RNA干扰Hsp70基因表达抑制胃癌细胞增殖

RNA interference-mediated silencing of the Hsp70 gene inhibits human gastric cancer cell growth

目的构建Hsp70的短发夹状RNA(shRNA),检测其在胃癌细胞株SGC-7901中对Hsp70基因表达的抑制作用以及对肿瘤细胞增殖的影响。方法根据Hsp70编码基因,设计并合成2对反向重复序列,同时设计1对与目的基因不相匹配的重复序列作为对照组分别定向克隆至载体pTZU6+1的U6转录启动子下,构建重组质粒phsp1-siRNA,phsp2-siRNA及对照组质粒phsp3-siRNA,分别转染胃癌细胞株SGC-7901,采用RT-PCR和Western blot检测phsp-siRNA对Hsp70基因表达的抑制作用。利用AlamarBlue法检测转染后对胃癌细胞生长的影响。结果酶切电泳分析和DNA测序证实重组质粒phsp-siRNA构建成功;证实phsp1-siRNA和phsp2-siRNA对内源性Hsp70基因的表达有明显的抑制作用,同时phsp1-siRNA和phsp2-siRNA转染组细胞的生长较对照组受到明显抑制。结论构建的重组质粒phsp1-siRNA和phsp2-siRNA能特异而有效抑制内源性Hsp70基因在SGC-7901细胞中的表达,并对细胞生长有明显抑制作用。

Objective To construct the plasmids containing short hairpin RNA of Hsp70 for knockdown of the expression of Hsp70 and investigate the effects on cell proliferation in gastric cancer cell line. Methods We designed two pairs of reversed duplicate sequence and a pair of reversed duplicate mismatched sequence along with a negative control, which were inserted into contrivance promotor U6 of pTZU6 ＋ / to generate recombinant plasmids, the phspl-siRNA, phsp2-siRNA and phsp3-siRNA, respectively. The recombinant plasmids were then transfected into human gastric carcinoma cell SGC-7901 for the detection of Hsp70 expression, respectively. The inhibition of cell proliferation was examined by AlamarBlue method. Results Electrophoretic analysis and sequencing showed that the recombinant plasmids of phspl-siRNA and phsp2-siRNA were successfully constructed. After the recombinant plasmids of phsp-siRNA were transfected into the cells of SGC-7901, the expressions of HspT0 in the phspl-siRNA and phsp2-siRNA group were down-regulated more significantly than those in the control ones, and the inhibition rate was higher than that in the control ones through AIamarBlue. Cone/usion The constructed recombinant plasmids of phspl-siRNA and phsp2-siRNA can efficiently suppress the expression of Hsp70 in SGC-7901 cells, which may be a basic foundation for the genetic therapy of gastric cancer in the future.