中山大学学报（医学科学版）2012，Vol.33Issue(5)：603-609,7.
慢病毒介导的Clusterin基因沉默抑制肾癌786-O细胞增殖并促进细胞凋亡
Lentivirus-mediated Clusterin Silence Inhibits Proliferation and Promotes Apoptosis in Human Renal Cell Cancer Line 786-O In Vitro
摘要
Abstract
[Objective]To testify the silence efficiency of a lentivirus-mediated vector for RNA interference (RNAi) targeting clusterin (CLU) and the influence of proliferation and apoptosis in human renal cancer cell line 786-0.[Methods] Lentiviral vectors for different short hairpin RNAs targeting the coding region of human CLU mRNA (CLU-RNAi-LV) were constructed. The recombinant lentiviral vectors were harvested from 293T cells and were used to transfect human renal cancer cell lines 786-0. There were five groups: CLU-RNAi-LV1 (KD1),CLU-RNAi-LV2 (KD2),and CLU-RNAi-LV3 (KD3) were renal cancer cells with lentiviral infection targeting CLU gene, NC were renal cancer cells with negative lentiviral infection, and CON were renal cancer cells 786-0 as blank control group. Expression of CLU mRNA and protein in the cells were detected by real time-PCR and Western blot, respectively. After that, MST-1 , wound healing assay, flow cytometry were applied to examine the effect of CLU silence on the proliferation and apoptosis in 786-0 cells. [ Results ] The lentiviral vectors CLU-RNAi-LV were constructed and confirmed by DNA sequencing. CLU-RNAi-LV resulted in obviously reduced expression of CLU mRNA and protein in the human renal cancer cell line 786-0 compared with the control vector. Real time-PCR showed that the CLU mRNA expression levels in KD1, KD2 and KD3 group after CLU-RNAi-LV infection by different MOI were down-regulated by 69.4%,58.7% ~ 96.5% and 0 respectively compared with NC group.Western blot showed that the CLU protein expression levels in KD1, KD2 and KD3 group decreased by 35.24%, 46.26% , and 58.91 % , respectively compared to CON group, and the expression of CLU gene in 786-0 cells was significantly inhibited in KD3 group. The wound healing assay showed the cell migration distance in KD3(si-CLU) group (408.43 ?25.92) was less than NC (101.35 ?6.05) and CON group (68.13 ?6.64), with statistically significant differences among groups (P < 0.05). WST-1 assay showed cell growth rate at 72h after transfection was significantly decreased in KD3 (si-CLU) group compared to NC and CON group (P<0.05) with statistically significant differences among groups (P < 0.05). FCM showed the apoptosis rate in KD3 (si-CLU), NC and CON group was (6.23 ?2.51)%, (1.05 ?0.30)%, and (1.17 ?0.29)%, respectively. The apoptosis rate in KD3 (si-CLU) group was increased compared to NC and CON group, with statistically significant differences among groups (P < 0.05). The proliferation and migration of 786-0 cell line were suppressed and apoptosis of 786-0 cell line was promoted. [Conclusion] siRNA sequences and renal cancer cell line 786-0 cell with stable interference of CLU gene expression were filtered out. Lentivirus-mediated CLU RNAi can specifically inhibit endogenous CLU expression in human renal carcinoma cell line 786-O. CLU-RNAi-LV can also effectively inhibit proliferation and promote apoptosis in human renal carcinoma cell line 786-O.关键词
RNA干扰/慢病毒/clusterin/肾细胞癌/786-OKey words
RNA interference/ lentivirus/ clusterin/ human renal cell carcinoma/ 786-0分类
医药卫生引用本文复制引用
石华,邓军洪,郑少斌,王铸,曹开源,周亮,万华..慢病毒介导的Clusterin基因沉默抑制肾癌786-O细胞增殖并促进细胞凋亡[J].中山大学学报（医学科学版）,2012,33(5):603-609,7.基金项目
广东省科技计划项目(2009B030801053) （2009B030801053）
广州市科技计划项目(2009Z1-E381-02) （2009Z1-E381-02）
