生长因子受体结合蛋白-2抑制肠癌细胞HT29增殖

摘要

目的 研究Grb2 shRNA转染对肠癌细胞HT29增殖相关信号通路的影响,探讨以Grb2为肠癌治疗靶点的可行性.方法 应用shRNA慢病毒质粒系统,将Grb2 shRNA转染至293T细胞,获得5株不同序列Grb2 shRNA慢病毒颗粒,并感染肠癌HT29细胞,分别获得的5株感染Grb2 shRNA慢病毒颗粒的肠癌HT29细胞系为实验组(即感染Grb2 shRNA的细胞HT29/shGrb2-69、HT29/shGrb2-70、HT29/shGrb2-71、HT29/shGrb2-72、HT29/shGrb2-73),以eGFP shRNA慢病毒感染肠癌HT29细胞为阴性对照组.MTT法检测细胞增殖情况,RT-PCR检测Grb2 mRNA表达,Western blot检测Grb2、P42/44 ERK、磷酸化P42/44 ERK、Akt、磷酸化Akt(P-Akt)和STAT5等信号通路分子表达的改变.结果 感染shGrb2病毒72 h,HT29/shGrb2-69、HT29/shGrb2-73两株细胞在mRNA和蛋白水平均出现抑制现象.HT29/shGrb2-69、HT29/shGrb2-73细胞在感染后24h A值分别为0.176±0.045,0.186±0.013,感染48 h后为0.347±0.048、0.382±0.041均显著高于空白对照、阴性对照(P ＜0.001),72 h为0.934±0.038、0.983±0.205高于空白对照、阴性对照(P＜0.01);感染后72 h,phospho-P42/44 ERK、Akt、phospho-Akt明显下降,而ERK、STAT5表达不受影响.结论 在HT29细胞,Grb2 mRNA和蛋白水平上明显抑制,可诱导HT29细胞增殖和相关信号传导通路分子表达下调.%Objective To analyze the effect of Grb2 inhibition by shRNA transfection on the proliferation of colon cancer cells HT29,and to estimate the feasibility of Grb2 as a target for the treatment of colorectal cancer.Methods The Grb2 shRNA was transfected to 293T cells by shRNA lentiviral vector system,5 strains of different sequence Grb2 shRNA lentiviral particles was obtained and infected colon cancer HT29 cells respectively.5 colon cancer HT29 cell lines infected with the Grb2 shRNA lentiviral particles for the experimental group eGFP shRNA slow virus infection colon cancer HT29 cell for the negative control group.MTF was used to determine the cell proliferation situation,the expression of Grb2 mRNA was detected by RT-PCR and Grb2 、P42/44 ERK、phosphorylation P42/44 ERK、serine/threonine protein (Akt)、phosphorylated Akt (P-Akt)、STAT5 and other molecules signaling pathway detected by Western blot.Results Seventy-two hours after infection,HT29/shGrb2-69,HT29/shGrb2-73 cell were suppression in the mRNA and protein levels.After 24h infection,HT29/shGrb2-69 、HT29/shGrb2-73 cell A values were 0.176 ±0.045、0.186 ±0.013,and 48 h for0.347 ±0.048,0.382 ±0.041 Significantly lower than that of the blank control and negative control(P ＜ 0.001),and 72 hours after infection were 0.934 ±0.038、0.983 ±0.205 (compare with the blank control,negative control,P ＜0.01) ; 72 h after infection,phospho-P42/44 ERK,Akt,phospho-Akt were decreased,while the ERK,STAT5 expression were not affected.Conclusions The application of a lentiviral vector system transfected of Grb2 shRNA to HT29 cells inhibites the level of Grb2 mRNA and protein significantly,induces HT29 cell proliferation and down-regulates signal transduction molecules expression.