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目的:运用RNA干扰(RNA interference,RNAi)技术沉默结肠癌细胞株LoVo中Livin基因的表达,研究Livin对LoVo细胞增殖和凋亡等生物学特性的影响。方法:构建针对Livin基因的干扰质粒pSilencer4.1-L1和pSilencer4.1-L2,转染LoVo细胞,通过RT-PCR、Western blotting检测转染后LoVo细胞Livin的表达,并用流式细胞术、克隆形成实验、MTT法检测转染后LoVo细胞的凋亡、细胞周期、克隆形成、增殖以及对顺铂的敏感性。结果:成功构建pSilencer4.1-L1、pSilencer4.1-L2干扰质粒,pSilencer4.1-L1可有效沉默LoVo细胞中Livin mRNA和蛋白的表达(P<0.01),但pSilencer4.1-L2效果不明显。与对照组相比,pSilencer4.1-L1转染后LoVo细胞的凋亡率增加[(24.2±3.2)%vs(8.1±1.4)%,P<0.01];细胞增殖明显抑制,转染后72 h增殖抑制率约70%;细胞的克隆形成率明显下降[(15±4.6)%vs(85±5.8)%,P<0.01];S期细胞明显增加[(45.7±4.9)%vs(28.0±3.0)%,P<0.01],G_1期细胞明显减少[(43.0±5.2)%vs(62.8±5.1)%,P<0.01];对顺铂的敏感性增加,细胞凋亡率增加[(65.3±6.2)%vs(43.4±6.9)%,P<0.01]。结论:pSilencer4.1-L1可有效沉默结肠癌LoVo细胞中Livin基因的表达,抑制细胞增殖和克隆形成、诱导细胞凋亡、增加细胞对顺铂的敏感性。
OBJECTIVE: To silence the expression of Livin gene in LoVo colon cancer cell line by using RNA interference (RNAi) technique and to study the effect of Livin on the biological characteristics of LoVo cell proliferation and apoptosis. Methods: LoVo cells were transfected with pSilencer4.1-L1 and pSilencer4.1-L2 targeting Livin gene. The expression of Livin in LoVo cells was detected by RT-PCR and Western blotting. Flow cytometry and cloning The formation of experiments, MTT assay LoVo cells after transfection apoptosis, cell cycle, the formation of clones, proliferation and sensitivity to cisplatin. Results: pSilencer4.1-L1 and pSilencer4.1-L1 were successfully constructed and pSilencer4.1-L1 could effectively silence the expression of Livin mRNA and protein in LoVo cells (P <0.01), but the effect of pSilencer4.1-L2 was not obvious . Compared with the control group, the apoptosis rate of LoVo cells was increased after transfected with pSilencer4.1-L1 [(24.2 ± 3.2)% vs (8.1 ± 1.4)%, P <0.01] h (45.7 ± 4.9)% vs (28.0%) (P <0.01). The rate of cell proliferation was significantly lower than that of control (P <0.01). The sensitivity to cisplatin was increased and the apoptosis rate was increased [(43.0 ± 5.2)% vs (62.8 ± 5.1)%, P < 65.3 ± 6.2)% vs (43.4 ± 6.9)%, P <0.01]. Conclusion: pSilencer4.1-L1 can effectively silence the expression of Livin gene in colon cancer LoVo cells, inhibit cell proliferation and colony formation, induce apoptosis and increase cell sensitivity to cisplatin.