微RNA-509-3p/干扰素刺激基因15轴对喉癌细胞血管生成及血管内皮生长因子蛋白表达的影响

Effect of miR-509-3p/ISG15 axis on angiogenesis and VEGF protein expression in laryngeal cancer cells

目的探究微RNA(miR)-509-3p/干扰素刺激基因15(ISG15)轴对喉癌细胞血管生成及血管内皮生长因子(VEGF)蛋白表达的影响。方法2022年1―9月,将喉癌M4E细胞分为Con组、miR-NC组、miR-509-3p组、pcDNA-ISG15组。逆转录聚合酶链式反应(RT-PCR)检测细胞中miR-509-3p表达;细胞计数试剂盒(CCK8)检测细胞增殖能力;Transwell检测细胞侵袭能力;划痕实验检测细胞迁移能力;流式细胞仪检测细胞凋亡率;Matrigel体外成管实验检测血管生成能力;蛋白质印迹法检测细胞中ISG15、VEGF蛋白表达;萤光素酶活性报告检测miR-509-3p和ISG15的靶向关系。结果与喉上皮细胞NP69中miR-509-3p表达(1.01±0.13)μg/L相比,喉癌细胞SCC-40、SCC-2、M4E中miR-509-3p表达[(0.61±0.08)、(0.45±0.07)、(0.18±0.07)μg/L]均降低,且M4E细胞中miR-509-3p表达低于SCC-40、SCC-2细胞(P<0.05)。与Con组相比,miR-509-3p组细胞中miR-509-3p表达增加[(1.17±0.05)μg/L比(118±0.03)μg/L];Con组细胞中miR-509-3p表达与miR-NC组相比差异无统计学意义(P>0.05)。与Con组相比,miR-509-3p组细胞增殖(1.71±0.02比3.09±0.07)、侵袭(55.33±2.52比150.67±2.52)、迁移(30.58±2.08比98.33±1.53)、形成小管数量(61.28±2.15比135.62±2.54)及细胞中ISG15(0.24±0.03比1.11±0.13)、VEGF(0.35±0.02比0.99±0.04)蛋白表达均降低,细胞凋亡率[(18.76±0.18)%比(5.61±0.08)%]增加(P<0.05);Con组细胞增殖、侵袭、迁移、形成小管数量、凋亡率及细胞中ISG15、VEGF蛋白表达和miR-NC组相比差异无统计学意义(P>0.05);与miR-509-3p组相比,pcDNA-ISG15组细胞增殖(3.25±0.06)、侵袭(144.00±2.65)、迁移(89.34±5.13)、形成小管数量(131.56±2.08)及细胞中ISG15(0.83±0.05)、VEGF蛋白(0.89±0.03)表达增加,细胞凋亡率降低(6.85±0.06)%(P<0.05)。结论过表达miR-509-3p可抑制喉癌细胞血管生成及VEGF蛋白表达。

Objective To investigate the effect of miR-509-3p/ISG15 axis on angiogenesis and VEGF protein expression in laryngeal cancer cells.Methods The study was conducted between January 2022 and September 2022.The laryngeal carcinoma M4E cells were divided into Con group,miR-NC group,miR-509-3p group,and pcDNA-ISG15 group.The rate of apoptosis;Matrigel in vitro tube formation assay to detect angiogenesis;protein immunoblotting to detect ISG15 and VEGF protein expression in cells;luciferase activity reporter to detect the targeting relationship between miR-509-3p and ISG15.Results Compared with the laryngeal epithelial cell NP69(1.01±0.13)μg/L,the expression of miR-509-3p in laryngeal cancer cells SCC-40,SCC-2 and M4E was decreased(0.61±0.08,0.45±0.07 and 0.18±0.07)μg/L,respectively.The expression of miR-509-3p in M4E cells was lower than that in SCC-40 and SCC-2 cells(P<0.05).Compared with the con group(118±0.03)μg/L,the expression of miR-509-3p in cells of the miR-509-3p group was increased(1.17±0.05)μg/L.The expression of miR-509-3p in Con group was not significantly different from that in miR-NC group(P>0.05).Compared with con group[3.09±0.07,150.67±2.52,98.33±1.53,135.62±2.54,(5.61±0.08)%],In miR-509-3p group,cell proliferation(1.71±0.02),invasion(55.33±2.52),migration(30.58±2.08),the number of tubules(1.28±2.15),ISG15(0.24±0.03)and VEGF(0.35±0.02)protein expression in cells were decreased.The apoptosis rate(18.76±0.18)%,P<0.05 was increased.There was no significant difference in cell proliferation,invasion,migration,the number of tubules formed,apoptosis rate and the expression of ISG15 and VEGF protein in cells between the Con group and the miR-NC group(P>0.05).Compared with the miR-509-3p group,In the pcDNAISG15 group,the cell proliferation(3.25±0.06),invasion(144.00±2.65),migration(89.34±5.13),the number of tubules formed(131.56±2.08),and the expression of ISG15(0.83±0.05)and VEGF protein(0.89±0.03)in the cells increased the apoptosis rate was decreased(6.85±0.06)%(P<0.05).Conclusion Overexpression of miR-509-3p inhibits angiogenesis and VEGF protein expression in laryngeal cancer cells.