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  

Lentivirus-mediated RNA interference suppresses APRIL expression and enhances chemosensitivity in colorectal cancer cells

作　　者：关婧[1] 孙爱民[2] 王莉慧[1] 何美蓉[1] 

机构地区：[1]南方医科大学南方医院消化内科,广东广州510515 [2]南方医科大学放疗科,广东广州510515

出　　处：《南方医科大学学报》2011年第9期1600-1604,共5页Journal of Southern Medical University

基　　金：广东省自然科学基金(8151051501000040);广东省自然科学基金(8151051501000511)

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10μg/ml5-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 μg/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,P〈0.01];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,P0.01].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.

关 键 词：增殖诱导配体 RNA干扰 LOVO细胞 5-氟尿嘧啶 

分 类 号：R735.34[医药卫生—肿瘤]