PKM2基因沉默对人乳腺癌MCF-7细胞增殖及侵袭的影响

Influence of PKM2 gene silencing on proliferation and invasion of human breast cancer MCF-7 cells

目的 :探讨M2型丙酮酸激酶(pyruvate kinase M2,PKM2)下调对人乳腺癌MCF-7细胞增殖及侵袭的影响。方法 :采用免疫荧光技术检测MCF-7细胞中PKM2蛋白的表达。通过脂质体法将构建有PKM2-sh RNA的重组质粒转入MCF-7细胞;分别采用实时荧光定量PCR法和蛋白质印迹法检测PKM2 m RNA和蛋白的表达水平;CCK-8法、FCM法和Transwell侵袭实验分别检测PKM 2基因沉默后对MCF-7细胞增殖、凋亡及侵袭能力的影响;蛋白质印迹法检测对增殖相关蛋白c-myc和cyclin D1以及凋亡相关蛋白Bcl-2和Bax表达的影响。结果:乳腺癌MCF-7细胞的细胞质及细胞核中均有PKM2的表达;PKM2-sh RNA质粒转染MCF-7细胞48 h后,可明显下调MCF-7细胞中PKM2 m RNA及蛋白的表达水平(P值均<0.01),并且明显抑制MCF-7细胞的增殖及侵袭能力,促进其凋亡(P值均<0.05)。沉默PKM2基因的表达,可下调c-myc、cyclin D1和Bcl-2蛋白的表达水平(P值均<0.05),而上调Bax蛋白的表达(P<0.05)。结论 :转染PKM2-sh RNA特异性干扰PKM2基因的表达后,能通过下调c-myc和cyclin D1的表达抑制细胞的增殖,通过下调Bcl-2的表达以及上调Bax的表达促进细胞的凋亡。

Objective： To investigate the effect of pyruvate kinase M2 （PKM2） silencing on proliferation and invasion of human breast cancer MCF-7 cells. Methods： The expression of PKM2 protein in MCF-7 cells was detected by immunofluorescence assay. MCF-7 cells were transfected with recombinant plasmid of PKM2-shRNA by liposome method. The expression levels of PKM2 mRNA and protein in MCF-7 cells transfected with PKM2-shRNA were detected by real-time fluorescent quantitative-PCR and Western blotting, respectively. The effects of PKM2 gene silencing on proliferation, apoptosis and invasion ability of MCF-7 cells were detected by CCK-8 method, FCM and Transwell invasion test, respectively. The expression levels of proliferation-related proteins c-myc and cyclin D1 and apoptosis-related proteins Bcl-2 and Bax were detected by Western blotting.Results： The PKM2 protein was located in cytoplasm and nucleus in MCF-7 cells. The expression levels of PKM2 mRNA and protein in MCF-7 cells at 48 h after transfection with PKM2-shRNA were significantly reduced （both P 〈 0.01）. The cell proliferation and invasion ability of MCF-7 cells were significantly inhibited （both P 〈 0.05）, and the apoptosis rate was significantly increased （P 〈 0.01）. The expression levels of c-myc, cyclin D1 and Bcl-2 proteins were significantly reduced （all P 〈 0.05）, and the expression level of Bax protein was significantly increased （P 〈 0.05）.Conclusion： Transfection with PKM2-shRNA can obviously decrease the expression of PKM2 gene in MCF-7 cells, then inhibit the cell proliferation via downregulating the expressions of c-myc and cyclin D1, and promote the apoptosis via downregulating the expression of Bcl-2 and upregulating the expression of Bax.