慢病毒介导RNAi抑制大肠癌细胞APRIL表达对化疗敏感性的影响

摘要

目的 探讨慢病毒介导的RNA干扰(RNA interference，RNAi)沉默APRIL基因对大肠癌细胞株LOVO化疗敏感性的影响。方法 实验分为3组:siRNA-APRIL组、非特异性序列组和空白对照组。制备siRNA-APRIL慢病毒表达载体，PCR筛选阳性克隆，测序鉴定。转染LOVO细胞48 h后，Western blot检测LOVO细胞的APRIL表达水平;同时用10 μg/ml 5-FU处理细胞，5-FU作用72 h后，采用流式细胞仪检测细胞凋亡和细胞周期变化，MTT法检测细胞增殖抑制率。结果PCR和测序证实，成功构建siRNA-APRIL慢病毒表达载体。Western blot检测表明siRNA - APRIL组的APRIL表达水平下调。5 -FU处理LOVO细胞后，流式细胞仪检测各组的凋亡率分别为(21.12±3.35)％、(13.06±1.92)％和(12.28±1.79)％，siRNA-APRIL组显著高于另两组(P＜0.01)，且G0/G1期细胞增多，G2/M期细胞减少(P＜0.05); MTT法检测显示各组的增殖抑制率分别为(59.67±5.03)％、(42.33±4.16)％、和(39.67±4.73)％，siRNA - APRIL组显著高于另两组(P＜0.01)。结论 以APRIL为靶标的RNA干扰能下调大肠癌细胞株LOVO中的APRIL表达，增强其对5-FU的化疗敏感性。%Objective To investigate the effects of lentivirus-mediated RNA interference (RNAi) targeting a proliferation-inducing ligand (APRIL) on the chemosensitivity to 5-FU of colorectal cancer cell line LoVo. Methods The lentiviral vector siRNA-APRIL was constructed and verified by PCR and DNA sequencing. LoVo cells were transfected with siRNA-APRIL plasmid, nontargeting siRNA plasmid, or empty plasmid. Forty-eight hours after the transfection, the cells were examined for APRIL expression using Western blot. Seventy-two hours after treatment with 10 ng/ml 5-FU, flow cytometry was used to detect the cell apoptosis and cell cycle changes. The cell growth inhibition rate following 5-FU exposure was detected by MTT assay. Results PCR analysis and DNA sequencing demonstrated that the RNAi sequence targeting APRIL gene was successfully inserted into the lentiviral vector. siRNA-APRIL transfection resulted in obviously reduced expression of APRIL in LoVo cells. After 5-FU exposure, the apoptosis rate of siRNA-APRIL-transfected cells were increased to (21.12±3.35)%, significantly higher than that in cells transfected with the nontargeting plasmid or the empty plasmid [(13.06±1.92)% and (12.28±1.79)%, respectively, PO.01]; the cell number in G0/G1 phase increased while that in G2/M phase decreased in siRNA-APRIL-transfected cells. The growth inhibition rate in siRNA-APRIL group was (59.67±5.03)%, significantly higher than that in the other two groups [(42.33+ 4.16)% and (39.67±4.73)%, respectively, PO.01]. Conclusion Lentivirus-mediated RNAi targeting APRIL can effectively suppress the expression of APRIL in LoVo cells and enhance the chemosensitivity of the cells to 5-FU.