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

【目的】应用慢病毒介导的RNA干扰技术,检测clusterin(CLU)基因沉默在人肾癌786-O细胞的干扰效果及其对人肾癌786-O细胞增殖、迁移和凋亡的影响。【方法】构建靶向CLU基因的慢病毒干扰载体,利用包装细胞293T获得重组慢病毒,感染人肾癌786-O细胞株。实验分5组:CLU-RNAi-LV1(KD1)、CLU-RNAi-LV2(KD2)、CLU-RNAi-LV3(KD3)为加入靶向CLU基因的慢病毒感染的肾癌细胞组,未处理的慢病毒感染的肾癌细胞组(NC组),肾癌786-O细胞为空白对照组(CON组)。应用Real time-PCR及Western blot检测不同组别干扰前后CLU mRNA及蛋白表达的变化。用细胞划痕实验、MST-1、流式细胞仪等方法检测CLU沉默后肾癌786-O细胞在增殖、迁移、凋亡等生物学行为的改变。【结果】成功构建CLU shRNA慢病毒载体clu-RNAi-LV并获得相应慢病毒。Real time-PCR显示不同感染复数CLU-RNAi-LV处理的KD1、KD2、KD3组CLU mRNA表达水平与对照组相比分别下调69.4%～96.5%和0。Western blot结果显示KD1、KD2、KD3组CLU蛋白表达水平与CON组相比分别下降35.24%、46.26%和58.91%,KD3能显著抑制786-O细胞中CLU基因的表达。划痕实验显示24 h时KD3(si-CLU)组细胞迁移相对距离(408.43±25.92)小于NC组(101.35±6.05)和CON组(68.13±6.64,P<0.05)。WST-1法检测转染后72 h KD3(si-CLU)组细胞生长速度较NC组及CON组明显下降(P<0.05),各组间差异(P<0.05),均有统计学意义。流式细胞仪检测KD3(si-CLU)组与NC组、CON组细胞凋亡率分别为(6.23±2.51)%、(1.05±0.30)%和(1.17±0.29)%,KD3(si-CLU)组与NC组、CON组相比凋亡率增加(P<0.05),差异有统计学意义。肾癌786-O细胞的增殖、迁移受到抑制,而凋亡率增加。【结论】筛选出能稳定干扰CLU基因表达的siRNA序列和肾癌786-O细胞株。CLU慢病毒干扰载体可有效沉默肾癌786-O细胞的内源性CLU基因,抑制肾癌786-O细胞的增殖、迁移并促进细胞凋亡。

[ 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 lenfiviral 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 coml6ared 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 786-0. CLU-RNAi-LV can also effectively inhibit proliferation and promote apoptosis in human renal carcinoma cell line 786-0.