Study Of Tumor Inhibitory Gene RECK On The Biologic Behavior Of Trophoblast Invasion

Part one Roles of RECK gene in shallow trophoblast invasion in preeclampsia[Objective] To study the mRNA,protein expression and protein expression localization of RECK gene in normal pregnancy and preeclampsia and its relation to the trophoblast invasiveness.[Methods] Immunohistochemistry, the semi-quantitative RT-PCR analysis and Western-blot were respectively used to detect RECK protein localizations, mRNA ,protein expression levels in the placental tissues of 45 cases of normal pregnancy undergoing selective cesarean section among which 22 cases were in early pregnancy, 23 cases in term pregnancy, and of preeclampsia patients(22 cases of mild and 20 cases of severe preeclampsia ) undergoing cesarean section.[Results] (1) Immunohistochemistry results showed RECK expression was found both in placental tissues of normal pregnancy and preeclampsisa, and mainly expressed in cell membrane and cytoplasm of cytotrophoblast, syneytiotrophoblast. RECK expression was obviously lower in cellular column (CC) having invasion abiliy in normal early pregnancy. RECK expression increased with gestational week in normal pregnancy. RECK protein expression of normal early pregnancy group was significantly lower than that of normal term pregnancy group (P<0.05). RECK protein expression of mild preeclampsia group and severe preeclampsia group were significantly higher than that of group term pregnancy (P<0.01,P<0.05,respectively). The optical density values of RECK protein in mild preeclampsia group was significantly higher than that in severe preeclampsia group (P<0.05 ). (2) RT-PCR showed mRNA of normal early pregnancy group was significantly lower than that of normal term pregnancy group (P<0.05). RECK mRNA of mild preeclampsia group and severe preeclampsia group were significantly higher than that of term pregnancy group (P<0.01,P<0.05,respectively ). The optical density values of RECK mRNA in mild preeclampsia group was significantly higher than that in severe preeclampsia group (P<0.05 ). (3) Western blot showed the optical density values of RECK protein expression in early pregnancy group and term pregnancy group were 0.15±0.02 and 0.68±0.06 ,respectively.The RECK protein expression level in early pregnancy group was significantly lower than that in the term placental tissues (P<0.05). RECK protein level of mild preeclampsia group and severe preeclampsia group were significantly higher than that of group term pregnancy (P < 0.01 , P <0.05,respectively ).The optical density values of RECK protein in mild preeclampsia group was significantly higher than that in severe preeclampsia group (P<0.05 ).[Conclusion] RECK expression increased with gestational week in normal pregnancy and increased significantly in preeclampsia , suggesting RECK may play an important role in shallow invasion of trophoblast in preeclampsia. Part two Transfection of RECK gene expressing plasmid reduces MMP-2 activation and invasion ability of cultured extravillous trophoblast[Objective] To investigate effect of RECK gene expression on MMP-2 activation and invasional ability of TEV-1 cell line.[Methods] The recombinant eukaryotic expression vector- -RECK inserted by the full length cDNA encoding human RECK gene or pcDNA3/control vector were stably transfected into sub-confluent TEV-1 by Lipofectamine? 2000 method following the manufacturer's protocol (Invitrogen Life Technologies, Gaithersburg, MD).Briefly,5×105 TEV-1 cells, seeded in an individual well of a 6-well culture plate, were transfected with 3μg of DNA and changed with complete DMEM/F12 after transfection 24 h, followed by G418 selection after transfection (800μg/ml) The cells were maintained in complete DMEM/F12 and 200μg/ml of G418 gradually. After 16 days culture, G418-resisitant TEV-1 were collected.mRNA and protein expression level of RECK in TEV-1 were assayed by RT-PCR and flow cytometric analysis respectively. MMP-2 activation and cell invasional ability were analyzed by gelatinase zymography and matrigel invasion assay, respectively. Cell proliferation was tested by MTT.[Results](1)mRNA expression and cell surface protein expression of RECK gene were both significantly higher in transfected cells than those in blank plasmid pcDNA3 group (all P<0.05).(2)Gelatinase zymography showed MMP-2 activation of transfected cells group was obviously less than that of blank plasmid pcDNA3 group (P<0.05). (3)Cell invasion assay showed cell number invading through Matrigel was dramatically decreased in transfected group compared with those in blank plasmid pcDNA3 group(P<0.01).(4)MTT showed no significant difference were found between transfected cells and blank plasmid pcDNA3 group and control group in comparing cell proliferation.[Conclusion] Over expression of RECK gene significantly inhibits MMP-2 activation and cell invasional ability of TEV-1 and has no impact on cell proliferation, suggesting RECK is a likely mediator of trophoblast invasion of early pregnancy.