RBMS3-AS3对宫颈癌细胞增殖、凋亡和侵袭的影响及作用机制

目的:探讨RNA结合基序单链相互作用蛋白3反义链3(RBMS3-AS3)对宫颈癌细胞增殖、凋亡和侵袭的影响及作用机制。方法:培养人宫颈永生化细胞Ect1/E6E7和宫颈癌HeLa、Caski和SiHa细胞,实时荧光定量PCR检测细胞中RBMS3-AS3表达水平,Western blot法检测RNA结合基序单链相互作用蛋白3(RBMS3)蛋白水平。将HeLa细胞分为对照组(细胞正常培养)、si-RBMS3-AS3组(转染RBMS3-AS3小干扰RNA)、阴性序列组(转染乱序无意义阴性序列)、si-RBMS3-AS3+si-RBMS3组(共转染RBMS3-AS3和RBMS3的小干扰RNA)和si-RBMS3-AS3+阴性序列组(共转染RBMS3-AS3与乱序无意义阴性序列),四甲基噻唑蓝染色法(MTT)检测细胞增殖率,流式细胞术检测细胞凋亡率,Transwell检测细胞侵袭能力,Western blot检测各组HeLa细胞中细胞周期蛋白D1(Cyclin D1)、B淋巴细胞瘤-2(Bcl-2)、B淋巴细胞瘤-2相关蛋白(Bax)和基质金属蛋白酶2(MMP-2)蛋白表达水平。结果:宫颈癌细胞系HeLa、Caski和SiHa中RBMS3-AS3表达水平均高于Ect1/E6E7细胞(P<0.05),而RBMS3蛋白表达低于Ect1/E6E7细胞(P<0.05)。与对照组或阴性序列组相比,si-RBMS3-AS3组HeLa细胞的活性、侵袭细胞数、Cyclin D1、Bcl-2和MMP-2蛋白表达水平降低(P<0.05),细胞凋亡率及RBMS3和Bax蛋白表达水平升高(P<0.05)。与si-RBMS3-AS3+阴性序列组比较,si-RBMS3-AS3+si-RBMS3组HeLa细胞活性、侵袭细胞数及Cyclin D1、Bcl-2和MMP-2蛋白表达水平升高(P<0.05),细胞凋亡率和Bax蛋白表达水平降低(P<0.05)。结论:抑制RBMS3-AS3表达可抑制宫颈癌细胞增殖和侵袭,并促进细胞凋亡,这可能与上调RBMS3表达有关。

Mechanism of RBMS3-S3 on the proliferation,apoptosis and invasion of cervical cancer cells

OBJECTIVE: To investigate effects from regulation of expression of RBMS3 on proliferation,apoptosis and invasion of cervical cancer cells. METHODS: Immortalized human cervical cells,Ect1/E6E7,and cervical cancer cells:HeLa,Caski and SiHa were cultured,then Real-time quantitative PCR was used to detect expression of RBMS3-AS3 in cells,and Western blot was used to detect protein expression of RBMS3. HeLa cells were divided into control (cells were cultured normally),si RBMS3 AS3 (transfected with RBMS3 AS3 small interfering RNA),negative sequence (transfected with disordered and meaningless negative sequences),si RBMS3 AS3+si RBMS3 (co transfected with small interfering RNAs of RBMS3 AS3 and of RBMS3) and si RBMS3 AS3+negative sequence group (co transfected with RBMS3 AS3 small interfering RNA and disordered meaningless negative sequences). MTT assay was used to detect proliferation rates of HeLa cells in each group;flow cytometry to detect apoptosis rates;Transwell to detect HeLa cell invasion;Western blot to detect protein expression levels of Cyclin D1,Bcl 2,Bax and MMP 2. RESULTS: Compared with Ect1/E6E7 cells,expression levels of RBMS3 AS3 in cervical cancer cell lines HeLa,Caski and SiHa were significantly increased (P < 0.05),and expression levels of RBMS3 protein were decreased (P < 0.05). Compared with the negative sequence group,the number of invasive HeLa cells and their expression levels of Cyclin D1,Bcl 2 and MMP-2 proteins in the si RBMS3 AS3 group were decreased (P < 0.05),apoptosis rate and the expression levels of RBMS3 and Bax protein were increased (P < 0.05). Compared with the si RBMS3 AS3+negative sequence group,the number of invasive HeLa cells and their expression levels of Cyclin D1,Bcl-2 and MMP-2 were increased in the si-RBMS3-AS3+si-RBMS3 group (P < 0.05),apoptosis rate and Bax protein expression levels were decreased (P < 0.05). CONCLUSION: Inhibition of RBMS3 AS3 expression inhibited proliferation and invasion of cervical cancer cells and promoted apoptosis,which might be related to up-regulation of RBMS3 expression.