The reinforcement of invasion in epithelial ovarian cancer cells by 17 beta-Estradiol is associated with up-regulation of Snail.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Ding
Jing-Xin
JX
Gynecologic and Obstetric Hospital, Fudan University, Department of Gynecology and Obstetrics, Shanghai Medical College, Fudan University, Shanghai, PR China.
Feng
You-Ji
YJ
Yao
Liang-Qing
LQ
Yu
Min
M
Jin
Hong-Yan
HY
Yin
Lian-Hua
LH
D018262:Adenocarcinoma, Clear Cell / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D015820:Cadherins / Q000096:biosynthesis / Q000235:genetics; D045744:Cell Line, Tumor; D018284:Cystadenocarcinoma, Serous / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D004847:Epithelial Cells / Q000473:pathology; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D020868:Gene Silencing; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D009361:Neoplasm Invasiveness; D010051:Ovarian Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011960:Receptors, Estrogen / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics; D015854:Up-Regulation
D015820:Cadherins; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors; D004958:Estradiol; D020778:Matrix Metalloproteinase 2