Objective: To investigate the molecular mechanism by which miR-20a-5p regulates HOXB13 gene expression and inhibits lung cancer cell proliferation.
Methods: The expression levels of HOXB13 mRNA and protein in lung cancer A549 cells transfected with HOXB13 overexpression plasmid or HOXB13 siRNA were detected with real-time fluorescence quantitative PCR (qRT-PCR) and Western blotting. CCK-8 and EdU assays were used to examine the effect of modulation of HOXB13 expression on cell proliferation. We screened possible binding miRNAs of HOXB13 by bioinformatics analysis. In A549 cells transfected with miR-20a-5p mimic or miR-20a-5p inhibitor, the expression level of miR-20a-5p was detected by qRT-PCR and the protein expression of HOXB13 was determined with Western blotting. CCK-8 and EdU assays were used to assess the effect of miR-20a-5p overexpression on the proliferation of A549 cells. miR-20a-5p mimic and HOXB13 overexpression plasmids were co-transfected into A549 cells, and the changes in cell proliferation were evaluated with CCK-8 and EdU assays.
Results: HOXB13 overexpression obviously promoted the proliferation of A549 cells (P < 0.05). miR-20a-5p was identified as the potential binding miRNA of HOXB13. Overexpression of miR-20a-5p in A549 cells significantly decreased the expression of HOXB13 protein (P < 0.05), while interference of miR-20a-5p obviously increased HOXB13 expression (P < 0.05). The results of cell proliferation experiment showed that miR-20a-5p and HOXB13 had opposite effects on cell proliferation, and the cells overexpressing both miR-20a-5p and HOXB13 showed a lower proliferation activity than the cells overexpressing HOXB13 but higher than the cells overexpressing miR-20a-5p alone (P < 0.05).
Conclusion: miR-20a-5p inhibits proliferation of lung cancer cells by down-regulating the expression of HOXB13.
目的: 探讨miR-20a-5p调控HOXB13基因表达抑制肺癌细胞系A549增殖的分子机制。
方法: 转染HOXB13过表达质粒与HOXB13 siRNA到肺癌细胞系A549，通过qRT-PCR及Western blot实验检测HOXB13 mRNA及蛋白的表达水平；CCK-8及EdU实验检测HOXB13对肺癌细胞系A549细胞增殖的影响；利用生物信息学分析筛选HOXB13可能结合的miRNA；通过qRT-PCR检测转染miR-20a-5p mimic与miR-20a-5p inhibitor到肺癌细胞系后miR-20a-5p的表达水平；Western blot实验检测HOXB13在肺癌细胞系中的表达情况；CCK-8及EdU实验检测miR-20a-5p肺癌细胞系A549细胞增殖的影响；在A549细胞中共转染miR-20a-5p mimic及HOXB13过表达质粒，CCK-8及EdU实验检测A549细胞增殖能力。
结果: HOXB13促进了A549细胞的增殖（P＜0.05）；生物信息学筛选出HOXB13可能结合的miRNA为miR-20a-5p；在肺癌细胞系中过表达miR-20a-5p后，HOXB13蛋白表达量降低（P＜0.05）；而干扰miR-20a-5p表达后，HOXB13蛋白的表达量升高（P＜0.05）；细胞增殖实验结果显示，miR-20a-5p与HOXB13对细胞增殖的影响相反，miR-20a-5p及HOXB13同时过表达，细胞增殖情况介于单独过表达miR-20a- 5p及单独过表达HOXB13组之间（P＜0.05）。
结论: miR-20a-5p调控HOXB13基因表达抑制肺癌细胞系A549的增殖。
Keywords: A549; HOXB13; cell proliferation; lung cancer; miR-20a-5p.