摘要
目的观察微小RNA-20a-5p(miR-20a-5p)靶向调控乳腺癌转移抑制基因1(BRMS1L)对人结直肠癌细胞株SW480增殖、凋亡能力的影响,并探讨其可能机制。方法取SW480细胞,分别将miR-20a-5p抑制物(anti-miR-20a-5p)、miR-20a-5p抑制物阴性对照(anti-miR-NC)、BRMS1L过表达载体(pcDNA-BRMS1L)、BRMS1L过表达载体阴性对照(pcDNA-NC)、miR-20a-5p模拟物(miR-20a-5p mimics)、miR-20a-5p模拟物阴性对照(miR-NC)转染至细胞中,CCK-8法测算细胞存活率,流式细胞术测算细胞凋亡率,Western blotting法检测细胞周期素D1(CyclinD1)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved-Caspase-3)、β-catenin蛋白。取SW480细胞,分别将miR-20a-5p mimics、miR-NC与WT-BRMS1L或MUT-BRMS1L共转染至细胞中,采用双荧光素酶报告基因检测系统检测细胞荧光素酶活性;另取SW480细胞,分别将miR-20a-5p mimics、miR-NC、anti-miR-20a-5p、anti-miR-NC分别转染至细胞中,采用Western blotting法检测细胞BRMS1L蛋白。结果抑制miR-20a-5p表达或过表达pcDNA-BRMS1L,SW480细胞存活率降低,细胞凋亡率升高,CyclinD1蛋白表达降低,Cleaved-Caspase-3蛋白表达升高(P均<0.05);抑制BRMS1L表达部分逆转anti-miR-20a-5p对SW480细胞增殖和凋亡的影响(P均<0.05)。抑制miR-20a-5p表达后SW480细胞中β-catenin蛋白的表达较低,抑制BRMS1L表达部分逆转anti-miR-20a-5p对β-catenin蛋白表达的影响(P均<0.05)。miR-20a-5p靶向BRMS1L,并负性调控其表达(P均<0.05)。结论miR-20a-5p靶向调控BRMS1L抑制SW480细胞增殖,促进细胞凋亡,其机制可能与抑制β-catenin蛋白表达有关。
Objective To observe the effects of miR-20a-5p(miR-20a-5p)on the proliferation and apoptosis of human colorectal cancer cell line SW480 by targeting breast cancer metastasis suppressor gene 1-like(BRMS1L),and to explore its possible mechanism.Methods SW480 cells were transfected with miR-20a-5p inhibitor(anti-miR-20a-5p),negative control of miR-20a-5p inhibitor(anti-miR-20a-5p),overexpression vector of BRMS1L(pcDNA-BRMS1L),negative control of BRMS1L overexpression vector(pcDNA-NC),miR-20a-5p mimics(miR-20a-5p mimics)and miR-20a-5p mimics negative control(miR-NC),respectively.CCK-8 was used to detect the cell viability;flow cytometry was used to detect the apoptosis;Western blotting was used to detect the expression levels of cyclin D1 and Cleaved-Caspase-3.The miR-20a-5p mimics,miR-NC and WT-BRMS1L or MUT-BRMS1L were co-transfected into SW480 cells,respectively.The dual luciferase reporter gene assay was used to detect the luciferase activity.In addition,SW480 cells were transfected with miR-20a-5p mimics,miR-NC,anti-miR-20a-5p,and anti-miR-NC,and the BRMS1L protein was detected by Western blotting.Results After inhibiting miR-20a-5p or over-expressing pcDNA-BRMS1L,the cell survival rate of SW480 cells was lower,and the apoptosis rate was higher,the expression level of CyclinD1 was lower,the expression level of Cleaved-Caspase-3 was higher(all P<0.05).Inhibiting BRMS1L partially reversed the effect of anti-miR-20a-5p on proliferation and apoptosis of SW480 cells(all P<0.05).After inhibiting the expression of miR-20a-5p,the expression level ofβ-catenin protein was lower in SW480 cells.Inhibition of BRMS1L partially reversed the effect of anti-miR-20a-5p onβ-catenin protein expression(P<0.05).The miR-20a-5p targeted BRMS1L and negatively regulated its expression(P<0.05).Conclusion The miR-20a-5p inhibits the cell proliferation and promote apoptosis of SW480 cells by targeting BRMS1L,and the mechanism may be related to the inhibition ofβ-catenin protein expression.
作者
陈杰
CHEN Jie(Hunan Cancer Hospital,Changsha 410000,China)
出处
《山东医药》
CAS
2020年第12期5-9,共5页
Shandong Medical Journal
