA(P<0.01):siRNAl(0.32±0.09),siR NA2(0.83±0.07),siRNA3(0.79±0.11). siRNAl was identified as the most po tent sequence which suppressed GRP78 mRNA expression.Western blot analysis also demonstrated a significant reduction in GRP78 protein level(P<0.01):siRNA1(0.21±0.03), siRNA2(0.79±0.03), siR NA3(0.86±0.04) when compared with that in blank control(1±0.03) and th at in scrambled siRNA(1±0.07). No statistically significance existed betwee n the expression of GRP78 mRNA and protein in blank control group an d that in scrambled siRNA group(P>0.05).In agreement with Realtime-PCR results,the expression of GRPS^ protein was down-regulated effectively by siRNA1.Conclusion Lentivirus siRNA-GRP78 can effectively inhibit the expression GRP78 mRNA and protein, in renal cancer cell 786-O.Subsequent experiments focused on the siRNA 1 because it was the most effective at inhibiting GRP78 mRNA and protein expression.Part 2RNA interferencing GRP78 inhibits the renal carcinoma 786-0 cells proliferation and invasion in vitroObjective To investigate the proliferation and invasion of RNA interferencing GRP78 inhibits the renal carcinoma 786-0 cells in vitro.Methods SiRNA-GRP78 were transfected with renal carcinoma cell 786-O.Checking the effects of siRNA targeting GRP78 on proliferation and invasiveness of 786-Ocells by MTT Assay,Matrigel Invasion Assay in vitro.Results MTT results compared with blank control and scrambled siRNA, siRNAl group was identified as the most potent sequence which suppressed renal cell carcinoma proliferation(P<0.01).Transwell analysis also demonstrated a significant reduction in siRNA 1 group which suppressed renal cell carcinoma invasion when compared with that in blank control and that in scrambled siRNA(P<0.01).No statistically significance existed between the blank control group and that in scrambled siRNA group(P>0.05).Conclusion The siRNA targeting GRP78 can significantly decrease the expression GRP78 mRNA and protein, in renal cancer cell 786-0. It can decrease cell grow,supressing cell proliferation and invasion. GRP78 may be involved in the carcinogenesis and progression. It suggested that GRP78 as a target for cancer gene therapy reliable theoretical and experimental basis.