| Background and Objectives:Mitogen-inducible gene 6(Mig-6)(also known as receptor-associated late transducer,RALT,gene 33)is the first verified negative feedback regulator of the epidermal growth factor receptor(EGFR)in mammalian cells,which suppresses various biology processes regulated by EGFR signaling pathway,including mitosis,apoptosis,stress and so on.Mig-6 is a tumor suppressor gene,down-regulation or loss of Mig-6 expression has been reported in several human cancers including lung cancer,breast cancer,liver cancer and thyroid cancer.Meanwhile,previous study has suggested significant low-expression of Mig-6 in endometrial carcinoma(EC).Endometrioid-type endometrial adenocarcinoma is a kind of hormone-dependent tumor,unopposed estrogen exposure may result in endometrial hyperplasia,even atypical hyperplasia,which increases the occurrence of EC.High-dose of progesterone(P4)has been applicted as a kind of effective conservative treatment in clinical practice.Results of gene chip demonstrated that MIG-6 was a downstream target gene of progesterone receptor(PR),it could regulate the effect of progesterone on endometrium,and adjust the transformation from proliferation phase to secretoiy phase.Other studies have demonstrated that Mig-6 is crucial for the effect of P4 in endometrium epithelial,lack of Mig-6 significantly increases the occurrence of EC in Mig-6-null mouse.During the conservative treatment of EC,P4 has low or even no curative effect on some patients,which is known as "progesterone resistance".This phenomenon exists not only in EC,but also in EMs and PCOS.Present studies suggest that progesterone resistance is also related to low expression of Mig-6.In this study,after confirming the down-regulation of Mig-6 protein in EC tissues,the expression of Mig-6 was up-regulated in Ishikawa cells by pCMV6-Mig-6 plasmid,and the cells were treated with E2 or E2+P4,then the effects of Mig-6 on proliferation,apoptosis,invasion ability of Ishikawa cells were tested,which may uncover the molecular mechanism that Mig-6 inhibited the occurrence of EC.In addition,this study explored the role Mig-6 played in progesterone resistance,which may guide the individualized management of EC,EMS and PCOS.Materials and Methods(1)EC specimen confirmed by pathology were obtained from patients diagnosed as endometrial carcinoma(IA and IB stage)in the first affiliated hospital of NanJing medical university from February 2013 to December 2014,normal endometria were taken from patients diagnose as uterine myoma who performed hysterectomy.The protein level of Mig-6 in EC tissue and normal endometrium were tested by Western Blot.(2)Ishikawa cells were transfected with Mig-6 overexpression plasmid(pCMV 6-ERRFI1)and negative control vector(pCMV6-entry)using lipofectamine 2000 according to the manufacturer’s recommendations,QPCR and Western Blot were used to check the transfection efficiency on mRNA and protein level.(3)After the plasmid transfection(with pCMV6-entry or pCMV6-ERRFI1),Ishikawa cells were treated with EtOH(control),17β-E2 only(10-7M)or 17β-E2(10"7M)+ progesterone(10-7M),then we used flow cytometry to examine the apoptosis rate of cells in each group.Additionally,the expression of cleaved-caspase3,Bax,Bcl2 protein which related to cell apoptosis were tested using Western Blot.(4)Treated as above,cell Immunofluorescence technique and flow cytometry were used to examine the BrdU incorporation rate of cells in each group,and Western Blot to test the expression of cyclinDl,pERK,ERK.(5)Treated as above,Transwell assay was used to examine the invasive potential of cells in each group,and Western Blot to test the expression of MMP2,MMP9.(6)Statistical Analysis:SPSS 20.0 was used for statistical analysis.Data were presented as mean ± standard deviation(SD),and checked by the normality assumption and the homogeneity of variances assumption in one-way analysis of variance(ANOVA).P<0.05 was considered as statistically significant.Results(1)Compared with normal endometrium tissues,the expression of Mig-6 protein in EC specimen were significantly decreased by 48.11%(P<0.05).(2)After the transfection with pCMV6-ERRFI1,expression of Mig-6 mRNA and protein were 24.2,2.8 fold of that in negative control(NC)group(P<0.05).(3)After Mig-6 over-expression,the early apoptosis rate of Ishikawa cells was 2.67 times of that in NC group(P<0.05),the pro-apoptotic activity of P4 was 1.39 times of that in NC group(P<0.05),both the expression of cleaved-caspase3 protein and Bax/Bcl2 ratio also elevated significantly(P<0.05).(4)After Mig-6 over-expression,BrdU incorporation rate was 70.28%of that in NC group(P<0.05),and E2 was not able to promote cell proliferation as in NC group,the ability of P4 to antagonize estrogen-mediated proliferation was increased by 37.90%(P<0.05).Meanwhile,the expression of cyclinDl,pERK/ERK were significantly decreased(P<0.05).(5)After Mig-6 over-expression,the amount of cells trans-membrane was 72.38%of that in NC group(P<0.05),the pro-invasion effect of E2 decreased by 32.37%,while the anti-invasion effect of P4 increased by 48.89%compared with that in NC group(P<0.05),and the expression of MMP2,MMP9 protein were significantly decreased(P<0.05),Conclusion(1)Mig-6 over-expression triggered apoptosis of EC cells,which may occur by activating the mitochondira apoptosis pathway.(2)Mig-6 over-expression inhibited proliferation of EC cells,which may correlate with RAS/RAF/ERK signaling pathway.(3)Mig-6 over-expression suppressed invasion of EC cells,which may be achieved by inhibiting the expression of MMP2,MMP9 protein.(4)Mig-6 over-expression synergizes the pro-apoptosis effect of P4,enhances the function of P4 to antagonize not only E2-mediated cell proliferation,but also E2-mediated cell invasion. |
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