GRIM-19 Involvement In WT1 Assosiated Epithelial-to-mesenchymal Transition In Adenomyosis

Background:Adenomyosis may cause infertility,dysmenorrhea,chronic pelvic pain,dyspareunia,abnormal uterine bleeding and other manifestations in women of premenopausal age.It gradually worsens with the menstrual cycle and there are no radical measures.The existence of endometrial glands and stroma in myometrium is one of the main characteristics of this disease,which shows the high invasion and migration ability of the ectopic endometrium.In a variety of cancers including liver cancer,kidney cancer and skin squamous cell carcinoma,gene associated with retinoid-interferon-induced mortality-19(GRIM-19)is low expression.The low expression of GRIM-19 is related to epithelial-to-mesenchymal transition(EMT)in some cancers.The low expression of GRIM-19 occurs in adenomyosis and the mechanism of differential expression of GRIM-19 on the progression of adenomyosis needs to be further studied.Objective:To explore the mechanism of low expression of GRIM-19 in promoting the progression of adenomyosis and the effect of GRIM-19 on EMT in adenomyosis.Methods:1.Differentially expressed genes in adenomyosis were screened in GEO database.Adenomyosis differential gene analysis andthe biological process analyses were performed.2.Clinical samples:Western blotting and qRT-PCR were used to detect the expression of GRIM-19,Wilms’ tumor-1(WT1),Snail and EMT markers in adenomyosis samples and normal endometria.Immunohistochemical staining was applied to detect the expression of WT1 in clinical samples.Cells experiment:(1)GRIM-19siRNA was transfected in Ishikawa cells and CCK-8 was applied to cell growth detection.Using Wound-healing assay and Transwell assay,changes in cell migration and invasion ability were known.(2)Western blotting and qRT-PCR were exploited to verify the changes of WT1,Snail and EMT markers in Ishikawa cells with low GRIM-19 expression.Animal experiment:GRIM-19 knockdown mice were sacrificed to verify the changes of WT1,Snail and EMT markers by Western blotting and qRT-PCR in uteri.WT1 immunohistochemical staining was done in mice uteri.Results:1.Clinical samples:(1)The protein level and mRNA level of GRIM-19 in ectopic adenomyosis were down-regulated compared with the normal endometria of the control group(P=0.0057 and P<0.0001).(2)WT1 protein was down-regulated in ectopic adenomyosis(P=0.0043)and WT1 mRNA expression was down expressed(P=0.006).Immunohistochemical staining of WT1 in the stromal cells of the ectopic endometria of adenomyosis and the whole adenomyosis lesions was lower than that in the control group(P=0.0009 and P=0.0024),while the level of WT1 staining was higher in glandular region(P=0.0045).(3)Compared with normal endometria,the mRNA level of KRT8 decreased in adenomyosis(P=0.0044),while the expression of stroma marker CDH2 mRNA increased(P=0.0183).The protein expression of epithelial marker E-cadherin decreased in ectopic adenomyosis(P<0.0001).2.Cell experiment:(1)CCK-8 analysis uncovered that the proliferation of Ishikawa cells increased after transferring the GRIM-19 siRNA(P=0.0449).The migration ability of Ishikawa cells was higher after the knockdown of GRIM-19(P=0.0025).The transwell assay was applied to test the migration and invasion of Ishikawa cells after GRIM-19 siRNA manipulation.Down-regulation of GRIM-19 expression increased the migration ability(P=0.0257)and invasion ability(P=0.0368)of Ishikawa cells.(2)The results of GRIM-19 siRNA transfection in Ishikawa cells showed that the expression of GRIM-19 was reduced,which was verified by Western blotting(P=0.0498)and qRT-PCR(P=0.0006).With the decreased expression of GRIM-19 in Ishikawa cells,WT1 mRNA expression was increased(P=0.0306)and the expression of WT1 protein was increased as well(P=0.0258).We also saw the expression of KRT8 mRNA decreased(P=0.0108).The mRNA expression of Snaill decreased(P=0.0009),while the change of SNAIL+SLUG protein expression was not statistically significant.With the down-regulation of GRIM-19,the expression of KRT8 protein was low(P=0.0174),E-cadherin protein expression decreased(P=0.0482)while N-cadherin protein expression increased(P=0.0325).3.Animal experiment:(1)GRIM-19+/-gene knockdown mice were fertile and had no obvious phenotypic abnormalities.The GRIM-19 gene knockdown was confirmed by qRT-PCR(P=0.0058)and Western blotting(P=0.0153).(2)WT1 mRNA was highly expressed in the uterus of GRIM-19+/-mice(P=0.0247).Western blotting analysis showed that the expression of WT1 increased(P=0.0128).Immunohistochemical analysis of WT1 staining of GRIM-19+/-mouse uterus showed that the expression of WT1 protein increased(P=0.0002).(3)E-cadherin as epithelial marker decreased in mRNA level(P=0.0133)and protein level(P=0.0352)in GRIM-19+/-mouse uterine and KRT8 decreased in mRNA level(P=0.0142).Conclusions:1.Differences in expression of GRIM-19 and WT1 in uterine adenopathy and the occurrence of EMT may be related to the development of adenomyosis.2.Low expression of GRIM-19 in endometrial cell promotes the expression of WT1 and the occurrence of EMT in participating in the development of uterine adenomyopathy.