The Study Of The Effect Of RNA Interference On HIF-2 In The 786-0 Renal Cancer Cell

Renal cell carcinoma (RCC)is one of the most common cancer, RCC are solid tumours that arise from the proximal convoluted tubules of the kidney and are characerized by abundant neovascularization and arterio-venous fistula formation, accounting for 85% of all renal cancers. Almost 20% to 30% of patients with newly diagnosed renal cell carcinoma have evidence of metastases at presentation and their median survival is 6 to 8 months. With metastatic progression, renal cell carcinoma is incurable, and existing systemic therapies are largely ineffective in impacting disease response or patient survival. At present there is lack of effective systemic therapy for metastatic renal cell carcinoma. So it is important to find new methods to improve the patient survival of renal cell carcinoma.There are three mammalian HIF isoforms. Hypoxia-inducible factor (HIF) is a critical transcriptional regulator of many hypoxia-associated genes, including VEGF and cellular growth, metastasis, and apoptosis and GLUT 1. Some observations show that HIF-2 plays an important role in the growth, progression and metastasis of solid tumour. The overexpression of HIF-2 has been demonstrated in many types of cancers, including lung cancer, ovarian cancer, and bladder cancer, the overexpression of HIF-2 has also been demonstrated in the RCC. Many studies have clearly demonstrated that the overexpression of HIF-2 is associated with the the growth and progression in the RCC. So the therapeutic approaches aimed at inhibiting the expression of HIF-2 can inhibit the growth of RCC. Weconstruct mammalian expression vector of HIF-2 RNAi and study the effect on renal cell carcinoma 786 —0. Our study can provide some experimental bases for the gene therapy of RCC and perhaps disclose the mechanism of the renal cell carcinoma.一 . The construction of HIF-2 RNA interference expression plasmid Objective: To construction of HIF-2 RNA interference expression plasmid.Methods:siRNA was designed. For each sequence, two ssDNA oligonucleo -tides were synthesized. The annealed products were ligated into the pSilencer 2.1-U6 neo vector. All constructs were verified by standard DNA sequencing. Using Western blot method to detect the protein expression of HIF-2 after transfection of RNAi vector using Lipofectamine.Results: DNA sequencing to verify the all constructs,and No land No 2 and No 3 can individually decrease the (70 + 4) % and (45 + 5) % and (30±5) % of expression of protein in the renal cancer cell.Conclusions: The HIF-2 RNAi mammalian expression vector pSilencer 2.1-U6 neo was successfully constructed.二 . The study of the effect of RNA interference on HIF-2 in the 786-0 renal cancer cell in vitro and in vivoObjective: To study of the effect of RNA interference on HIF-2 in the 786-0 renal cancer cell in vitro and in vivo.Methods: The 786-0 human renal cell cancer cell-line was cultured. Colonies were produced after transfection of HIF-2 RNAi vector or control vector using Lipofectamine 2000.Transfected colonies were selected in the G418. Using RT-PCR and Western blot and immunohistochemical methodto detect the expression of HIF-2 in the RNAi cell groups and control cell groups and empty cell groups. Using MTT method to study growth of the RNAi cell groups and control cell groups and empty cell groups. FCM to measure cell cycle and cell apoptosis of the RNAi cell groups and control cell groups and empty cell groups. Nude mouse xenograft assays were done, viable cells were injected in the flanks of female nude mice with both control cell groups and RNAi cell groups. At the time of sacrifice tumors were excised and weighed and tumor volume was calculated.Results: Compared with control cell groups and empty cell groups, the amount of HIF-2 mRNA expression are decreased in the RNAi group cell. Their difference is significant (P< 0. 01). There is no significant between control cell groups and empty cell groups in the the amount of HIF-2 mRNA expression (P > 0. 05) . Compared with control cell groups and empty cell groups, the amount of HIF-2 protein expression are lower in the RNAi cell groups. Their difference is significant (P< 0.01) . There is no significant between control cell groups and empty cell groups in the the amount of HIF-2 protein expression (P > 0. 05) . The growth of RNAi cell groups are slower than that of control cell groups and empty cell groups. The cell cycle of RNAi cells be inhibited in the G2/M phase. The percent of cell apoptosis in RNAi cell groups increase than that of control cell groups and empty cell groups. Compared with empty cell groups, the growth of tumor are slower in the RNAi cell groups in the nude mice.Conclusions: The RNAi on HIF-2 gene can greatly decrease the HIF-2 expression and slow the growth of renal cell carcinoma 786-0 very much. It can affect the cell cycle and increase the percent of cell apoptosis and slow the growth of tumor in the nude mice.jn. The study of VEGF A and VEGF C expression of renal cancer cells byHIF-2 RNA interference in vitroObjective: To study the VEGF A and VEGF C expression of renal cancer cells by HIF-2 RNA interference in vitro.Methods:Using RT-PCR and Western blot and immunohistochemical method to detect the expression of VEGF A in the RNAi cell groups and control cell groups and empty cell groups. Using RT-PCR and Western blot and immunofluorescence method to detect the expression of VEGF C in the RNAi cell groups and control cell groups and empty cell groups.Results: Compared with control cell groups and empty cell groups, the amount of VEGF A mRNA expression are decreased in the RNAi cell groups. Their difference is significant ( P< 0.01) .There is no significant between control cell groups and empty cell groups in the the amount of VEGF A mRNA expression (P>0. 05) . Compared with control cell groups and empty cell groups, the amount of VEGF A protein expression are lower in the RNAi cell groups. Their difference is significant (P<0. 01). There is no significant between control cell groups and empty cell groups in the the amount of VEGF A protein expression (P>0. 05) .Compared with control cell groups and empty cell groups, the amount of VEGF C mRNA expression are decreased in the RNAi cell groups. Their difference is significant (P<0.01) .There is no significant between control cell groups and empty cell groups in the the amount of VEGF C mRNA expression (P > 0. 05) . Compared with control cell groups and empty cell groups, the amount of VEGF C protein expression are lower in the RNAi cell groups. Their difference is significant (P< 0.01) . There is no significant between control cell groups and empty cell groups in the the amount of VEGF C protein expression (P>0. 05) .Conclusions: The RNAi on HIF-2 gene can greatly decrease the VEGF A expression and increase the the VEGF C expression in the renal cancercell.