miR-300通过LEF-1调控卵巢癌SKOV3细胞增殖、凋亡的实验研究

Experimental Study on miR-300 Regulating Proliferation and Apoptosis of Ovarian Cancer SKOV3 Cells by LEF-1

目的探究miR-300介导LKF-1调控卵巢癌细胞增殖和凋亡的作用的机制。方法体外培养卵巢癌SK0V3细胞及正常卵巢上皮细胞,使用RT-PCR检测卵巢癌SK0V3细胞及正常卵巢上皮细胞中LEF-1及miR-300的表达情况;利用生物信息学软件预测LEF-1为miR-300的靶基因,并通过双荧光素酶等实验进行验证;将卵巢癌细胞分为空白对照组、空白转染组和miR-300抑制剂组。空白转染组使用miR-NC转染卵巢癌SKOV3细胞;miR-300抑制剂组采用miR-300 inhibitor转染细胞。使用CCK-8检测细胞增殖情况;使用蛋A质印记检测各组细胞的增殖相关蛋白Ki-67表达情况;划痕实验法检测各组细胞迁移能力;使用蛋白质印记法检测迁移相关蛋白N-cadherin、E-cadherin表达情况;使用流式细胞仪检测各组细胞凋亡情况;使用蛋A质印记法检测凋亡相关蛋白Bcl-2、Bax表达情况。结果卵巢癌SK0V3细胞中LEF-1及miR-300的表达显著高于正常卵巢上皮细胞(T=4.528,P=0.017;T=6.328,P=0.017);相比空白对照组,空白转染组卵巢癌细胞增殖、迁移能力均无显著改变(P>0.05);相比空白转染组,miR-300抑制剂组Ki-67蛋内相对表达(T15.687;P=0.001)、增长率(T=9.732;P=0.024)、划痕愈合率(T=11.558;P=O.005)、E-cadherin蛋白相对表达(T=18.558;P=0.003)、Bcl-2蛋白相对表达(T=11.001;P=0.035)均显著降低;N-cadherin蛋白相对表达(T=22.478;P=0.001)、B ax蛋白相对表达(T=18.528;P=0.004)、细胞凋亡率(T=19.975;P=0.000)均显著升高。结论miR-300可以介导LEF-1抑制卵巢癌细胞增殖并促进其凋亡,其作用机制与调控增殖、凋亡相关蛋白和调控EMT过程相关。

Objective To examine the mechanism by which miR-300 regulates the proliferation and apoptosis of ovarian cancer cells via LEF-1.Methods The ovarian cancer SK0V3 cells and normal ovarian epithelial cells were cultured in vitro.The expression of LEF-1 and miR-300 in the two kinds of cells was detected by RT-PCR.The relationship between LEF-1 and miR-300 was predicted by using hioinformatics software and verified by dual luciferase assay and other experiments.The ovarian cancer cells were divided into a blank control group,a blank transfection group and an miR-300 inhibitor group.In Wank transfection group,ovarian cancer SK0V3 cells were transfected with miR-NC.In miR-300 inhibitor group,cells were transfected with miR-300 inhibitor.The cell proliferation was detected by CCK-8 assay.The expression of proliferation-related protein(Ki-67)in each group was detected by Western blotting.The cell migration ability in each group was tested by scratch test.The expression of migration-related proteins(N-cadherin,E-cadherin)was detected by Western blot analysis.The apoptosis in each group was detected by flow cytometry.The expression apoptosis-related proteins(Bcl-2,Bax)was detected by Western blot ting.Results The expression of LEF-1 and miR-300 in ovarian cancer SKOV3 cells was significantly higher than in normal ovarian epithelial cells(T=4.528,P=0.017.T=6.328,P=0.017).Compared with blank control group,no significant changes in proliferation and migration abilities of ovarian cancer cells were found in blank transfection group(P>0.05).Compared with blank transfection group,relative expression of Ki-67 protein(T=15.687.P=0.001),growth rate(T=9.732;P=0.024),cell healing rate(T=11.558.P=0.005),relative expression of E-cadherin protein(T=18.558.P=0.003)and relative expression of Bcl-2 protein(T=11.001.P=0.035)were significantly decreased in miR-300 inhibitor group,while relative expression of Ncadherin protein(T-22.478.P=0.001),relative expression of Bax protein(T=18.528.P=0.004)and apoptosis rate(T=19.975.P=0.000)were significantly increased.Conclusion MiR-300 can inhibit proliferation of ovarian cancer cells,and promote their apoptosis by mediating LEF-1.And the effect is achieved by regulating the proliferation-and apoptosis-related proteins and EMT.