LncRNA NEAT1调控miR-195对宫颈癌细胞SiHa放射敏感性的影响

Effect of lncRNA NEAT1 on the radiosensitivity of cervical cancer SiHa cells by regulating miR-195

目的研究长链非编码RNA(lncRNA)核富集转录本1(NEAT1)对宫颈癌SiHa细胞放射敏感性的影响和潜在的分子机制。方法体外培养宫颈癌细胞SiHa和正常宫颈细胞Ect1/E6E7,使用实时荧光定量PCR(RT-qPCR)法检测细胞中NEAT1和miR-195的表达水平。放射处理(0、2、4、6、8 Gy)的宫颈癌SiHa细胞中,RT-qPCR检测NEAT1和miR-195的表达水平。双荧光素酶报告系统验证SiHa细胞中NEAT1与miR-195的调控关系。转染si-NEAT1或共转染si-NEAT1与anti-miR-195至SiHa细胞,克隆形成实验测定细胞存活分数、四氮唑蓝(BTC)法测定细胞增殖,流式细胞术检测细胞凋亡率,蛋白印迹(Western blot)检测Cleaved caspase-3蛋白的表达水平。结果宫颈癌SiHa细胞中NEAT1的表达高于正常宫颈细胞Ect1/E6E7(P<0.05),而miR-195的表达低于正常宫颈细胞Ect1/E6E7(P<0.05)。在放射处理(2、4、6、8 Gy)的宫颈癌SiHa细胞中,NEAT1的表达量显著升高(P<0.05),而miR-195的表达量显著下降(P<0.05);双荧光素酶报告系统结果显示,NEAT1靶向负调控miR-195的表达。抑制NEAT1表达可抑制宫颈癌SiHa细胞的增殖,并促进其凋亡,增强SiHa细胞的放射敏感性;敲除miR-195则会逆转抑制NEAT1表达对SiHa细胞的增殖、凋亡及放射敏感性。结论抑制NEAT1可能通过靶向miR-195增强SiHa细胞的放射敏感性,NEAT1有望成为提高宫颈癌临床放射敏感性的分子靶点。

Objective To investigate the effects of lncRNA NEAT1 on radiosensitivity in cervical cancer SiHa cells and the underlying mechanism.Methods Cervical cancer cells SiHa and normal cervical cells Ect1/E6E7 were cultured in vitro,and the expression levels of NEAT1 and miR-195 in the cells were detected by RT-qPCR.The expression levels of NEAT1 and miR-195 in SiHa cells treated with irradiation(0,2,4,6,8 Gy)were measured by RT-qPCR.Dual luciferase reporter system was used to verify the regulatory relationship between NEAT1 and miR-195 in SiHa cells.After si-NEAT1 was transfected into SiHa cells or si-NEAT1 and anti-miR-195 were co-transfectedinto SiHa cells,cell viability was measured by colony formation assay,cell proliferation was measured by BTC assay,apoptosis rate was detected by flow cytometry,and the protein expression of Cleaved-caspase-3was detected by Western blot.Results The expression of NEAT1 in cervical cancer SiHa cells was higher than that in normal cervical cells Ect1/E6E7(P<0.05),while the expression of miR-195 was lower than that of Ect1/E6E7 cells(P<0.05).The expression levels of NEAT1 in cervical cancer SiHa cells treated with radiation(2,4,6,8 Gy)increased significantly(P<0.05),while the expression levels of miR-195 decreased significantly(P<0.05).The results of dual-luciferase reporter assay system showed that NEAT1 targeting negatively regulated the expression of miR-195.Inhibition of NEAT1 expression can inhibit the proliferation of cervical cancer SiHa cells,promote their apoptosis,and enhance the radiosensitivity of SiHa cells;knocking out miR-195 will reverse the inhibition of NEAT1 expression on the proliferation,apoptosis and radiosensitivity of SiHa cells.Conclusions NEAT1 enhances the radiosensitivity of SiHa cells by targeting miR-195.NEAT1 is expected to be a molecular target for improving clinical radiosensitivity of cervical cancer.