miR-302b-3p对食管癌EC-109细胞增殖和凋亡的影响及其机制

doi:10.13481/j.1671-587x.20190626

摘要/Abstract

摘要： 目的：探讨miR-302b-3p对食管癌EC-109细胞增殖和凋亡的影响，阐明miR-302b-3p抑制食管癌细胞增殖和诱导细胞凋亡的相关机制。方法：选取体外培养的食管癌EC-109细胞和正常食管上皮HET-1A细胞，采用实时荧光定量PCR法检测EC-109细胞和HET-1A细胞中miR-302b-3p表达水平。对EC-109细胞进行瞬时转染，分为空白对照组（未转染）、miR-NC组（转染miR-302b-3p mimics阴性对照）、miR-302b-3p组（转染miR-302b-3p mimics）、anti-miR-NC组（转染miR-302b-3p inhibitor阴性对照）和anti-miR-302b-3p组（转染miR-302b-3p inhibitor）。MTT法检测各组EC-109细胞活性，流式细胞术检测各组不同周期EC-109细胞百分比和细胞凋亡率，Western blotting法检测各组EC-109细胞中CyclinD1、CDK2、Bax和Cleaved caspase-3蛋白表达水平，双荧光素酶报告基因实验和Western blotting法检测各组EC-109细胞的荧光素酶活性和细胞中上皮细胞转化序列2（ECT2）蛋白的表达水平。结果：与HET-1A细胞比较，EC-109细胞中miR-302b-3p表达水平明显降低（P<0.05）。与空白对照组比较，miR-302b-3p组EC-109细胞活性、S期细胞百分比和EC-109细胞中CyclinD1及CDK2蛋白表达水平明显降低（P<0.05），而G₀/G₁期细胞百分比、细胞凋亡率和细胞中Bax及Cleaved caspase-3蛋白表达水平均明显升高（P<0.05）；与anti-miR-NC组比较，anti-miR-302b-3p组细胞活性、S期细胞百分比和细胞中CyclinD1及CDK2蛋白表达水平均明显升高（P<0.05），而G₀/G₁期细胞百分比、细胞凋亡率和细胞中Bax及Cleaved caspase-3蛋白表达水平均明显降低（P<0.05）。与miR-NC+ECT2-WT组比较，miR-302b-3p+ECT2-WT组EC-109细胞的荧光素酶活性降低（P<0.05）；与anti-miR-NC+ECT2-WT组比较，anti-miR-302b-3p+ECT2-WT组EC-109细胞的荧光素酶活性明显升高（P<0.05）。结论：miR-302b-3p可抑制EC-109细胞增殖并诱导凋亡，其作用机制可能与靶向调控ECT2蛋白表达有关。

关键词: miR-302b-3p, 食管肿瘤, 细胞增殖, 细胞凋亡, 上皮细胞转化序列2基因

Abstract: Objective: To investigate the effect of miR-302b-3p on the proliferation and apoptosis of the esophageal cancer EC-109 cells,and to elucidate the related mechanism of miR-302b-3p inhibiting the proliferation and inducing apoptosis of the EC-109 cells. Methods: The esophageal cancer EC-109 cells and normal esophageal epithelium HET-1A cells were cultured in vitro.The expression levels of miR-302b-3p in the EC-109 cells and HET-1A cells were detected by real-time fluorescence quantitative PCR method.The EC-109 cells were transfected instantaneously and divided into blank control group(non-transfection),miR-NC group (transfected with negative control of miR-302b-3p mimics),miR-302b-3p group (transfected with miR-302b-3p mimics),anti-miR-NC group (transfected with negative control of miR-302b-3p inhibitor) and anti-miR-302b-3p group (transfected with miR-302b-3p inhibitor).The viabilities of the EC-109 cells in various groups were detected by MTT assay;the percentages of EC-109 cells at different cell cycles and the apoptotic rates of EC-109 cells were detected by flow cytometry;the expression levels of CyclinD1,CDK2,Bax,and Cleaved caspase-3 in the EC-109 cells in various groups were detected by Western blotting method;the luciferase activities of EC-109 cells in various groups were detected by double luciferase reporter gene assay and Western blotting method was used to detect the expression level of epithelial cell transformation sequence 2(ECT2) protein in the EC-109 cells in various groups. Results: Compared with the HET-1A cells,the expression level of miR-302b-3p in the EC-109 cells was significantly decreased (P<0.05). Compared with blank control group,the viability of EC-109 cells,the percentage of cells at S phase and the expression levels of CyclinD1 and CDK2 protein in the EC-109 cells in miR-302b-3p group were significantly decreased(P<0.05),while the percentage of the cells at G₀/G₁ phase,the apoptotic rate,and the expression levels of Bax and Cleaved caspase-3 proteins in the EC-109 cells were significantly increased(P<0.05).Compared with anti-miR-NC group, the cell viability,the percentage of the cells at S phase and the expression levels of Cyclin D1 and CDK2 protein in the EC-109 cells in anti-miR-302b-3p group were significantly increased(P<0.05),and the percentage of the cells at G₀/G₁ phase,the apoptotic rate and the expression levels of Bax and Cleaved caspase-3 proteins in the EC-109 cells were significantly decreased (P<0.05).Compared with miR-NC+ECT2-WT group,the luciferase activity of the EC-109 cells in miR-302b-3p+ECT2-WT group was decreased(P<0.05);compared with anti-miR-NC+ECT2-WT group,the luciferase activity of the EC-109 cells in anti-miR-302b-3p+ECT2-WT group was increased(P<0.05). Conclusion: miR-302b-3p can inhibit the proliferation and induce the apoptosis of EC-109 cells,and its mechanism may be related to the targeting regulation of ECT2 protein expression.

Key words: miR-302b-3p, esophageal neoplasms, cell proliferation, apoptosis, epithelial cell transformation sequence 2 gene

中图分类号:

R735.1

刘翼, 祝琳, 康敏. miR-302b-3p对食管癌EC-109细胞增殖和凋亡的影响及其机制[J]. 吉林大学学报(医学版), 2019, 45(06): 1346-1352.

LIU Yi, ZHU Lin, KANG Min. Effect of miR-302b-3p on proliferation and apoptosis of esophageal cancer EC-109 cells and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2019, 45(06): 1346-1352.

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