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本实验探究乳腺癌MCF-7细胞中干扰PGI的表达对HIF-1α的调控以及对细胞凋亡的影响。首先,使用慢病毒质粒转染构建干扰PGI(PGI-si RNA)的稳转细胞株,然后使用Hoechst33258染色和流式细胞术(FCM)检测细胞的凋亡,随后,采用RT-PCR检测PGI、HIF-1α和LDHA基因的表达,Western blotting检测Bcl-2、Bax、PGI、LDHA、HIF-1α和ERK蛋白的表达。成功构建PGI-si RNA的MCF-7稳转细胞株后,Hoechst33258染色实验结果显示,PGI-si RNA稳转细胞株中出现核固缩、染色质凝集等形态学改变,FCM结果亦表明PGI-si RNA稳转细胞株发生细胞凋亡。RT-PCR结果显示,PGI-si RNA稳转细胞株中PGI、LDHA和HIF-1α基因表达下调;Western blotting结果显示,PGI-si RNA稳转细胞株中LDHA、HIF-1α、ERK和Bcl-2蛋白表达下调,Bax蛋白表达上调。以上实验结果表明:在乳腺癌MCF-7细胞中,干扰PGI可抑制HIF-1α的表达,同时促进MCF-7细胞凋亡。
This experiment explored the regulation of HIF-1α and the effect of apoptosis on the expression of PGI in breast cancer MCF-7 cells. First, a stably transfected cell line that interferes with PGI (PGI-si RNA) was constructed using lentiviral plasmid transfection, and then apoptosis was detected using Hoechst 33258 staining and flow cytometry (FCM). Subsequently, PGI was detected by RT-PCR. The expression of HIF-1α and LDHA genes were detected. Western blotting was used to detect the expression of Bcl-2, Bax, PGI, LDHA, HIF-1α and ERK proteins. After the successful construction of MCF-7 stably transfected cell line with PGI-si RNA, Hoechst33258 staining results showed that morphological changes such as nuclear condensation and chromatin condensation were observed in the PGI-si RNA-transformed cell line. FCM results also indicated that PGI- The si RNA stabilizes the cell line and undergoes apoptosis. RT-PCR results showed that the PGI, LDHA and HIF-1α gene expression was down-regulated in the PGI-si RNA stably transfected cell line; Western blotting results showed that PGI-si RNA stably transfected LDHA, HIF-1α, ERK and Bcl- 2 The protein expression was down-regulated and the expression of Bax protein was up-regulated. The above experimental results show that: in breast cancer MCF-7 cells, interference with PGI can inhibit the expression of HIF-1α and promote apoptosis of MCF-7 cells.