生长因子受体结合蛋白-2抑制肠癌细胞HT29增殖

基础医学与临床 ›› 2013, Vol. 33 ›› Issue (9): 1199-1204.

生长因子受体结合蛋白-2抑制肠癌细胞HT29增殖

陈丽妹,叶韵斌,刘枋,陈慧菁,李洁羽,苏光建,林巧燕

福建省肿瘤医院

收稿日期:2013-02-06 修回日期:2013-05-24 出版日期:2013-09-05 发布日期:2013-08-28
通讯作者: 叶韵斌 E-mail:zjyunbin@189.cn

Growth factor receptor binding protein-2 inhibits colon cancer HT29 cell proliferation

Received:2013-02-06 Revised:2013-05-24 Online:2013-09-05 Published:2013-08-28

摘要/Abstract

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细胞株为实验组（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72h，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48h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),72h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72h，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转导通路分子表达下调。

关键词: [关键词]结直肠癌, Grb2, RNA干扰, 慢病毒, 信号通路

Abstract: 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. Method 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 (i.e. infected the Grb2 shRNA cells HT29/shGrb2-69,HT29/shGrb2-70,HT29/shGrb2-71, HT29/shGrb2-72,HT29/shGr-b2-73),eGFP shRNA slow virus infection colon cancer HT29 cell for the negative control group. MTT method 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 72h 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 48h for 0.347±0.048,0.382±0.041 (Significantly lower than the blank control, 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); 72h after infection，Phospho-P42/44 ERK, Akt, Phospho-Akt were decreased, while the ERK,STAT5 expression were not affected. Conclusion The application of a lentiviral vector system transfected of Grb2 shRNA to HT29 cells inhibited the levels of Grb2 mRNA and protein significantly, and induced HT29 cell proliferation and down-regulated signal transduction molecules expression.

Key words: colorectal cancer, Grb2, RNAi, lentivirus, signaling pathway

陈丽妹叶韵斌刘枋陈慧菁李洁羽苏光建林巧燕. 生长因子受体结合蛋白-2抑制肠癌细胞HT29增殖[J]. 基础医学与临床, 2013, 33(9): 1199-1204.

[1]	郭雄飞王挺汤立新. miR-29a对类风湿关节炎成纤维样滑膜细胞增殖和凋亡的影响[J]. , 2020, 40(1): 9-15.
[2]	陈民佳杜娟袁丹凤张蜀黄宏朱方强. 姜黄素抑制TGF-β1诱导的人肾小管上皮细胞系表型转化[J]. , 2020, 40(1): 48-53.
[3]	洪雯丽黄瑶琪祝费隐郑月慧李佳戴峻张立霞. PTEN抑制大鼠原始卵泡启动和生长[J]. 基础医学与临床, 2019, 39(8): 1077-1084.
[4]	吴艳张铭鲁开智陈杨. 腺苷A2b受体激活减轻小鼠肾缺血再灌注致肺损伤[J]. 基础医学与临床, 2019, 39(8): 1102-1107.
[5]	余伟张秀王清唐立华吴杰. 人CYP4Z1基因重组慢病毒载体的构建、鉴定及抑制乳腺癌细胞增殖[J]. 基础医学与临床, 2019, 39(2): 175-181.
[6]	刘雅林谢兰兰王丽云赵瑞杰许峰王文胜. 丁苯酞减轻阿尔兹海默病大鼠海马神经元凋亡[J]. , 2019, 39(12): 1746-1751.
[7]	张军峰徐曦徐仓宝张一博房智超郝佳晖赵朝华. 银胶菊内酯减轻氧糖剥夺/再恢复诱导的PC12细胞损伤[J]. 基础医学与临床, 2018, 38(9): 1286-1291.
[8]	沈冰冰张松朱启仁王志荣张卓琦. 羟基红花黄色素A减轻大鼠心肌细胞缺氧/复氧损伤[J]. 基础医学与临床, 2018, 38(4): 480-484.
[9]	刘树根李泽玲罗新辉魏桂林. 氯化两面针碱对糖尿病肾病模型大鼠肾脏损伤的保护作用[J]. 基础医学与临床, 2018, 38(10): 1422-1427.
[10]	任静李毅杨慧鲁玲玲. 慢病毒载体介导下DJ-1过表达细胞模型的建立[J]. 基础医学与临床, 2018, 38(1): 1-6.
[11]	秦青青李婷婷郑永钦贺军栋. microRNA在甲状腺乳头状癌中作用机制的研究进展[J]. 基础医学与临床, 2017, 37(8): 1184-1189.
[12]	常海平宋淑芳任杰. 靶向TPX2基因shRNA慢病毒载体的构建及鉴定[J]. 基础医学与临床, 2017, 37(7): 945-952.
[13]	王彬彬武承凤张方信. 自噬在肠黏膜屏障功能作用机制的研究进展[J]. 基础医学与临床, 2017, 37(3): 405-409.
[14]	丁博月韩春. 结直肠癌干细胞表面标志物和信号通路研究进展[J]. 基础医学与临床, 2017, 37(1): 133-137.
[15]	王丽妍刁宗礼张启东刘文虎. Apelin抑制TGF-β诱导的人肾小管上皮-间充质细胞转换[J]. 基础医学与临床, 2016, 36(8): 1051-1056.