慢病毒携带shRNA对乳腺癌HPA基因表达的抑制作用

摘要

目的:以乙酰肝素酶(HPA)基因为靶点,构建短发夹RNA(shRNA)慢病毒表达载体,研究慢病毒携带的shRNA对乳腺癌HPA基因表达的抑制作用.方法:以乳腺癌HPA基因为靶基因,根据RNA干扰(RNAi)序列设计原则,通过构建3对shRNA重组慢病毒的表达载体,转染乳腺癌MDA-MB-231细胞,在体外采用蛋白质印迹(Western blot)法检测HPA基因蛋白表达水平;在体内构建乳腺癌模型,通过免疫组化检测HPA基因蛋白的表达.结果:在体外实验中,HPA-shRNA-1组和HPA-shRNA-2组均有效抑制了人乳腺癌MDA-MB-231细胞HPA的表达.在体内实验中,HPA-shRNA-2组HPA基因蛋白的表达明显低于阴性对照组及空白对照组,HPA-shRNA-2组阳性率与空白对照组比较,差异有统计学意义(x2=12.504,P<0.05).表明在体内实验中HPA-shRNA-2组可以下调HPA基因蛋白表达.结论:以HPA为靶点构建shRNA重组慢病毒载体,体内外实验均能下调乳腺癌HPA基因的表达,可为乳腺癌靶向治疗提供新的靶点.%Objective:To construct short hairpin RNA (shRNA) lentiviral expression vector by choosing HPA genes as the targeting point so as to investigate the inhibitive effect of lentiviral vector carryed shRNA for the expression level of heparanase (HPA) genes of breast cancer.Methods: The HPA genes were chosen as target gene, 3 pairs expression vectors of recombinant lentivirus carried shRNA were constructed according to the sequence designed principle of interfering RNA (RNAi) were transfected in MDA-MB-231 cell of breast cancer. Western Blot was used to detect the expression level of HPA gene in vitro, and the breast cancer model was constructed for vivo verification, and then the expression level of HPA gene was detected by using immunohistochemistry.Results: In vitro experiment, both of the HPA-shRNA-1 group and HPA-shRNA-2 group effectively inhibited the expression of HPA gene in MDA-MB-231 cells of breast cancer. In vivo experiment, the expression of HPA gene of HPA-shRNA-2 group was obviously lower than that of negative group and blank group. The difference of positive rate between HPA-shRNA-2 group and blank group was statistically significant (x2=12.504,P<0.05). And these results revealed that the expression of HPA gene in HPA-shRNA-2 group could be down-regulated in vivo experiment.Conclusion: The HPA is chosen as the targeting point to construct recombinant lentiviral vector carried shRNA, and the results of vitro and vivo experiment reveals that the expression of HPA gene can be down-regulated in the two groups. This result verifies that the new method can provide new targeting point for targeted therapy of breast cancer.