shRNA介导的热休克蛋白70敲低对人结肠癌细胞在裸鼠体内生长的影响

目的:探讨热休克蛋白70(HSP70)敲低对结肠癌细胞在裸鼠体内生长的影响。方法:构建两种干扰HSP70的shRNA(HSP70 shRNA-1和HSP70 shRNA-2)质粒表达载体,分别转染到结肠癌HT29细胞,并以空质粒转染(阴性对照)和未转染的HT29细胞(空白对照)为对照,用RT-PCR和Western blot方法检测各组HT29细胞HSP70基因和蛋白的表达;将HSP70shRNA-2转染、空质粒转染及未转染的HT29细胞分别接种至裸鼠皮下建立移3组植瘤模型,观察肿瘤生长情况,3周后剥离瘤块,用HE染色、免疫组化和TUNEL法分别检测移植瘤组织形态学、增殖细胞核抗原(PCNA)的表达及细胞凋亡情况。结果:RT-PCR和Western blot结果显示HSP70 shRNA-1和HSP70 shRNA-2均能明显抑制HT29细胞HSP70基因和蛋白的表达,且HSP70 shRNA-2的抑制作用更为明显,空质粒转染对HSP70的表达无明显影响;与空白对照组比较,HSP70 shRNA-2转染组裸鼠移植瘤生长明显较明显减慢(P<0.01),瘤组织中心出现明显坏死,PCNA表达明显降低,细胞凋亡率明显增加(P<0.01),而空质粒转染组无明显上述改变(P>0.05)。结论:HSP70沉默能抑制结肠癌HT29细胞裸鼠移植瘤的生长,促进细胞凋亡,HSP70可能是治疗结肠癌有效靶点。

Effect of shRNA-mediated HSP70 knockdown on growth of colon cancer cells in nude mice

Objective: To investigate the influence of heat shock protein (HSP70) knock down on the growth of colon cancer cells in nude mice. Methods: Two shRNA plasmid vectors against HSP70 (HSP70 shRNA-1 and HSP70 shRNA-2) were constructed and were transfected into colon cancer HT29 cells respectively, and the empty plasmid transfected (negative control) and untransfected HT29 cells (blank control) served as control. The gene and protein expressions of HSP70 in the cells of each group were determined by RT-PCR and Western blot analysis, respectively. Three groups of xenograft tumor model in nude mice were created by subcutaneous inoculation with HSP70 shRNA-2 transfected, empty plasmid transfected and untransfected HT29 cells respectively, and then the growth of the xenograft tumors was observed. The lumps in the nude mice were stripped off after three weeks and the H&E, immunohistochemical staining and TUNEL assay were performed to determine the morphological condition, proliferating cell nuclear antigen (PCNA) expression and apoptosis in the tumor tissues, respectively. Results: The results of RT-PCR and Western blot showed that the gene and protein expressions of HSP70 in HT29 cells were significantly inhibited by either HSP70 shRNA-1 or HSP70 shRNA-2 transfection, which was more evident in HSP70 shRNA-2 transfected cells. Compared with blank control group, in HSP70 shRNA-2 transfection group, the growth of the xenograft tumor was significantly reduced (P<0.01), marked necrosis in the central area of the tumor was observed, the PCNA expression significantly decreased and apoptosis significantly increased in the tumor tissues (P<0.01), while none of the obvious above changes were noted in empty plasmid transfection group. Conclusion: HSP70 silencing can inhibit the growth and enhance the apoptosis of xenograft tumor of colon cancer HT29 cells in nude mice. HSP70 is probably an effective target for colon cancer.