miRNA-143对宫颈癌细胞增殖及凋亡的影响及机制研究

Study on the effect and its mechanism of miRNA-143 on proliferation and apoptosis of cervical cancer cells

目的探讨miRNA-143对宫颈癌细胞增殖及凋亡的影响及机制。方法实时荧光定量聚合酶链式反应(RT-PCR)检测宫颈癌组织、癌旁组织及宫颈癌C-33A、He La、Ca Ski细胞中miRNA-143的表达水平。将miRNA-143 mimics、miRNA-143 inhibitor及HIF-1α-siRNA转染至He La细胞中,未转染的作为对照组。Western blot检测转染48 h后miRNA-143 mimics组、miRNA-143 inhibitor组和对照组的HIF-1α蛋白表达水平。将miRNA-NC+miRNA-143 mimics、Wt-HIF-1α+miRNA-143 mimics、Mut-HIF-1α+miRNA-143 mimics转染He La细胞,检测荧光素酶活性;后续实验分为对照组、miRNA-143 mimics组及HIF-1α-siRNA组,各组细胞转染48 h后,CCK8试验检测细胞增殖,流式细胞仪检测细胞凋亡,Western blot检测Ki-67、cleaved caspase 3、β-catenin、cyclin D1蛋白表达水平。结果宫颈癌组织中miRNA-143的表达水平明显低于癌旁组织(P﹤0.01),人宫颈癌Ca Ski细胞中的miRNA-143表达水平高于人宫颈癌He La细胞和C-33A细胞(P﹤0.05)。miRNA-143 mimics组的HIF-1α蛋白表达水平低于对照组,miRNA-143 inhibitor组的HIF-1α蛋白表达水平高于对照组,差异均有统计学意义(P﹤0.05)。WtHIF-1α+miRNA-143 mimics组的荧光素酶活性低于miRNA-NC+miRNA-143 mimics组和Mut-HIF-1α+miRNA-143mimics组,差异均有统计学意义(P﹤0.05);而Mut-HIF-1α+miRNA-143 mimics组与miRNA-NC+miRNA-143 mimics组的荧光素酶活性比较,差异无统计学意义(P﹥0.05)。miRNA-143 mimics和HIF-1α-siRNA组的细胞存活率及Ki-67、β-catenin、cyclin D1蛋白表达水平均低于对照组,细胞凋亡率及cleaved caspase 3蛋白表达水平均高于对照组,差异均有统计学意义(P﹤0.05)。结论 miRNA-143在宫颈癌中低表达,miRNA-143可通过下调HIF-1α的表达及抑制Wnt/β-catenin信号通路,抑制人宫颈癌He La细胞增殖并促进其凋亡。

Objective To investigate the effect and its mechanism of miRNA-143 on proliferation and apoptosis of cervical cancer cells. Method The expression of miRNA-143 in cervical cancer tissues, para-carcinoma tissue and cervical cancer cells C-33 A, He La and Ca Ski were detected by reverse transcription-polymerase chain reaction（RT-PCR）.miRNA-143 mimics, miRNA-143 inhibitor and HIF-1α-siRNA were transfected into He La cells, without transfection as the control group. The expression of HIF-1α in miRNA-143 mimics group, miRNA-143 inhibitor group and control group were detected after transfected for 48 h by Western blot. miRNA-NC＋miRNA-143 mimics, Wt-HIF-1α＋miRNA-143 mimics, Mut-HIF-1α＋miRNA-143 mimics were transfected into He La cells, luciferase activity was detected; the followup experiment was divided into control group, miRNA-143 mimics group and HIF-1α-siRNA group, cells were transfected for 48 h, cell proliferation was detected by CCK8 test, apoptosis was detected by flow cytometry, the expression of Ki-67, cleaved caspase 3, β-catenin and cyclin D1 protein were detected by Western blot. Result The expression of miRNA-143 in cervical cancer tissues was significantly lower than that in adjacent tissues（P〈0.01）; the expression of miRNA-143 in human cervical cancer Ca Ski cells was higher than those in human cervical cancer He La and C-33 A cells（P〈0.05）. The expression of HIF-1α protein in miRNA-143 mimics group was obviously lower than that in the control group, was obviously higher in miRNA-143 inhibitor group than that in the control group（P〈0.05）. Luciferase activity in Wt-HIF-1α ＋ miRNA-143 mimics group was obviously lower than those in miRNA-NC ＋ miRNA-143 mimics group and in Mut-HIF-1α＋miRNA-143 mimics group（P〈0.05）, while there were no significant difference in luciferase activity between Mut-HIF-1α ＋miRNA-143 mimics group and miRNA-NC＋miRNA-143 mimics group（P〉0.05）. Cell survival rate and expressions of Ki-67, β-catenin and cyclin D1 protein in miRNA-143 mimics group and HIF-1α-siRNA group were obviously lower than those in the control group, the apoptosis rate and the expression of cleaved caspase3 protein in miRNA-143 mimics group and HIF-1α-siRNA group were obviously higher than those in the control group（P〈0.05）. Conclusion miRNA-143 is lowly expressed in cervical cancer, which can inhibit the proliferation and promote apoptosis of human cervical cancer cell line HeLa by down regulating HIF-1α and inhibiting Wnt/β-catenin signaling pathway.