miR-3607-3p介导PFN2表达抑制宫颈癌细胞的恶性生物学行为

miR-3607-3p inhibits the malignant biological behavior of cervical cancer cells by mediating the expression of profilin 2

目的探讨miR-3607-3p对宫颈癌细胞的恶性表型的作用及相关作用机制。方法OncoLnc生物信息学网站分析miR-3607-3p的表达与宫颈癌患者预后的关系。采用实时荧光定量PCR(qRT-PCR)检测人正常宫颈上皮细胞(H8)和宫颈癌细胞系(Hela、HCC94、SiHa、C33A)中miR-3607-3p的表达情况。SiHa细胞体外转染阴性对照核苷酸序列为阴性对照(NC)组,转染miR-3607-3p模拟序列为miR-3607-3p组。qRT-PCR检测转染后的SiHa细胞中miR-3607-3p表达变化。采用CCK-8法和划痕实验检测转染前后SiHa细胞的恶性生物学行为变化。qRT-PCR和Western blot检测转染前后肌动蛋白结合蛋白2(PFN2)基因表达变化,双荧光素酶报告基因实验检测miR-3607-3p与PFN2的靶向关系。结果miR-3607-3p表达水平高的宫颈癌患者生存期显著长于miR-3607-3p低表达患者(P<0.01)。与H8细胞相比,miR-3607-3p在人宫颈癌细胞系中异常低表达(P<0.05),其中SiHa细胞系中表达最低(P<0.01)。NC组和miR-3607-3p组miR-3607-3p表达分别为(1.04±0.31)和(9.28±1.76),转染miR-3607-3p模拟序列后SiHa细胞miR-3607-3p表达水平明显高于NC组(P<0.01),增殖活性和划痕愈合率明显低于NC组(均P<0.01)。双荧光素酶报告基因实验证实miR-3607-3p直接靶向结合PFN2(P<0.01)。qRT-PCR和Western blot结果显示,miR-3607-3p组的PFN2 mRNA和蛋白表达均低于NC组,增殖蛋白CDK3、CDK2表达低于NC组,上皮表型相关蛋白Claudin-1、ZO-1表达高于NC组(均P<0.01)。结论miR-3607-3p与宫颈癌患者的生存期相关,上调miR-3607-3p表达可抑制宫颈癌SiHa细胞的增殖和迁移,其机制可能与靶向抑制PFN2有关。

Objective To explore the effect of miR-3607-3p on the malignant phenotype of cervical cancer cells and related mechanisms.Methods The OncoLnc bioinformatics website was used to analyze the relationship between the expression of miR-3607-3p and the prognosis of cervical cancer patients.Real time fluorescent quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression of miR-3607-3p in human normal cervical epithelial cells(H8)and cervical cancer cell lines(Hela,HCC94,SiHa,C33A).SiHa cells transfected with negative control nucleotide sequence in vitro were defined as negative control(NC)group,and SiHa cells transfected with miR-3607-3p mimicked sequence was defined as miR-3607-3p group.qRT-PCR was used to detect the expression changes of miR-3607-3p in SiHa cells.Cell counting kit-8(CCK-8)method and scratch test were used to detect the malignant biological behavior changes of SiHa cells.qRT-PCR and Western blot were used to detect the expression changes of profilin 2(PFN2)gene,and the dual luciferase reporter gene experiment was used to detect the targeting relationship between miR-3607-3p and PFN2.Results The survival time of patients with high expression of miR-3607-3p was significantly higher than that of patients with low expression of miR-3607-3p(P<0.01).Compared with H8 cells,the expression of miR-3607-3p was abnormally low in human cervical cancer cell lines(P<0.05),and the expression of miR-3607-3p was the lowest in the SiHa cell line(P<0.01).The expression of miR-3607-3p in the NC group and miR-3607-3p group was(1.04±0.31)and(9.28±1.76),respectively.The expression level of miR-3607-3p in SiHa cells transfected with miR-3607-3p mimic sequence was significantly higher than that in NC group,and the proliferation activity and scratch healing rate were significantly lower than that in NC group(all P<0.01).Dual-luciferase reporter gene assay confirmed that miR-3607-3p directly targeted and binded to PFN2(P<0.01).qRT-PCR and Western blot results showed that the expression of PFN2 mRNA and protein in miR-3607-3p group was lower than that in NC group;Western blot results showed the expression of proliferating proteins CDK3 and CDK2 in miR-3607-3p group was lower than that in NC group(all P<0.05),and the expression of epithelial phenotype related proteins Claudin-1 and ZO-1 in miR-3607-3p group was higher than that in NC group(all P<0.05).Conclusions miR-3607-3p is positively correlated with the survival of cervical cancer patients.Up-regulating the expression of miR-3607-3p can inhibit the proliferation and migration of cervical cancer SiHa cells.The mechanism may be related to the targeted inhibition of PFN2.