miR-133b靶向PKM2基因对肺癌A549干细胞增殖及药物敏感性的影响

MiR-133b Affect the Proliferation and Drug Sensitivity in A549 Lung Cancer Stem Cells by Targeting PKM2

背景与目的已有研究表明miR-133b可抑制肺癌细胞生长,miR-133b表达水平在肺癌组织及患者血清中显著降低,miR-133b通过靶向丙酮酸激酶M2型(pyruvate kinase isozyme type M2,PKM2)基因提高鳞癌细胞的放疗敏感性,但其机制尚未明了。本研究通过提取非小细胞肺癌细胞株A549的CD133^+/CD34^+干细胞,研究miR-133b对其增殖及顺铂类药物(cisplatin,DDP)敏感性的影响,探讨miR-133b与PKM2基因的关系,以及它们在肺癌干细胞中的作用。方法使用miRBase和miRNAMap数据库进行miR-133b与PKM2基因的序列比对。应用免疫磁珠分选法从A549细胞中分选出CD133^+/CD34^+肺癌干细胞,流式细胞仪检测纯度。转染miR-133b进入CD133^+/CD34^+细胞,qRT-PCR检测验证miR-133b表达情况,CCK8法检测细胞增殖情况;15μg/mL DDP处理转染miR-133b细胞,检测0 h、12 h、24 h、72 h的细胞凋亡情况;采用Western blot检测PKM2蛋白水平的表达。结果PKM2基因可能为miR-133b的靶基因;流式结果显示CD133^+/CD34^+细胞的纯度为(92.15+4.27)%。qRT-PCR结果显示,与对照组相比,过表达miR-133b后,miR-133b明显上调,miR-133b抑制表达后,miR-133b明显下调(P<0.05)。细胞增殖实验及Western blot结果显示,与对照组相比,miR-133b mimics组细胞的增殖能力显著降低(P<0.05),PKM2蛋白水平明显降低(P<0.05);而抑制miR-133b表达则明显增加细胞的增殖能力及PKM2蛋白水平(P<0.05)。DDP处理12 h后miR-133b mimics组细胞的凋亡持续明显高于对照组(P<0.05)。miR-133b mimics+DDP组PKM2蛋白的表达明显低于对照组及miR-133b inhibitor组(P<0.05)。结论过表达miR-133b能够通过下调PKM2而抑制肺癌干细胞的生长和增殖,并且可以增强肺癌干细胞对DDP的敏感性。

Background and objective It has been proven that miR-133 b could inhibit cancer cell growth, the expression level of miR-133 b was significant reduction in lung cancer tissue and serum of patients, and increase the radiation sensitivity of squamous cell carcinoma by targeting PKM2, but the exist mechanisms is not clear. The aim of this study is to explore the effect of miR-133 b on proliferation in A549 lung cancer stem cells and drug sensitivity in DDP, and to explore the relationship between miR-133 b and PKM2 gene, as well as the effect of cancer stem cells. Methods Using miRBase and miRNAMap database to sequence comparison miR-133 b and PKM2 gene. Using immune magnetic separation method to select the CD133^＋/CD34^＋ lung cancer stem cells from A549 cells, and using flow cytometry to detect the purity. The expression of miR-133 b m RNA was detected by real-time fluorescence quantitative PCR（q RT-PCR）. Cell proliferation was detected by CCK8 assay. 15 μg/m L DDP was treated to cells which was transfected with miR-133 b, and apoptosis was detected by flow Cytometry at 0 h, 12 h, 24 h, 72 h. The expression of PKM2 protein was detected by Western blot. Results Gene binding site report that PKM2 gene may be the target gene of miR-133b; the results of flow cytometry showed that the purity of CD133^＋/CD34^＋ stem cells was（92.15±4.27）%.q RT-PCR results showed that compared with the control group, after overexpression of miR-133 b, miR-133 b was upregulated and miR-133 b was down regulated after miR-133 b inhibition（P〈0.05）. Compared with the control group, cell proliferation of miR-133 b mimics group was significantly decreased（P〈0.05）, PKM2 protein levels were significantly lower（P〈0.05）; and cell proliferation of the miR-133 b inhibitor group and PKM2 level was increased（P〈0.05）. The apoptosis of miR-133 b mimics group was significantly higher than that of control group（P〈0.05） after DDP treatment with 12 h. The expression of PKM2 protein in miR-133 b mimics＋DDP group was significantly lower than that in control group（P〈0.05）. Conclusion Overexpression of miR-133 b can inhibit the growth and proliferation of lung cancer stem cells by down regulating PKM2, and can enhance the sensitivity of lung cancer stem cells to DDP.