LncRNA SNHG1通过miR-377-3p/AKT2轴对宫颈癌细胞增殖、凋亡和迁移的影响

Influences of lncRNA SNHG1 on proliferation,apoptosis and migration of cervical cancer cells via the miR-377-3p/AKT2 axis

目的探讨长链非编码RNA小核仁RNA宿主基因1(lncRNA SNHG1)对宫颈癌(CC)细胞增殖、凋亡和迁移的调控机制。方法体外培养人CC细胞系SiHa、Hela、CaSki和人正常宫颈上皮细胞HUCEC,实时荧光定量PCR(RT-qPCR)检测细胞中SNHG1、微小RNA-377-3p(miR-377-3p)和丝氨酸/苏氨酸激酶2(AKT2)mRNA的表达水平。将SNHG1小分子干扰RNA(si-SNHG1)转染Hela细胞构建SNHG1敲低细胞,并在SNHG1敲低细胞系中下调miR-377-3p表达进行验证。实验分为对照组(NC组)、si-NC组、si-SNHG1组、si-SNHG1+miR-377-3p inhibitor阴性对照组(inhibitor-NC)、si-SNHG1+miR-377-3p inhibitor组。转染48 h后,RT-qPCR检测转染效果;Western Blot检测细胞AKT2蛋白表达水平;MTT法检测细胞增殖能力;流式细胞术检测细胞凋亡率;Transwell小室实验检测细胞侵袭能力;划痕愈合实验检测细胞迁移能力。双荧光素酶报告基因检测和RNA结合蛋白免疫沉淀(RIP)分析Hela细胞中SNHG1、miR-377-3p和AKT2的调控作用。裸鼠异种移植瘤实验、免疫组织化学染色探究敲低SNHG1对CC细胞体内生长的影响。结果与HUCEC细胞相比,不同CC细胞系中SNHG1和AKT2 mRNA表达水平显著升高(P<0.05),miR-377-3p表达水平显著降低(P<0.05);敲低SNHG1表达可显著上调miR-377-3p的表达水平,下调AKT2的mRNA和蛋白水平,降低Hela细胞的增殖活性、迁移与侵袭能力,并诱导细胞凋亡(P<0.05)。双荧光素酶报告基因检测和RIP实验证实SNHG1可靶向负调控miR-377-3p的表达,AKT2是miR-377-3p的靶标。裸鼠异种移植瘤实验结果显示,敲低SNHG1可显著抑制体内移植瘤生长(P<0.05);下调miR-377-3p可显著减弱敲低SNHG1对Hela细胞增殖、迁移和侵袭能力并可抑制体内移植瘤生长(P<0.05)。结论SNHG1可能通过海绵miR-377-3p调节AKT2表达,参与CC进展。

Objective To investigate the regulatory mechanism of long non-coding RNA small nucleolar RNA host gene 1(lncRNA SNHG1)on the proliferation,apoptosis and migration of cervical cancer(CC)cells.Methods Human CC cell lines SiHa,Hela,CaSki and human normal cervical epithelial cells HUCEC were cultured in vitro,and real-time quantitative PCR(RT-qPCR)was performed to detect the expression levels of SNHG1,microRNA-377-3p(miR-377-3p)and AKT2 mRNA in the cells.The SNHG1 small interfering RNA(si-SNHG1)was transfected into Hela cells to construct SNHG1 knockdown cells,and the expression of miR-377-3p was down-regulated in the SNHG1 knockdown cell line for verification.The experiments were separated into control group(NC group),si-NC group,si-SNHG1 group,si-SNHG1+miR-377-3p inhibitor negative control(inhibitor-NC)group,and si-SNHG1+miR-377-3p inhibitor group.Forty-eight hours after transfection,RT-qPCR was performed to detect the transfection effect;Western Blot was performed to detect the expression of AKT2 protein in cells;MTT method was performed to detect cell proliferation ability;flow cytometry was performed to detect cell apoptosis rate;Transwell chamber assay was performed to detect cell invasion ability;scratch healing assay was performed to detect cell migration ability.The regulatory roles of SNHG1,miR-377-3p and AKT2 in Hela cells were analyzed by dual-luciferase reporter gene and RNA-binding protein immunoprecipitation(RIP).Nude mouse xenograft experiments and immunohistochemical staining were performed to explore the effect of SNHG1 knockdown on the growth of CC cells in vivo.Results Compared with HUCEC cells,the expression levels of SNHG1 and AKT2 mRNA in different CC cell lines were significantly increased(P<0.05),and the expression level of miR-377-3p was significantly decreased(P<0.05).Knocking down the expression of SNHG1 was able to significantly up-regulate the expression level of miR-377-3p,down-regulate the levels of AKT2 mRNA and protein,reduce the proliferation activity,migration and invasion abilities of Hela cells,and induce cell apoptosis(P<0.05).Dual-luciferase reporter gene and RIP experiments confirmed that SNHG1 could target and negatively regulate miR-377-3p expression,and AKT2 was a target of miR-377-3p.The results of xenograft tumor experiments in nude mice showed that knockdown of SNHG1 could significantly inhibit the growth of xenograft tumors in vivo(P<0.05).Down-regulation of miR-377-3p significantly attenuate the inhibitory effects of SNHG1 knockdown on Hela cell proliferation,migration,invasion,and xenograft growth in vivo(P<0.05).Conclusions The SNHG1 may regulate AKT2 expression and participate in CC progressionthrough sponging miR-377-3p.