| Background: Thyroid dysfunction is a common endocrine disease during pregnancy which is closely related to abortion,premature delivery,eclampsia and other adverse pregnancy outcomes.A number of clinical epidemiological studies have indicated that pregnant women with elevated TSH have an increased risk of abortion during early pregnancy.However,the specific mechanism of elevated TSH related spontaneous abortion(SA)is not yet clear.The successful pregnancy is a series of fine regulation processes,it requires the cooperation of the endometrium and the embryo.Regulated by a variety of hormones,the endometrium is receptive to embryo adhesion and implantation during ‘implantation window period'.Studies have shown that endometrial receptive developmental disorders can lead to SA.In addition to uterine receptivity,the migration of extravillious trophoblasts and the remodeling of spiral arteries are also critical to pregnancy,and its obstacles can lead to pregnancy-related complications such as abortion.Micro RNA(mi RNA)is a type of single-stranded non-coding RNA molecule with a length of approximately 22 nucleotides encoded by endogenous genes.Articles have showed that mi RNA is associated with the occurrence and development of many diseases.In recent years,many studies have performed mi RNA array in the decidua,villi and serum of abortion patients,and indicated that mi RNA abnormal expression is involved in uterine receptivity,trophoblast invasion.Our previous research showed that compared with euthyroid SA patients and normal pregnant women,mi R-940 and mi R-486-5p was increased in the serum of subclinical hypothyroidism(SCH)patients with SA.mi R-17-5p can regulate the proliferation,invasion and apoptosis of a variety of tumor cells,and it is downregulated in endometrium of porcine arresting conceptus.mi R-29c-5p changes dynamically with the menstrual cycle and can inhibit the proliferation of endometrial cancer,while mi R-34b-5p is also related to endometriosis and other gynecological diseases.Zinc finger factor 367(zinc finger factor,ZNF367)belongs to the ZNF transcription factor family and has multiple functions,including DNA recognition,RNA packaging,and transcription activation.Recent studies have shown that zinc finger proteins are involved in the invasion and differentiation of trophoblast cells.The aim of this research is to explore the differentially expressed mi RNAs in the villi and decidua tissues of TSH>2.5m U/L patients with SA and their main functions in the endometrium and trophoblast development.Method: Part ?: From September 2017 to April 2019,the serum,villi and decidua of TSH>2.5m U/L combined SA,TSH>2.5m U/L pregnant women,euthyroid SA patients and normal pregnant women at the Gynecology Clinic of the First Affiliated Hospital of China Medical University were collected.General information of all participants were recorded.The villi and decidua of three participants in each group were selected for mi RNA sequencing,and the differentially expressed mi RNAs in the villi and decidual tissues were screened by P and FC values.Chorionic villi and decidua tissues other than sequencing tissue were selected to verify differential expressed mi RNA using q RT-PCR;Spearman correlation test was used to analyze the correlation between TSH and target mi RNA.Part ?: Different concentration TSH stimulated the trophoblast cell line(HTR-8/SVneo)to explore the effect of TSH on the migration and invasion of the trophoblast by wound healing and the Transwell assays.The effects of TSH on the mi R-17-5p expression and invasion-related proteins in trophoblast were observed through q RT-PCR and Western blotting.mi R-17-5p inhibitor and mi R-17-5p mimics were transfected with HTR-8/SVneo to knockdown and overexpress mi R-17-5p,and transfection efficiency were verified by q RT-PCR.CCK8,wound healing,Transwell assays and Western blotting were used to detect the effects of mi R-17-5p on the proliferation,migration,invasion and function-related proteins of HTR-8/SVne cells.Target Scan 7.2,mi RDB,mi RTar Base,mi Randa were used to predict the potential targets of mi R-17-5p.m RNA and protein expression level of ZNF367 were determined by q RT-PCR and Western blotting in mi R-17-5p knockdown and overexpress trophoblasts as well as four groups of villi tissues.Luciferase assay was used to prove whether mi R-17-5p can directly bind to ZNF367.Transfection si-ZNF367 down-regulated the expression of ZNF367 and verified the transfection efficiency by q RT-PCR.CCK8,wound healing,Transwell assays and Western blotting were used to explore the effect of ZNF367 on the proliferation,migration,invasion and related proteins of trophoblast cells.mi R-17-5p inhibitor and siZNF367 were co-transfected to observe the effect of ZNF367 knockdown on the mi R-17-5p biological function.Part ?: mi R-29c-5p and mi R-34b-5p mimics/ inhibitors were transfected with endometrial cancer cell line(Ishikawa)to overexpress and knockdown mi R-29c-5p and mi R-34b-5p,and verify the transfection by q RT-PCR.CCK8,5-Ethynyl-2'-deoxyuridine(edu)and adhesion assays were used to determine the effects of mi R-29c-5p and mi R-34b-5p on the proliferation,receptivity and biological function-related protein.q RT-PCR was used to detect the expression level of predicted target gene in mi R-34b-5p knockdown and overexpression Ishikawa.Results: Part ?: Compared with the normal pregnant women group,there were 298 differentially expressed mi RNAs in the chorionic villi of TSH>2.5m U/L+SA group,mi R-17-5p was decreased and was negatively correlated with TSH.In the decidual tissue,there were 311 abnormally expressed mi RNAs,mi R-29c-5p and mi R-34b-5p were increased in the decidua of TSH>2.5m U/L+SA patients with SA and positively related to serum TSH.Part ?: 10 m IU/ml TSH inhibited the mi R-17-5p expression,migration,invasion and matrix metalloprotein(MMP)protein of trophoblast cells.mi R-17-5p overexpression promoted the proliferation,migration,invasion of trophoblast cells and the expression of proliferating cell nuclear antigen(PCNA)and MMP protein,while mi R-17-5p knockdown showed an opposite effect.mi R-17-5p targeted inhibited the expression of ZNF367 m RNA and protein.In addition,ZNF367 interference partially reversed the biological function of the mi R-17-5p inhibitor on HTR-8/SVneo cells.Part ?: In the decidua of the TSH>2.5mU/L+SA group,the expression of miR-29c-5p and mi R-34b-5p increased and were positively correlated with TSH.mi R-29c-5p and mi R-34b-5p overexpression inhibited the proliferation and receptivity of Ishikawa as well as protein expression of PCNA and integrin.mi R-34b-5p can inhibit the expression of indicators related to receptivity including insulin-like growth factor binding protein1(IGFBP1),caveolin(CAV)and homeobox C8(HOXC8).Conclusion: 1.In the villi of TSH>2.5m U/L+SA group,mi R-17-5p expression decreased and high concentration of TSH inhibited the migration and invasion of trophoblast cells and the expression of mi R-17-5p.mi R-17-5p inhibited the proliferation,migration and invasion of trophoblast cells via ZNF367.2.In the decidua of the TSH>2.5m U/L+SA group,the expression of mi R-29c-5p and mi R-34b-5p were increased and were positively correlated with TSH.mi R-29c-5p and mi R-34b-5p inhibited the proliferation and receptivity of Ishikawa.The inhibitory effects of mi R-29c-5p and mi R-34b-5p on proliferation and receptivity of Ishikawa may via integrins ?V and ?1. |
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