miR-27a-3p抑制β-1,4-半乳糖基转移酶Ⅲ表达对胃癌SGC-7901细胞的影响

miR-27a-3p promotes the malignant behaviors of gastric cancer SGC-7901 cells by inhibiting B4GALT3

目的目前关于胃癌中miRNAs靶向调控β-1,4-半乳糖基转移酶Ⅲ(B4GALT3)表达的具体机制尚不明确。文中旨在分析miR-27a-3p对胃癌SGC-7901细胞增殖、迁移及侵袭的影响,探讨其分子机制。方法生物信息学预测B4GALT3基因的靶miRNAs,针对B4GALT3′UTR与靶miRNA结合位点构建荧光素酶报告基因载体,检测其相对荧光素酶的活性;将miR-27a-3p mimic、mimic NC、control(空白对照)、miR-27a-3p inhibitor、inhibitor NC分别瞬转入SGC-7901细胞后,Western blot检测各转染细胞B4GALT3的表达情况;采用MTT、迁移、侵袭实验检测细胞的增殖、迁移、侵袭能力。结果与正常胃黏膜GES-1细胞比较,B4GALT3在胃癌各个细胞系中表达显著上调(P<0.05)。双荧光素酶报告基因检测结果显示,miR-27b-3p能使野生型B4GALT3的荧光素酶的活性显著性降低(P<0.001),说明miR-27a-3p与B4GALT3存在靶向关系。Western blot检测结果显示,瞬转miR-27a-3p mimic后,B4GALT3蛋白水平下降(P<0.05);瞬转miR-27a-3p inhibitor后,B4GALT3表达升高(P<0.01),说明miR-27a-3p对B4GALT3有负性调控作用。24、48、72、96、120 h,与mimic NC、空白对照比较,miR-27a-3p mimic的细胞增殖显著升高(P<0.05);与inhibitor NC、空白对照比较,miR-27a-3p inhibitor的细胞增殖显著降低(P<0.05)。miR-27a-3p mimic的迁移细胞数量[(319.3±13.9)个]较mimic NC[(218.0±10.7)个]明显增加(P<0.000);侵袭细胞数量[(402.0±15.0)个]较mimic NC[(241.0±17.3)个]明显增加(P<0.000)。miR-27a-3p inhibitor迁移细胞数量[(115.6±13.8)个]较inhibitor NC[(214.6±6.8)个]明显减少,迁移能力明显降低(P<0.000)。结论miR-27a-3p通过抑制B4GALT3表达,促进胃癌SGC-7901细胞增殖、迁移和侵袭能力,为寻找胃癌治疗靶点提供了实验依据。

Objective At present,the specific mechanism of miRNAs targeted regulation of B4GALT3 in gastric cancer is still unclear.The purpose of this article is to analyze the effects of miR-27a-3p on the proliferation,migration and invasion of gastric cancer SGC-7901 cellsand their molecular mechanisms.Methods Bioinformatics predicts the target miRNAs of B4GALT3 gene,constructs a luciferase reporter gene vector at the binding site of B4GALT3'UTR and target miRNA,and detects its relative luciferase activity;miR-27a-3pmimic,mimic NC,control(Blank control),miR-27a-3p inhibitor,and inhibitor NC were transferred into SGC-7901 cells,and Westernblot was used to detect the expression of B4GALT3;MTT,migration,and invasion experiments were used to detect cell proliferation,migration,and invasion capabilities.Results Compared with normal gastric mucosal cellsGES-1,the expression of B4GALT3 was significantly up-regulated in various gastric cancer cell lines(P<0.05).The results of dual luciferase reporter gene detection showed that miR-27b-3p can significantly reduce the luciferase activity of wild-type B4GALT3(P<0.001),indicating that miR-27a-3p has a targeting relationship with B4GALT3.Western blot showed that the B4GALT3 protein level decreased(P<0.05)after the transient miR-27a-3p mimic,and increased(P<0.05)after the miR-27a-3p inhibitor transiently changed.It shows that miR-27a-3p has a negative regulatory effect on B4GALT3.At 24,48,72,96,120 h,compared with mimic NC and blank control,the cell proliferation of miR-27a-3ppm was significantly increased(P<0.05);compared with inhibitor NC and blank control,miR-27a-3pinhibitor's cell proliferation was significantly reduced(P<0.05).The number of migratory cells in miR-27a-3pmimic(319.3±13.9)was significantly higher than that of mimic NC(218.0±10.7)(P<0.000);the number of invaded cells(402.0±15.0)were significantly increased compared with mimic NC(241.0±17.3)(P<0.001).The number of miR-27a-3pin inhibitor migrating cells(115.6±13.8)is significantly reduced compared with that of inhibitor NC(214.6±6.8),and the migration ability is significantly reduced(P<0.000).Conclusion miR-27a-3p promotes the proliferation,migration and invasion of gastric cancer SGC-7901 cells by inhibiting the expression of B4GALT3,providing experimental evidence for finding therapeutic targets for gastric cancer.