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目的 :探讨微RNA-21(microRNA-21,miR-21)水平下调对个旧肺鳞癌YTMLC-90细胞增殖、迁移及凋亡的影响,以及其作用机制是否与人第10号染色体缺失的磷酸酶及张力蛋白同源基因(phosphatase and tensin homolog deleted on chromosome ten,PTEN)、抗凋亡基因Bcl-2和基质金属蛋白酶抑制基因RECK(reversioninducing-cysteine-rich protein with kazal motifs)表达调控有关。方法 :首先采用实时荧光定量PCR法检测miR-21在正常肺上皮细胞和不同类型肺癌细胞中的表达量,验证个旧肺鳞癌YTMLC-90细胞中miR-21相对高表达。采用蛋白质印迹法及实时荧光定量PCR法检测转染pGCMV/EGFP-hsa-anti-miR-21干扰质粒后,个旧肺鳞癌细胞YTMLC-90中miR-21靶基因PTEN、RECK及Bcl-2表达水平的变化。MTS法及细胞划痕实验分别检测转染后YTMLC-90细胞的增殖及迁移能力。透射电子显微镜和FCM法分别对转染后YTMLC-90细胞的凋亡进行定性和定量分析。结果:相较于正常肺上皮细胞BEAS-2B,个旧肺鳞癌YTMLC-90细胞中miR-21表达水平明显较高(P<0.05)。经pGCMV/EGFP-hsa-anti-miR-21干扰质粒转染后,YTMLC-90细胞中miR-21水平下调,而其下游作用靶点PTEN及RECK蛋白水平上调(P<0.05),Bcl-2蛋白水平下调(P<0.05)。与未转染对照组相比,转染后YTMLC-90细胞的增殖及迁移能力均明显降低(P<0.05),而细胞凋亡率明显增加(P<0.05)。结论:miR-21可能通过调节靶基因PTEN、RECK及Bcl-2的表达促进个旧肺鳞癌细胞的生长和迁移,抑制细胞凋亡,提示miR-21可能是治疗个旧肺鳞癌的一个潜在靶点。此外,从分子遗传学的角度否定了个旧肺鳞癌是一种特殊种类的肺癌。
Objective: To investigate the effect of down-regulation of microRNA-21 (miR-21) on the proliferation, migration and apoptosis of lung cancer cell line YTMLC-90 in Gejiu old patients and whether its mechanism of action is related to the phosphorylation of human chromosome 10 PTEN, Bcl-2, and RECK (reversioninducing-cysteine-rich protein with kazal motifs) expression and regulation. Methods: The expression of miR-21 in normal lung epithelial cells and different types of lung cancer cells was detected by real-time fluorescence quantitative PCR, and the relative expression of miR-21 in YTMLC-90 cells was examined. Western blotting and real-time fluorescence quantitative PCR were used to detect the expression of PTEN, RECK and Bcl-2 in Gejiu lung squamous carcinoma cell line YTMLC-90 after transfection with pGCMV / EGFP-hsa-anti-miR- Horizontal changes. MTS assay and cell scratch assay were used to detect the proliferation and migration of YTMLC-90 cells after transfection. Transmission electron microscopy and FCM were used to qualitatively and quantitatively analyze the apoptosis of YTMLC-90 cells after transfection. Results: Compared with normal lung epithelial cells BEAS-2B, miR-21 expression was significantly higher in Gejiu lung squamous cell carcinoma YTMLC-90 cells (P <0.05). After transfection with pGCMV / EGFP-hsa-anti-miR-21 plasmid, the level of miR-21 in YTMLC-90 cells was down-regulated and the downstream targets of PTEN and RECK were upregulated (P <0.05) Protein levels were down-regulated (P <0.05). Compared with untransfected control group, the proliferation and migration ability of YTMLC-90 cells were significantly decreased (P <0.05), while the apoptosis rate was significantly increased (P <0.05). Conclusion: miR-21 may promote the growth and migration of old lung squamous cell carcinoma and inhibit apoptosis by regulating the expression of target genes PTEN, RECK and Bcl-2, suggesting that miR-21 may be a potential target for treating old squamous cell carcinoma point. In addition, from the point of view of molecular genetics, old lung squamous cell carcinoma is a special kind of lung cancer.