微小RNA-508-3p靶向调控配对盒基因2影响肺癌细胞生物学特性的分子机制研究

Study on the molecular mechanism of miR-508-3p targeted regulation of paired box gene 2 affecting the biological characteristics of lung cancer cells

  • 摘要:
      目的  探讨微小RNA-508-3p(miR-508-3p)在肺癌组织中的表达及其对肺癌细胞增殖活力、侵袭能力、凋亡率的影响与机制。
      方法  采用定量聚合酶链反应(qPCR)法和蛋白质印迹法(Western blotting)检测肺癌组织中miR-508-3p和配对盒基因2(PAX2)表达,分析两者的相关性。分别用miR-508-3p模拟物(miR-508-3p组)、模拟物阴性对照miR-NC(miR-NC组)、PAX2 siRNA si-PAX2 (si-PAX2组)、siRNA阴性对照(si-NC组)、miR-508-3p抑制剂anti-miR-508-3p(anti-miR-508-3p组)、抑制剂阴性对照anti-miR-NC(anti-miR-NC组)、共转染miR-508-3p和PAX2过表达质粒pcDNA-PAX2 (miR-508-3p+pcDNA- PAX2组)、共转染miR-508-3p和空白质粒pcDNA-NC(miR-508-3p+pcDNA-NC组)转染肺癌A549细胞,并设置空白组。采用噻唑蓝(MTT)法检测24~72 h的细胞增殖活力; 采用Transwell小室及流式细胞术分别检测细胞侵袭能力及凋亡率,采用Western blotting检测A549细胞基质金属蛋白酶-2(MMP-2)、B淋巴细胞瘤-2(Bcl-2)和Bcl-2相关X蛋白(Bax)蛋白表达。根据双荧光素酶活性检测结果评估miR-508-3p对PAX2的靶向调控。
      结果  肺癌组织miR-508-3p表达低于癌旁组织, PAX2 mRNA和PAX2蛋白表达高于癌旁组织,差异有统计学意义(P < 0.05); PAX2蛋白和miR-508-3p的表达呈显著负相关(P < 0.05)。与空白组比较, miR-508-3p组Bax表达和细胞凋亡率均升高, MMP-2、Bcl-2蛋白表达和增殖活力、侵袭能力均降低,差异有统计学意义(P < 0.05); 与si-NC组比较, si- PAX2组PAX2、MMP-2、Bcl-2表达降低, Bax表达、凋亡率升高,增殖活力和侵袭能力降低,差异有统计学意义(P < 0.05)。miR-508-3p靶向PAX2基因,调控PAX2蛋白的表达。PAX2可以逆转miR-508-3p对A549细胞增殖活力、侵袭能力以及细胞凋亡的影响。
      结论  miR-508-3p在肺癌组织低表达,其过表达可降低A549细胞增殖活力及侵袭能力,诱导细胞凋亡,机制与靶向调控PAX2基因有关。

     

    Abstract:
      Objective  To investigate the expression of miR-508-3p in lung cancer tissues, explore the effects of proliferation activity, invasion ability and apoptosis rate and mechanisms.
      Methods  Quantitative polymerase chain reaction (qPCR) and western blotting were used to detect the expressions of miR-508-3p and paired box gene 2(PAX2) in lung cancer tissues, and the correlation between them was analyzed. miR-508-3p mimics (miR-508-3p group), mimic negative control miR-NC (miR-NC group), PAX2 siRNA si- PAX2 (si- PAX2 group), siRNA negative control (si-NC group), miR-508-3p inhibitor anti-miR-508-3p (anti-miR-508-3p group), inhibitor negative control anti-miR-NC (anti-miR-NC group), co-transfected miR-508-3p and PAX2 overexpression plasmid pcDNA- PAX2 (miR-508-3p+pcDNA- PAX2 group), co-transfected miR-508-3p and blank plasmid pcDNA-NC (miR-508-3p+pcDNA-NC group) were transfected into lung cancer A549 cells and blank (NC) group was established. Methyl tetrazolium (MTT) assay was used to detect cell proliferation activity in 24 to 72 h; transwell chamber and flow cytometry were used to detect cell invasion ability and apoptosis rate, western blotting was used to detect A549 cell invasion-related matrix metalloproteinase-2 (MMP-2) and apoptosis-related B lymphocytoma-2(Bcl-2) and Bcl-2 associated X protein(Bax). The dual luciferase activity was used to evaluate the actions of targeted regulation of PAX2 by miR-508-3p.
      Results  The expression of miR-508-3p in lung cancer tissue was lower than that in adjacent tissues, and the expression of PAX2 mRNA and PAX2 protein was significantly higher than that in adjacent tissues (P < 0.05). The expression of PAX2 protein and miR-508-3p was negatively correlated (P < 0.05). Compared with the blank group, the expression of Bax and apoptosis rate of the miR-508-3p group were increased, and the expressions of MMP-2 and Bcl-2 proteins, proliferation activity and invasion ability were decreased (P < 0.05). Compared with si-NC group, the expressions of PAX2, MMP-2 and Bcl-2 in the si- PAX2 group were decreased, the expression of Bax and apoptosis rate were increased, and the proliferation activity and invasion ability were decreased (P < 0.05). MiR-508-3p targeted PAX2 and regulated the expression of PAX2. PAX2 could reverse the effects of miR-508-3p on the proliferation, invasion and apoptosis of A549 cells.
      Conclusion  MiR-508-3p is under-expressed in lung cancer tissues, its overexpression can reduce the proliferation and invasion of A549 cells and induce cell apoptosis. The mechanism is related to the targeted regulation of PAX2 gene.

     

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