中电导钙激活钾离子通道对子宫内膜癌细胞生物学行为的影响及调控机制

【作者】 张英丽；

【导师】 丰有吉；

【作者基本信息】 复旦大学 ， 妇产科学， 2009， 博士

【摘要】 子宫内膜癌(endometrial carcinoma,EC)是女性生殖系统中常见的恶性肿瘤,发病率大约为15-20/100,000,严重威胁妇女的健康和生命。随着科技和医疗的发展,子宫内膜癌的治愈率逐渐提高,但是特殊病理类型和晚期子宫内膜癌五年的存活率还是很低。尽管对子宫内膜癌的病因和发病机制有许多学说,但目前为止,人们对于子宫内膜癌的具体病因和发病机制还不清楚。研究发现,包括钾离子通道在内的离子通道在肿瘤细胞的增殖中发挥了重要作用,本课题组的前期研究则显示中电导钙激活性钾离子通道(intermediate-conductance Ca2+-activated K~+channels,KCa3.1)在子宫内膜癌组织中高表达,并能促进子宫内膜癌细胞的增殖,在此基础上,本课题将进一步研究中电导钙激活钾离子通道对子宫内膜癌细胞株生物学行为的影响,并探讨可能的分子作用机制。本课题共分三部分:①中电导钙激活钾离子通道对子宫内膜癌细胞增殖、周期调控及凋亡的影响;②中电导钙激活钾离子通道对子宫内膜癌细胞迁移和侵袭的影响;③表皮生长因子对中电导钙激活钾离子通道表达的影响及其相关分子机制。第一部分中电导钙激活钾离子通道对子宫内膜癌细胞增殖、周期调控及凋亡的影响目的利用KCa3.1 siRNA表达质粒及其特异性阻断剂TRAM-34抑制KCa3.1的表达和活性,探讨KCa3.1表达及活性的改变对子宫内膜癌细胞增殖、周期调控及凋亡的影响。方法将携有KCa3.1干扰RNA基因片断的质粒转染子宫内膜癌细胞株HEC-1-A、Ishikawa细胞,以转染阴性对照质粒及未转染(空白对照)的细胞作为对照,用Real time-RT-PCR技术及Western blot法检测KCa3.1的表达变化。应用MTT比色法、BrdU掺入法、流式细胞仪、Real time-PCR及Western blot法研究KCa3.1相对特异性阻断剂TRAM-34和KCa3.1 siRNA表达质粒对子宫内膜癌细胞株增殖、周期调控及凋亡的影响。结果KCa3.1 siRNA表达质粒可以显著抑制KCa3.1基因及蛋白的表达,与阴性对照质粒转染组及空白对照组相比差异有统计学意义;加入TRAM-34及转染KCa3.1 siRNA表达质粒可以显著抑制HEC-1-A、Ishikawa细胞的增殖;2株细胞的G0/G1百分比上升,S期百分比下降,与对照组相比差异有统计学意义(P＜0.05),而细胞凋亡率与对照组相比却无显著差异(P＞0.05);细胞蛋白Cyclin D1、Cyclin E及Survivin的表达水平降低,与对照组相比差异具有统计学意义。结论抑制KCa3.1通道表达及活性可以抑制细胞增殖,阻碍子宫内膜癌细胞周期进展,但对细胞凋亡无显著影响。第二部分中电导钙激活钾离子通道对子宫内膜癌细胞迁移和侵袭的影响目的利用特异性阻断剂TRAM-34抑制KCa3.1的活性,探讨钙激活性中电导钾离子通道(KCa3.1)活性改变对子宫内膜癌细胞运动和侵袭的影响。方法利用特异性阻断剂TRAM-34,应用划痕实验、运动实验、Transwell小室侵袭实验了解KCa3.1对子宫内膜癌细胞运动和侵袭能力的影响,并用RT-PCR及Western blot法检测TRAM-34作用前后MMP-2表达的改变。结果(1)TRAM-34可以显著抑制HEC-1-A、Ishikawa细胞的运动和侵袭能力,与对照组相比差异有统计学意义(P＜0.01);(2)与对照组相比,TRAM-34处理后MMP-2的基因和蛋白表达水平降低,差异具有统计学意义(P＜0.01)。结论抑制KCa3.1通道活性可以降低子宫内膜癌细胞的运动和侵袭能力,这可能与下调MMP-2的表达有关。提示KCa3.1可以促进子宫内膜癌细胞的运动和侵袭,它的表达和/或功能异常可能在子宫内膜癌的转移中起重要作用。第三部分EGF对子宫内膜癌细胞KCa3.1表达的影响及其相关分子机制目的研究EGF对子宫内膜癌细胞KCa3.1表达的影响及其相关分子机制。方法将EGF以不同浓度(0、1、10、20ng/ml)分别作用于HEC-1-A细胞或Ishikawa细胞不同时间(0、12、24、48、72h),采用Westernblot法检测KCa3.1表达水平。以10ng/ml EGF分别作用于HEC-1-A细胞或Ishikawa细胞不同时间(0、5、15、30、60、120min),分别测定AKT和ERK1/2的磷酸化水平,并应用特异性的MAPK通路阻断剂PD98059抑制ERK1/2的磷酸化、特异性的PI3K/AKT通路阻断剂LY294002抑制AKT的磷酸化和相对特异性阻断剂TRAM-34降低KCa3.1的活性,研究信号通路MAPK、PI3K/AKT与KCa3.1之间的相互关系。结果(1)随着EGF浓度的增加,HEC-1-A细胞或Ishikawa细胞的KCa3.1的蛋白表达明显增加,与对照组相比,EGF浓度达10ng/ml时,KCa3.1的表达变化具有统计学差异(P＜0.05),其后略有下降;随着EGF作用时间的延长,HEC-1-A细胞和Ishikawa细胞的KCa3.1蛋白表达水平逐渐增强,48小时达高峰,此后KCa3.1表达水平呈下降趋势。(2)10ng/ml EGF可以引起HEC-1-A细胞和Ishikawa细胞的p-ERK1/2和pAKT表达的明显增强,ERK1/2和AKT的表达无明显变化。MAPK信号通路的阻断剂PD98059和PI3K/AKT信号通路的阻断剂LY294002分别可抑制EGF引起的ERK1/2和AKT的磷酸化,进而引起KCa3.1的表达下降。(3)KCa3.1通道的相对特异性阻断剂TRAM-34可以阻断EGF引起的ERK1/2的磷酸化,对AKT的磷酸化水平没有影响。结论EGF通过MAPK和PI3K/AKT通路促进子宫内膜癌细胞KCa3.1的表达;KCa3.1在EGF激活MAPK信号通路过程中发挥一定作用。综上所述,KCa3.1可促进子宫内膜癌细胞株HEC-1-A和Ishikawa细胞的增殖、周期进展、迁移和侵袭。EGF可以通过MAPK和PI3K/AKT信号通路促进子宫内膜癌细胞KCa3.1的表达,同时,KCa3.1的活性改变对ERK1/2的激活具有一定的调节作用。本课题的研究为进一步研究子宫内膜癌的发病机制提供了新的思路,为子宫内膜癌的药物治疗提供了新的思路和靶点。更多还原

【Abstract】 Endometrial cancer（endometrial carcinoma,EC） is common in malignant tumors of female reproductive system,the incidence is at 15-20 per 100000 women per year.Owing to the development of technology and medical treatment,the curability of EC raised,but for the tumors with particular morphological variants, adverse histopathological features and/or advanced stage,the five years survival rate are still very low.Despite extensive etiopathogenisis and pathogenetic studies,the molecular mechanisms of EC remain elusive.The ion channel has been found to play an important role in the proliferation of cancer cells,and our previous study show that intermediate-conductance Ca2+-activated K⁺ channels is highly expressed in endometrial carcinoma tissues and it can promote the proliferation of endometrial carcinoma cells.We will further study the effects of KCa3.1 on endometrial carcinoma cells and to explore the possible molecular mechanisms.Our study contains three parts:（1）The effects of KCa3.1 on the proliferation, cell cycle and apoptosis of endometrial carcinoma cells;（2）The impact of KCa3.1 on the migration and invasion of endometrial carcinoma cells;（3） The effects of EGF on KCa3.1 expression and the possible mechanisms.SectionⅠEffects of KCa3.1 on proliferation,cell cycle and apoptosis of human endometrial carcinoma cellsObjective The aim of the present study was to explore the effect of KCa3.1 on cell proliferation,cell cycle and apoptosis in endometrial cancer cells.Methods The recombinant plasmid containing small interference RNA of KCa3.1 gene was transfected into endometrial cancer cell lines,HEC-1-A and Ishikawa.Real time-PCR and Western blot were used to examine the gene and protein expression of KCa3.1 channels,while un-transfected cells and transfected negative plasmid cells were served as control groups.The function of KCa3.1 channels in KCa3.1 siRNA transfected endometrial cancer cells was analyzed through MTT、BrdU uptake test,flow cytometry,Real time-RT-PCR and Western blot.In addition, the TRAM-34 was added as inhibitor of KCa3.1.Results KCa3.1 siRNA transfected cells inhibited the mRNA and protein expression of KCa3.1 channels compared with control groups（P＜0.01）.TRAM-34 and KCa3.1 siRNA transfectants suppressed the cell proliferation dramatically. Compared with control groups,the ratios of G0-G1 phase cells in experimental groups increased,while the ratios of S phase cells decreased and the apoptotic rate had no significant changes（P＞0.05） among the groups.The protein expressions of Cyclin D1、Cyclin E and survivin had significantly decreased in the experiment groups by Western blot analysis.Conclusion The blockage of the function or expression of KCa3.1 channels could inhibit cell proliferation and cell cycle progression,while may not involved in apoptosis in endometrial cancer cells.SectionⅡThe impact of KCa3.1 on the migration and invasion of human endometrial carcinoma cellsObjective To explore the impacts of intermediate-conductance Ca²⁺-activated K⁺ channel on migration and invasion of endometrial carcinoma cells.Methods With TRAM-34,the specific blocker of KCa3.1,we detected the changes of migration and invasion of endometrial carcinoma cells through scratch experiment, migration experiment and Transwell chamber invasion assay.At the same time,we studied the changes of MMP-2 expression before and after treatment with TRAM-34, using RT-PCR and Western blot.Results（1） TRAM-34 can significantly inhibit the ability to migrate and invade in HEC-1-A and Ishikawa cells,compared with the control group（P＜0.01）;（2） TRAM-34 could cause the decline of MMP-2 expression and the difference was significant（P＜0.01）.Conclusion The inhibition of KCa3.1 channel activity can reduce the ability to migrate and invade in endometrial carcinoma cells.These phenomenon may be related to reduced MMP-2 expression.In endometfial carcinoma cells,KCa3.1 probably can prompted the migration and invasion,and its expression and/or dysfunction maybe plays an important role in the metastasis of endometrial carcinoma.SectionⅢEffect of EGF on KCa3.1 expression and the possible mechanisms Objective To study the effects of EGF on the expression of KCa3.1 and the possible mechanisms.Methods Cultured HEC-1-A and Ishikawa were treated by EGF with different concentration for different time.The cells were then harvested and the mRNA or proteins of KCa3.1 were extracted for RT-PCR or Western blot analysis,and also the expression of p-ERK1/2 and p-AKT with 10ng/ml EGF after different time（0,5, 15,30,60,120min）.Moreover,to investigate the molecular mechanism of EGF,the specific inhibitor of MAPK pathway PD98059 or/and the specific inhibitor of PI3K/AKT pathway LY294002 or/and the specific inhibitor of KCa3.1 TRAM-34 were also used to inhibit the phosphorylation of ERK1/2 or AKT or the KCa3.1 and to study the relations in all these molecules.Results（1） With the increase in EGF concentration,HEC-1-A cells or Ishikawa cells KCa3.1 protein expression was significantly increased,compared with the control group;when the dose arrived at 10ng/ml,KCa3.1 expression arrived at the peak with significant difference（P＜0.01）;with time extension,KCa3.1 protein expression level gradually increased,peaked at 48 hours,after which the expression level of KCa3.1 was downward trend in HEC-1-A or Ishikawa cells.（2） 10ng/ml EGF can cause significant enhancement of p-ERK1/2 and p-AKT expression,which reached the peak after 60min,while the ERK1/2 and AKT expression had no significant changes,the specific inhibitor of MAPK pathway PD98059 or/and the specific inhibitor of PI3K/AKT pathway LY294002,respectively,inhibited EGF-induced ERK1/2 and AKT phosphorylation,thereby causing the expression of KCa3.1 decline compared with the control group statistically（P＜0.01）.（3）The relative specific blocker of KCa3.1 channel TRAM-34 can block the majority of EGF-induced ERK1/2 phosphorylation,but not the AKT phosphorylation or the expression of ERK1/2 or AKT.Conclusion EGF can promote the expression of KCa3.1 through the MAPK and PI3K/AKT signaling pathways;KCa3.1 plays a role in EGF-activated MAPK signaling pathway.Overall,KCa3.1 can promote the proliferation,cell cycle,migration and invasion in endometrial cancer cell lines HEC-1-A and Ishikawa.EGF can up-regulate the expression of KCa3.1 through MAPK and PI3K/AKT signaling pathways,meanwhile,KCa3.1 activity changes plays a regulatory role in the activation of MAPK signaling pathway.Our study provide a new sight for study of pathogenesis and a new target for drug therapy of endometrial cancer in the future.更多还原