雌激素调控子宫内膜癌Ishikawa细胞中LRP16基因表达及其意义

Expression regulation of LRP16 gene by 17β-estradiol and its significance in human endometrial cancer Ishikawa cells

目的:探讨子宫内膜癌Ishikawa细胞中雌激素(E2)对LRP16基因表达的调控作用,LRP16基因过表达对Ishikawa细胞增殖及侵袭生长能力的影响以及可能的分子机制.方法:采用Northernblot方法检测细胞中LRP16基因mRNA表达水平.双荧光报告系统检测相对荧光素酶活性.胎盘蓝死活细胞计数方法观察LRP16过表达对Ishikawa细胞增殖的影响,Matrigel包被的Transwell方法观察LRP16过表达对Ishikawa细胞侵袭生长能力的影响.Westernblot方法检测Ishikawa细胞中的蛋白水平.结果:雌二醇诱导了Ishikawa细胞中LRP16基因mRNA水平表达上调;而ICI182780则Ishikawa细胞中LRP16mRNA水平下调.增加ERα表达量促使Ishikawa细胞中LRP16基因上调.pGL3S5与ERα共转染Ishikawa细胞的相对荧光素酶活性较单纯pGL3S5转染组细胞升高30倍.我们没有观察到LRP16基因过表达对Ishikawa细胞的促增殖效应.Transwell结果显示LRP16基因过表达Ishikawa细胞的侵袭率较对照组细胞增加了30%.LRP16基因过表达的Ishikawa细胞中E钙粘合素的mRNA以及蛋白水平较对照组细胞下调了3倍,而没有检测到MMP2,MMP9以及CD44蛋白水平的变化.结论:我结果表明E2通过激活ERα上调子宫内膜癌Ishikawa细胞中LRP16基因mRNA水平,LRP16表达水平依赖于雌激素.LRP16基因表达上调没有促进子宫内膜癌细胞的增殖,但可能通过抑制E钙粘合素表达水平增加了细胞的侵袭能力.

AIM： To explore the regulation of LRP16 gene expression by 17β-estradiol （E2） in ERa-positive human endometrial cancer Ishikawa cell line, and to investigate the effect of LRP16 overexpression on the proliferative potential and invasive growth of Ishikawa and the possible molecular mechanism. METHODS： The LRP16 mRNA level in Ishiwaka cells was determined by Northern blot analysis. The relative luciferase activity was measured using Dual-luciferase reporter assay system. The effect of LRP16 overexpression on Ishiwaka proliferation was examined by the Trypan Blue exclusion method. The invasiveness of Ishikawa was evaluated by using the Matrigel-coated Transwell assay. The protein levels in Ishikawa were determined by Western blot analysis. In addition, the E-cadherin mRNA level was also examined by Nothern blot. RESULTS： 17β-E2 induced an increase in LRP16 mRNA levels in Ishikawa cells, whereas, its pure antagonist, ICI 182 780 reduced the LRP16 mRNA level. Ectopic ERα transfection in Ishikawa increased the expression of LRP16 gene. The significant increase of the relative luciferase activity in Ishikawa cells cotransfected by pGL3-S5 and ERa plasmids was observed compared with that in the control cells transfected by pGL3-S5 alone. Stimulative effect of LRP16 overexpression on the proliferation of Ishikawa calls was not observed in this study. Thirty-persent increase of the invasive capacity was observed in Ishikawa cells with LRP16 overexpression. The mRNA and protein levels of E-cadberin gene was nearly 3-fold decreased in Ishikawa cells with the overexpression of LRP16, while the protein levels of MMP2, MMP9 and CD44 were not different between LRP16-overexpressed Ishikawa cells and the control cells. CONCLUSION： Estrogen up-regulated the LRP16 mRNA level by activation of ERα and the LRP16 expression was dependent on the estrogen in ERα-pesitive endometrial cancer cells. Up-regulation of LRP16 did not promote the proliferation of the endometrial cancer cells, but increased its invasive potential possibly by suppressing the E-cadherin expression level.