摘要
目的研究微小RNA-135a(microRNA-135a, miR-135a)靶向十号染色体上缺失的磷酸酶和张力蛋白同源物(phosphatase and tensin homolog deleted on chromosome ten, PTEN)对宫颈癌细胞增殖和转移能力的影响及作用机制。方法采用qRT-PCR检测宫颈癌细胞C33A、Caski、HeLa中miR-135a和PTEN mRNA的表达水平,选择miR-135a表达较高的宫颈癌细胞进行后续实验。Targetscan 7.1预测软件及双荧光素酶报道基因试验检测miR-135a对PTEN基因的靶向调控作用。常规培养宫颈癌细胞,将其分为NC组、miR-135a inhibitor组和miR-135a inhibitor+si-PTEN组,采用Lip2000进行各组小分子化合物的转染。MTT实验检测3组细胞的增殖能力,流式细胞仪检测3组细胞的凋亡率,Transwell实验检测3组细胞的转移能力。Western blot检测3组细胞中Akt/mTOR信号通路的活性。结果与永生化宫颈上皮细胞H8相比,miR-135a在宫颈癌细胞系中的表达增加,在宫颈癌细胞Caski中的表达最高(P<0.05)。与永生化宫颈上皮细胞H8相比,PTEN在宫颈癌细胞系中的表达降低(P<0.05)。miR-135a靶向调控PTEN。与NC组相比,miR-135a inhibitor组和miR-135a inhibitor+si-PTEN组宫颈癌细胞增殖和转移能力降低、宫颈癌细胞凋亡率增加(P<0.05)。而与miR-135a inhibitor组相比,miR-135a inhibitor+si-PTEN组宫颈癌细胞增殖和转移能力增加、宫颈癌细胞凋亡率降低(P<0.05)。与NC组相比,miR-135a inhibitor组和miR-135a inhibitor+si-PTEN组细胞中pAkt和pmTOR蛋白表达降低,而与miR-135a inhibitor组相比,miR-135a inhibitor+si-PTEN组pAkt和pmTOR蛋白表达增加(P<0.05)。结论 miR-135a靶向调控PTEN表达促进宫颈癌细胞增殖和转移能力,抑制细胞凋亡。
Objective To study the effect and mechanism of microRNA-135a(miR-135a)on proliferation and metastasis of cervical cancer cells by targeting of phosphatase and tensin homolog deleted on chromosome ten(PTEN).Methods The expression levels of miR-135a and PTEN mRNA in cervical cancer cells C33A,Caski,HeLa were detected by qRT-PCR.Cervical cancer cells with higher miR-135a expression were selected for subsequent experiments.Targetscan7.1 prediction software and dual luciferase reporter gene test were used to detect the targeted regulation of miR-135a on PTEN gene.Cervical cancer cells were routinely cultured and divided into NC group,miR-135a inhibitor group and miR-135a inhibitor+si-PTEN group.Lip2000 was used to transfect small molecule compounds in each group.The MTT test was used to detect the cell proliferation ability of each group,the flow cytometry test was used to detect the apoptosis rate of each group of cells,and the Transwell test was used to detect the cell migration ability of each group.Western-blot was used to detect the activity of Akt/mTOR signaling pathway in each group of cells.Results Compared with the immortalized cervical epithelial cell H8,the expression of miR-135a was increased in cervical cancer cell lines,and the expression was highest in Caski cervical cancer cells(P<0.05).Compared with immortalized cervical epithelial cell H8,PTEN expression was reduced in cervical cancer cell lines(P<0.05),and there was targeted regulation of miR-135a on PTEN gene.Compared with NC group,proliferation and metastasis ability of cervical cancer cells in miR-135a inhibitor group and miR-135a inhibitor+si-PTEN group were reduced,and apoptosis rate of cervical cancer cells was increased(P<0.05).Compared with the miR-135a inhibitor group,proliferation and metastasis capacity of cervical cancer cells in the miR-135a inhibitor+si-PTEN group were increased,while cancer cell apoptosis rate were reduced(P<0.05).Compared with the NC group,the expression of pAkt and pmTOR proteins in miR-135a inhibitor group and miR-135a inhibitor+si-PTEN group were reduced.Compared with miR-135a inhibitor group,the expression of pAkt and pmTOR protein in miR-135a inhibitor+si-PTEN group were increased(P<0.05).Conclusion miR-135a targets and regulates PTEN expression to promote the proliferation and metastasis of cervical cancer cells and inhibit apoptosis.
作者
黄天舒
裴丽鹏
HUANG Tian-shu;PEI Li-peng(Department of Obstetrics and Gynecology,North Theater General Hospital,Shenyang 110003,China)
出处
《解放军医药杂志》
CAS
2021年第1期20-26,共7页
Medical & Pharmaceutical Journal of Chinese People’s Liberation Army
基金
辽宁省2016年第一批科技计划项目(2016SZ0017)。
