lncRNA ZNF674-AS1通过miR-510-5p/REPS2轴调控宫颈癌细胞HCC94的增殖和迁移

lncRNA ZNF674-AS1 regulates the proliferation and migration of cervical cancer HCC94 cells via miR-510-5p/REPS2 axis

目的:探讨lncRNA ZNF674-AS1在宫颈癌中的作用及其分子机制。方法:用qPCR法检测ZNF674-AS1在31例2019年1月至2020年7月在武汉儿童医院接受手术治疗患者的宫颈癌组织和对应的癌旁组织、宫颈癌细胞系(SiHa、HeLa、C33A和HCC94)和永生化子宫颈上皮细胞系中的表达。转染过表达ZNF674-AS1质粒及其阴性对照质粒至ZNF674-AS1表达最少的HCC94细胞,CCK-8法和Transwell实验检测过表达ZNF674-AS1对HCC94细胞增殖活性和迁移能力的影响。生物信息学方法预测和双荧光素酶报告基因实验验证ZNF674-AS1和miR-510-5p、REPS2三者间的互补结合关系。qPCR检测过表达ZNF674-AS1对miR-510-5p与REPS2表达的影响,WB法检测过表达ZNF674-AS对细胞增殖和迁移相关因子表达的影响。结果:与癌旁组织相比,ZNF674-AS1在宫颈癌组织中呈明显低表达(P<0.01);与永生化子宫上皮细胞相比,ZNF674-AS1在宫颈癌细胞系中也呈明显低表达(P<0.05或P<0.01),以HCC94细胞中表达最低(P<0.01)。过表达ZNF674-AS1能明显抑制HCC94细胞的增殖(P<0.05)和迁移(P<0.01)。与ZNF674-AS1相互作用的miRNA是miR-510-5p,与miR-510-5p相互作用的基因是REPS2。过表达ZNF674-AS1导致HCC94细胞中miR-510-5p的表达水平降低(P<0.01)而REPS2基因的表达水平升高(P<0.01),同时引起细胞增殖和迁移相关的多种因子(CDK2、cyclin D3、vimentin和twist)上调或下调。结论:lncRNA ZNF674-AS1在宫颈癌组织和细胞中呈低表达,可能通过竞争性结合miR-510-5p而上调REPS2的表达,从而抑制宫颈癌HCC94细胞的增殖和迁移。

Objective:To explore the role of lnc RNA ZNF674-AS1 in cervical cancer and its molecular mechanism. Methods: q PCR was used to detect the expression of ZNF674-AS1 in 31 pairs of cervical cancer tissues and corresponding para-cancerous tissues that obtained from patients who had received surgical treatment during January 2019 and July 2020 in Wuhan Children’s Hospital, as well as in cervical cancer cell lines(Si Ha, He La, C33 A and HCC94) and immortalized cervical epithelial cell lines. The NF674-AS1 over-expression plasmids or the negative control plasmids were transfected into the HCC94 cell line with the least expression of ZNF674-AS1. CCK-8 method and Transwell assay were used to detect the effect of ZNF674-AS1 over-expression on the proliferation and migration of HCC94 cells.Bioinformatics methods and Dual luciferase reporter gene experiment were respectively used to predict and verify the complementary binding relationship among ZNF674-AS1, mi R-510-5 p and REPS2(RALBP1 associated Eps domain containing 2). q PCR was conducted to examine the effect of ZNF674-AS1 over-expression on the levels of mi R-510-5 p and REPS2, and WB assay was used to detect the effect of ZNF674-AS1 over-expression on the level of molecules that related with cell proliferation and migration. Results: Compared with para-cancerous tissues, ZNF674-AS1 was significantly down-regulated in cervical cancer tissues(P<0.01). Compared with immortalized cervical epithelial cells, ZNF674-AS1 was also obviously down-regulated in cervical cancer cell lines(P<0.05 or P<0.01), with the lowest expression in HCC94 cells(P<0.01). Over-expression of ZNF674-AS1 could significantly inhibit the proliferation(P<0.05) and migration(P<0.01) of HCC94 cells. miR-510-5 p was proved to interact with ZNF674-AS1, and REPS2 was proved to interact with miR-510-5 p. Over-expression of ZNF674-AS1 caused a decrease in the expression level of mi R-510-5 p(P<0.01). but an increase in the expression level of REPS2 gene(P<0.01) in HCC94 cells, as well as the up-or down-regulation of multiple molecules(CDK2, cyclin D3, vimentin and twist) that related with cell proliferation and migration. Conclusions:lnc RNA ZNF674-AS1 is low-expressed in cervical cancer tissues and cell lines and may up-regulate the expression of REPS2 gene by competitively binding mi R-510-5 p, thereby inhibiting the proliferation and migration of cervical cancer HCC94 cells.