miR-20b通过靶向VEGFA对鼻咽癌细胞增殖凋亡和侵袭的调控作用

Regulatory Effect of miR-20b on the Proliferation, Apoptosis and In-vasion of Nasopharyngeal Carcinoma Cells by Targeting VEGFA

目的 探究miR-20b通过靶向VEGFA对鼻咽癌细胞增殖凋亡和侵袭的调控的机制.方法 使用RT-PCR检测鼻咽癌组织样本和细胞株中miR-20b和VEGFA表达水平;将鼻咽癌CNE细胞分为空白对照组、阴性对照组和miR-20b转染组;阴性对照组和miR-20b转染组分别使用空白质粒和miR-20b转染;RT-PCR检测各组VEGFA基因表达水平;荧光色酶报告基因实验分析miR-20b和VEGFA靶向作用关系;将鼻咽癌CNE细胞分阴性对照组、 miR-20b转染组、 VEGFA组和miR-20b+pc-VEGFA组, 使用蛋白质印迹法检测各组VEGFA蛋白表达;使用CCK-8检测细胞增殖情况;使用流式检测细胞凋亡情况;使用蛋白质印迹法检测增殖、凋亡、侵袭相关蛋白表达;使用Transwell法检测细胞侵袭情况.结果 鼻咽癌组织样本中miR-20b表达水平显著低于正常组织, VEGFA表达显著高于正常组织 (P<0.05);荧光素酶报告实验表明miR-20b上有VEGFA的结合位点;相比阴性对照组, miR-20b转染组miR-20b表达、细胞凋亡率、 Bax表达、 E-cadherin表达显著升高;VEGFA表达、细胞增殖率、 Ki67表达、 PCNA表达、 Bcl-2表达、细胞侵袭能力、 Vimentin表达、 Fibronectin表达显著降低 (P<0.05);相比VEGFA组, miR-20b+pc-VEGFA组miR-20b表达、细胞凋亡率、 Bax表达、 E-cadherin表达显著升高;VEGFA表达、细胞增殖率、 Ki67表达、 PC-NA表达、 Bcl-2表达、细胞侵袭能力、 Vimentin表达、 Fibronectin表达显著降低 ( P<0.05).结论 miR-20b通过靶向VEGFA促进鼻咽癌细胞的凋亡, 抑制其增殖和侵袭迁移能力.

Objective To explore the regulatory mechanism of miR-20b on the proliferation, apoptosis and invasion of nasopharyngeal carcinoma ( NPC) cells by targeting VEGFA.Methods RT-PCR was applied to detect the expression levels of miR-20b and VEGFA in NPC tissue samples and cell lines.NPC CNE cells were divided into blank control group, negative control group and miR-20b transfection group.Cells in the negative control group and miR-20b transfection group were transfected with blank plasmid and miR-20b, respectively.The expression level of VEGFA gene in each group was detected by RT-PCR.The luciferase reporter gene test was applied to analyze the tar-geting relationship between miR-20b and VEGFA.NPC CNE cells were divided into negative control group, miR-20b transfection group, VEGFA group and miR-20b +pc-VEGFA group.The expres-sion of VEGFA protein in each group was detected by Western blotting.Cell proliferation was detec-ted by CCK-8.Apoptosis was detected by flow cytometry.The expression of proteins related to cell proliferation, apoptosis and invasion was detected by Western blotting.Cell invasion was detected by Transwell method. Results The expression level of miR-20b in NPC tissue samples was signifi-cantly lower than that in normal tissues, while expression of VEGFA was significantly higher than that in normal tissues ( P <0.05 ).The luciferase reporter assay showed that there were binding sites of VEGFA on miR-20b.Compared with those in the negative control group, the miR-20b ex-pression, apoptosis rate, expression of Bax and E-cadherin were significantly increased, while the VEGFA expression, cell proliferation rate, expression of Ki67, PCNA and Bcl-2, cell invasive a-bility, expression of vimentin and fibronectin were significantly decreased in miR-20b transfection group (P <0.05 ).Compared with those in VEGFA group, the miR-20b expression, apoptosis rate, expression of Bax and E-cadherin were significantly increased, while the VEGFA expression, cell proliferation rate, expression of Ki67, PCNA and Bcl-2, cell invasive ability, expression of vimentin and fibronectin were significantly decreased in miR-20b+pc-VEGFA group ( P<0.05) . Conclusion miR-20b can promote the apoptosis of NPC cells and inhibit their proliferation, inva-sion and migration by targeting VEGFA.