LncRNA NEAT1/miR-101-3p/RAC1在宫颈癌细胞增殖、迁移和侵袭中的作用及机制研究

Study on the role and mechanism of lncRNA NEAT1/miR-101-3p/RAC1 in the proliferation,migration and invasion of cervical cancer cells

目的 探讨lncRNA NEAT1在宫颈癌细胞增殖、迁移和侵袭中的作用机制。方法 采用RT-qPCR法检测宫颈腺癌细胞系HeLa、宫颈鳞癌细胞系SiHa和人子宫内膜上皮细胞系EM中lncRNA NEAT1的表达;干扰或过表达lncRNA NEAT1后,通过CCK-8和Transwell观察宫颈癌细胞增殖、迁移和侵袭能力的变化,扫描电镜观察细胞侵袭性伪足的变化;采用双荧光素酶报告基因实验检测lncRNA NEAT1与miR-101-3p的靶向关系;通过免疫荧光、Transwell、CCK-8等方法检测lncRNA NEAT1/miR-101-3p/RAC1对SiHa和HeLa细胞增殖、迁移和侵袭能力的影响。结果 相比于EM细胞(0.99±0.04),宫颈癌SiHa(7.59±0.10,P<0.01)和HeLa(1.50±0.07,P<0.05)细胞中lncRNA NEAT1的表达明显升高。相比于sh-NC组,sh-NEAT1组(加入NEAT1敲低质粒)细胞增殖、迁移、侵袭及侵袭性伪足数量明显减少(P<0.05);相比于OE-NC组,OE-NEAT1组(加入NEAT1过表达质粒)SiHa、HeLa细胞增殖、迁移、侵袭及侵袭性伪足数量显著增加(P<0.05)。miR-101-3p与突变的NEAT1共转染后,相比于对照组(3.10±0.16)荧光素酶活性无显著变化(2.99±0.11,P>0.05),而与野生型NEAT1共转染后,相比于对照组(2.97±0.13)荧光素酶活性显著降低(1.15±0.05,P<0.01),提示lncRNA NEAT1与miR-101-3p靶向结合。RT-qPCR结果显示,与sh-NC组(SiHa,0.97±0.09;HeLa,1.07±0.06)相比,sh-NEAT1组miR-101-3p mRNA表达明显升高(SiHa,3.86±0.39;HeLa,2.62±0.51,P<0.01);与OE-NC组(SiHa,0.98±0.20;HeLa,1.05±0.18)相比,OE-NEAT1组miR-101-3p mRNA表达显著降低(SiHa,0.35±0.05;HeLa,0.14±0.03,P<0.05),提示lncRNA NEAT1与miR-101-3p的表达呈负相关。CCK-8、Transwell结果显示,siRNA-RAC1、miR-101-3p mimic使SiHa和HeLa细胞的增殖、迁移和侵袭受到抑制(P<0.05),上调lncRNA NEAT1可部分逆转以上现象,使细胞增殖、迁移和侵袭数目增加(P<0.05)。结论 宫颈癌细胞中lncRNA NEAT1的表达显著升高,它作为miR-101-3p的竞争性内源RNA(ceRNA),通过竞争性结合miR-101-3p并部分控制其对RAC1的调控,进而调节宫颈癌细胞侵袭性伪足的形成并诱导细胞增殖、迁移和侵袭。

Objective To investigate the mechanism of lncRNA NEAT1 in cervical cancer cells proliferation,migration and invasion.Methods The expression of lncRNA NEAT1 in the cervical adenocarcinoma cell line HeLa,the cervical squamous carcinoma cell line SiHa and the human endometrial epithelial cell line EM was detected by RT-qPCR.Changes in proliferation,migration and invasion ability of cervical cancer cells were observed by CCK-8 and Transwell after interfering or overexpressing lncRNA NEAT1,and scanning electron microscopy to observe changes in cellular invadopodia.A dual luciferase reporter gene assay was used to detect the targeting relationship between lncRNA NEAT1 and miR-101-3p.The effects of lncRNA NEAT1/miR-101-3p/RAC1 on invasion,migration and proliferation of SiHa and HeLa cells were detected by immunofluorescence,Transwell,and CCK-8.Results Expression of lncRNA NEAT1 was significantly higher in cervical cancer HeLa(1.50±0.07,P<0.05) and SiHa cells(7.59±0.10,P<0.01) compared to control EM cells(0.99±0.04).Compared with the sh-NC group,cell proliferation,migration,invasion and the number of invadopodias were significantly reduced in the sh-NEAT1 group(with the addition of NEAT1 knockdown plasmid)(P<0.05).Compared with OE-NC group,the number of proliferation,migration,invasion and invadopodias were significantly increased in SiHa and HeLa cells in OE-NEAT1 group(with the addition of NEAT1 overexpression plasmid)(P<0.05).There was no significant change in luciferase activity after co-transfection of miR-101-3p with mutant NEAT1(2.99±0.11,P>0.05) compared to control(3.10±0.16),whereas co-transfection of miR-101-3p with wild-type NEAT1(1.15±0.05) resulted in a significant decrease in luciferase activity compared to control(2.97±0.13,P<0.01),suggested that lncRNA NEAT1 binds to miR-101-3p target.miR-101-3p gene expression was significantly higher in the sh-NEAT1 group(SiHa,3.86±0.39;HeLa,2.62±0.51,P<0.01) compared to the sh-NC group(SiHa,0.97±0.09;HeLa,1.07±0.06),and significantly lower in the OE-NEAT1 group(SiHa,0.98±0.20;HeLa,1.05±0.18),miR-101-3p gene expression was significantly lower in OE-NEAT1 group(SiHa,0.35±0.05;HeLa,0.14±0.03,P<0.05).Compared to OE-NEAT1 group,suggested that lncRNA NEAT1 is negatively correlated with miR-101-3p expression.The proliferation,migration and invasion of SiHa and HeLa cells were inhibited by siRNA-RAC1,miR-101-3p mimic(P<0.05),and up-regulation of lncRNA NEAT1 partially reversed the above phenomena,resulting in an increase in the number of the cells proliferating,migrating and invading(P<0.05).Conclusion The expression of lncRNA NEAT1 in cervical cancer cells is significantly increased.As a competitive endogenous RNA(ceRNA) of miR-101-3p,lncRNA can competitively bind miR-101-3p and partially control its regulation of RAC1.Furthermore,it regulates the formation of invasive pseudopodia of cervical cancer cells and induces cell proliferation,migration and invasion.