RNA干扰抑制磷脂酰肌醇3-激酶p110β的表达增加氟尿嘧啶诱导人胃癌细胞凋亡的能力  

RNA interfering PI3Kp110β increases the sensitivity of fluorouracil induced apoptosis in human gastric cancer cells

作　　者：许慈[1] 蔡懿婷[1] 董晴晴[1] 王碧君[1] 戴强[1] 朱黎明[1] 

机构地区：[1]上海交通大学医学院附属第三人民医院消化科,上海201999

出　　处：《解剖学研究》2016年第1期1-6,共6页Anatomy Research

基　　金：上海交通大学医学院新华集团基金(13XJ22012)

摘　　要：目的探讨沉默磷脂酰肌醇3-激酶(PI3K)p110β因子的表达增加氟尿嘧啶(FU)诱导人胃癌细胞凋亡的能力。方法运用RNA干扰(shRNA)技术构建携带靶向抑制PI3Kp110β分子的慢病毒载体LV-sh PI3Kp110β并感染人胃癌细胞株AGS(sh PI3Kp110β组),未转染的细胞为空白对照组(Con组),转染无义序列的对照慢病毒载体LV-shRNA为阴性对照组(sh NC组),荧光显微镜观察慢病毒的感染效率,RT-PCR和Western blot方法验证慢病毒感染后PI3Kp110β mRNA和蛋白在Con、shNC和shPI3Kp110β各组细胞中的表达水平,并用嘌呤霉素筛选PI3Kp110β静默的稳转细胞。在稳转细胞中和对照细胞中加入不同浓度FU分别作用24、48和72 h,通过MTT法检测Con、shNC和shPI3Kp110β各组细胞在加入氟尿嘧啶后细胞增殖的情况,并用Western blot方法检测可能涉及的凋亡相关信号分子的变化。结果慢病毒干扰RNA(shRNA)感染人胃癌细胞株AGS后,能有效下调PI3Kp110β在细胞内的表达水平并成功筛选PI3Kp110β静默的AGS稳转细胞;FU可抑制AGS细胞的增殖,其作用呈现浓度的依赖性;在该细胞中下调PI3Kp110β的表达可增加该细胞对FU抑制细胞生长的敏感性;下调胃癌细胞AGS内shPI3Kp110β分子的表达可显著增加FU诱导凋亡相关蛋白PARP的裂解,抑制Bcl-2蛋白的表达,从而促进胃癌细胞的凋亡。结论 PI3Kp110β shRNA转染人胃癌细胞AGS可有效下调该细胞中PI3Kp110β的表达,增加5-Fu抑制人胃癌细胞生长及促进其凋亡的敏感性,促进凋亡信号通路相关蛋白的活化。Objective To study the effect of PI3Kp110β silencing on enhancing the ability of fluorouraci（lFU）to induce the apoptosis of human gastric cancer cells in vitro. Methods RNA interfering the sequence targeted by PI3Kp110β was transfected into lenti-virus carrier（LV-sh PI3Kp110β）and then it infected AGS gastric cancer cells for 72 hours（sh-PI3Kp110β group）. Other two groups including uninfected cells（Con group）and lenti-virus containing nonsense sh RNA sequence（LV-sh NC）infected cells（sh-NC group）. The efficiency of infection was observed by fluorescence microscope. The m RNA and protein expression of PI3Kp110β were determined by reverse transcriptase polymerase chain reaction（RT-PCR）and Western blotting. The stable cells of PI3Kp110β silencing expression and NC by lentivirus-mediated RNA interference were selected by puromycin. Then two groups of stable lentivirus infected cells（sh-PI3Kp110β and sh-NC）and Con group were treated with different concentrations of FU for 24 h,48 h and 72 h,respectively. Cellular proliferation of each group was measured by MTT assay. Western blotting was used to determine the relative signal molecules it may concern after the three groups was treated with or without FU for 72 h. Results Compared with Con and sh-NC groups,the expression level of PI3Kp110β was downregulated significantly in sh-PI3Kp110β group in AGS cell lines after LV-sh PI3Kp110β affected the cells 72 hours later. The stable cells of PI3Kp110β silencing expression and NC were selected. FU could inhibit the proliferation of AGS cell lines,with concentration dependence in three time points. Silencing of PI3Kp110β expression in AGS cells could improve the inhibitory effect by FU. Also,downregulation of PI3Kp110β expression by RNA interference in AGScells significantly increase the ability of FU inducing the cleavage of PARP and inhibited the expression of Bcl-2 and PI3Kp110β to induce cell apoptosis. Conclusion The expression of PI3Kp110β in gastric cancer cells were effective

关 键 词：磷脂酰肌醇3-激酶p110β RNA干扰 胃肿瘤 增殖 凋亡 

分 类 号：R735.2[医药卫生—肿瘤]