KCa3．1表达改变对子宫内膜癌细胞增殖、细胞周期及凋亡的影响

Effect of expression of intermediate-conductance Ca^(2+) -activated K^+ channels on proliferation,cell cycle,and apoptosis of human endometrial carcinoma

目的:探讨钙激活性中电导钾离子通道(intermediate-conductance Ca2+-activated K+channels,KCa3.1)表达水平和活性改变对子宫内膜癌细胞增殖、细胞周期及凋亡的影响。方法:将携有KCa3.1RNA干扰(RNA interference,RNAi)片段的质粒转染子宫内膜癌HEC-1-A和Ishikawa细胞,应用实时荧光定量PCR(real time fluorogentic quantitative PCR,RFQ-PCR)及Western印迹法检测KCa3.1的表达变化;应用MTT法、BrdU掺入法、FCM及Western印迹法检测KCa3.1特异性阻断剂TRAM-34和KCa3.1RNAi表达质粒干扰后,子宫内膜癌细胞株增殖、细胞周期、凋亡及cyclin D1、cyclin E及survivin蛋白表达的改变。结果:KCa3.1RNAi表达质粒可以明显抑制KCa3.1mRNA及蛋白的表达,与对照组相比差异有统计学意义(P<0.01);TRAM-34及KCa3.1RNAi可以明显抑制HEC-1-A和Ishikawa细胞的增殖,2株细胞的G0/G1期细胞百分比上升,S期细胞百分比下降,与对照组相比差异有统计学意义(P<0.05),但细胞凋亡率与对照组相比无明显差异(P>0.05);cyclin D1、cyclin E及survivin的蛋白表达水平降低,与对照组相比差异有统计学意义(P<0.05)。结论:抑制KCa3.1的表达及活性可以抑制子宫内膜癌细胞增殖,阻碍细胞周期进展,但对细胞凋亡无明显影响。

Objective ：To study the effects of expression level and activity alteration of intermediate-conductance Ca2＋ -activated K＋ （ KCa3.1 ） channels on the cell proliferation, cell cycle, and apoptosis of endometrial carcinoma cells. Methods： The recombinant plasmid containing small interference RNA of KCa3.1 gene was transfected into endometrial cancer cell lines, HEC-1-A cell line and Ishikawa cell line. Real-time fluorogentic quantitative RT-PCR and Western blotting were used to examine the gene and protein expressions of KCa3.1 channels. Un-transfected cells and the cells transfected with negative plasmids served as control groups. The prolifera- tion, cell cycle, apoptosis, and expressions of cyclin D1, cyclin E, survivin proteins of endometrial cancer cells were determined by MTT assay, BrdU incoporation test, flow cytometry, and Western blotting, respectively, after interference by KCa31 sepcific antagonist TRAM-34 or KCa3.1 RNAi plasmid. Results： KCa3.1 RNAi interference significantly inhibited the mRNA and protein expressions of KCa3.1 channels compared with control groups （P 〈0.01 ）. TRAM-34 and KCa3.1 RNAi interference markedly suppressed the cell proliferation of HEC-1-A and Ishikawa cells. Compared with control groups, the proportion of cells in G0/G1 phase of the two experi- ment cell lines was increased, while the proportion of cells in S phase was decreased. The difference was significant （P 〈 0.05）. The apoptotic rataio had no significant changes in the two cell lines after treatment compared with control group （P 〉 0.05 ）. The expression levels of cyclin D1, cyclin E, and surAvin protein were significantly decreased in the experiment groups. The difference was significant compared with control group （ P 〈 0.05 ）. Conclusion ： Inhibition of the expression and activity of KCa3.1 channels suppress cell proliferation, blocked cell cycle progression, but have no effect on apoptosis of endometrial cancer cells.