下调HOXA-AS2/miR-20a通路促进皮肤鳞癌细胞凋亡并抑制其侵袭迁移

Down-regulating HOXA-AS2/miR-20a pathway promotes apoptosis and inhibits invasion and migration of skin squamous carcinoma cells

目的研究下调长链非编码RNA(lncRNA)HOXA-AS2/miR-20a通路对皮肤鳞癌细胞凋亡和侵袭的影响。方法qRT-PCR检测皮肤鳞癌组织中HOXA-AS2的表达变化。在皮肤鳞癌细胞A431中转染HOXA-AS2 siRNA,MTT测定细胞增殖,克隆形成实验测定细胞克隆形成能力,流式细胞仪测定细胞凋亡,Transwell小室实验检测细胞迁移和侵袭,采用Western blot检测MMP-2、MMP-9、C-Caspase-3蛋白表达。生物信息学软件预测HOXA-AS2和miR-20a有互补结合位点,荧光素酶报告系统鉴定二者的靶向关系。qRT-PCR检测皮肤鳞癌组织中miR-20a的表达变化。在皮肤鳞癌细胞A431中共转染HOXA-AS2 siRNA、miR-20a inhibitor,检测细胞增殖、克隆、凋亡、迁移、侵袭能力的变化。结果HOXA-AS2在皮肤鳞癌组织中的表达升高。转染HOXA-AS2 siRNA后的皮肤鳞癌细胞A431增殖、克隆、侵袭、迁移能力下降,细胞凋亡水平升高,MMP-2、MMP-9蛋白表达减少,C-Caspase-3蛋白表达增高。下调HOXA-AS2靶向促进miR-20a表达。miR-20a在皮肤鳞癌组织中的表达与HOXA-AS2负相关。miR-20a inhibitor可以逆转HOXA-AS2 siRNA对皮肤鳞癌细胞A431增殖、克隆、侵袭、迁移和凋亡作用。结论下调HOXA-AS2/miR-20a通路降低皮肤鳞癌细胞增殖、克隆、侵袭、迁移能力,诱导细胞凋亡。

Objective To study the effect of down-regulation of long non-coding RNA(lncRNA)HOXA cluster antisense RNA 2(HOXA-AS2)/miR-20 a pathway on apoptosis and invasion of skin squamous cell carcinoma cells in vitro.Methods qRT-PCR was used to detect the expression of HOXA-AS2 and miR-20 a in 50 clinical specimens of skin squamous cell carcinoma and paired adjacent tissues.Skin squamous carcinoma cell line A431 was transfected with HOXA-AS2 siRNA,and the changes in cell proliferation,clonogenic ability,apoptosis,migration and invasion were evaluated by using MTT assay,clone formation experiment,flow cytometry,and Transwell assay respectively.Western blotting was performed to detect the expression of MMP-2,MMP-9,and cleaved caspase-3 proteins in the cells.Bioinformatics software predicted that HOXA-AS2 and miR-20 a had complementary binding sites,and a luciferase reporter system was employed to verify their targeting relationship.A431 cells were co-transfected with HOXA-AS2 siRNA and miR-20 a inhibitor,and the changes in cell proliferation,clone formation,apoptosis,migration and invasion were assessed.Results The expression level of HOXA-AS2 was increased in skin squamous cell carcinoma tissues.Transfection of A431 cells with HOXA-AS2 siRNA significantly suppressed the cell proliferation,clone formation,invasion and migration,promoted cell apoptosis,decreased the expression of MMP-2 and MMP-9 proteins,and increased the expression of cleaved caspase-3 protein.Down-regulation of HOXA-AS2 significantly up-regulated miR-20 a expression.The expression level of miR-20 a in skin squamous cell carcinoma tissues was negatively correlated with that of HOXA-AS2.In A431 cells,miR-20 a inhibitor obviously reversed the effects of HOXA-AS2 siRNA on proliferation,clone formation,invasion,migration and apoptosis of the cells.Conclusion Down-regulation of HOXA-AS2/miR-20 a pathway can promote the expression of miR-20 a,suppress the proliferation,clone formation,invasion and migration,and induce apoptosis of skin squamous cell carcinoma cells in vitro.