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

【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?

Lentivirus-mediated Clusterin Silence Inhibits Proliferation and Promotes Apoptosis in Human Renal Cell Cancer Line 786-O In Vitro

【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-O. 【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-O. 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-O 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-O 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-O 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-O 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-O cell line were suppressed and apoptosis of 786-O cell line was promoted. 【Conclusion】 siRNA sequences and renal cancer cell line 786-O 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.