Ding Jing-Xin, Feng You-Ji, Yao Liang-Qing, Yu Min, Jin Hong-Yan, Yin Lian-Hua
Gynecologic and Obstetric Hospital, Fudan University, Department of Gynecology and Obstetrics, Shanghai Medical College, Fudan University, Shanghai, PR China.
Gynecol Oncol. 2006 Nov;103(2):623-30. doi: 10.1016/j.ygyno.2006.04.023. Epub 2006 Jun 27.
The transcription factor Snail, which is implicated in the triggering of epithelial-mesenchymal transitions (EMT), plays an important role in adhesion, invasion and metastasis of tumor cells. In the present study, we assessed 17beta-Estradiol (E2)'s effect on Snail, E-cadherin and MMP-2 expression of epithelial ovarian cancer cell line ES-2 and SKOV3. Then we induced Snail gene silencing by RNA interference to explore the effect of E2 on E-cadherin and MMP-2 expression when Snail gene expression was blocked.
Treated by 10(-8) M E2, Snail, E-cadherin and MMP-2 mRNA expression of the cells was measured by RT-PCR; Snail, MMP-2 protein expression was detected by IHC; and MMP-2 activity was determined by Zymography. E-cadherin protein level was measured by Western blot. We constructed the small interfering dsRNA expression vector (pRNAT-U6.1/Neo-Snail) targeting Snail gene, as well as a negative control vector (pRNAT-U6.1/Neo-Neg). Then the cells were transiently transfected with the vectors. Western blot and zymography were conducted to determine E-cadherin protein level and matrix metalloproteinase activity of the cells transfected with pRNAT-U6.1/Neo-Snail or pRNAT-U6.1/Neo-Neg after treated with E2 for 24 h.
The expression of ER alpha mRNA and protein was negative in ES-2 cells and positive in SKOV3 cells, and ER beta expression was positive in both cell lines. 10(-8) mol/l E2 elevated expression of Snail and MMP-2 mRNA and protein in both ES-2 and SKOV3 cells, and reduced expression of E-cadherin mRNA and protein in SKOV3 cells. While in the RNAi group transfected with the small interfering dsRNA expression vector (pRNAT-U6.1/Neo-Snail) targeting Snail gene, E2 treatment did not have a significant effect on MMP-2 activity or E-cadherin protein in ES-2 and SKOV3 cells.
17beta-Estradiol increased Snail expression in both ER alpha-negative ES-2 cells and ER alpha-positive SKOV3 cells independent of the existence of ER alpha. The increase of MMP-2 expression in ES-2 and SKOV3 cells and decrease of E-cadherin expression in SKOV3 cells induced by E2 were associated with up-regulation of Snail.
转录因子Snail参与上皮-间质转化（EMT）的触发，在肿瘤细胞的黏附、侵袭和转移中起重要作用。在本研究中，我们评估了17β-雌二醇（E2）对上皮性卵巢癌细胞系ES-2和SKOV3中Snail、E-钙黏蛋白和MMP-2表达的影响。然后我们通过RNA干扰诱导Snail基因沉默，以探讨当Snail基因表达被阻断时E2对E-钙黏蛋白和MMP-2表达的影响。
用10⁻⁸ M E2处理细胞后，通过RT-PCR检测细胞中Snail、E-钙黏蛋白和MMP-2 mRNA的表达；通过免疫组化检测Snail、MMP-2蛋白的表达；通过明胶酶谱法测定MMP-2活性。通过蛋白质免疫印迹法检测E-钙黏蛋白的蛋白水平。我们构建了靶向Snail基因的小干扰双链RNA表达载体（pRNAT-U6.1/Neo-Snail）以及阴性对照载体（pRNAT-U6.1/Neo-Neg）。然后将细胞用这些载体进行瞬时转染。在用E2处理24小时后，对用pRNAT-U6.1/Neo-Snail或pRNAT-U6.1/Neo-Neg转染的细胞进行蛋白质免疫印迹法和明胶酶谱法检测，以确定细胞中E-钙黏蛋白的蛋白水平和基质金属蛋白酶活性。
ES-2细胞中ERα mRNA和蛋白表达为阴性，SKOV3细胞中为阳性，两种细胞系中ERβ表达均为阳性。10⁻⁸ mol/l E2可使ES-2和SKOV3细胞中Snail和MMP-2 mRNA及蛋白表达升高，使SKOV3细胞中E-钙黏蛋白mRNA和蛋白表达降低。而在转染了靶向Snail基因的小干扰双链RNA表达载体（pRNAT-U6.1/Neo-Snail）的RNAi组中，E2处理对ES-2和SKOV3细胞中的MMP-2活性或E-钙黏蛋白蛋白没有显著影响。
17β-雌二醇在ERα阴性的ES-2细胞和ERα阳性的SKOV3细胞中均增加Snail表达，且与ERα的存在无关。E2诱导的ES-2和SKOV3细胞中MMP-2表达增加以及SKOV3细胞中E-钙黏蛋白表达降低与Snail的上调有关。
