Journal of Jilin University(Medicine Edition) ›› 2022, Vol. 48 ›› Issue (4): 1018-1027.doi: 10.13481/j.1671-587X.20220423

• Research in clinical medicine • Previous Articles    

Expression of miR-27a in villi tissue of patients with recurrent abortion and its effects on trophoblast cell proliferation and apoptosis and their mechanisms

Lihua ZHOU(),Ying HU,Hui ZOU   

  1. Department of Reproductive Medicine,Second Affiliated Hospital,Hainan Medical College,Haikou 570311,China
  • Received:2021-09-24 Online:2022-07-28 Published:2022-07-26
  • Contact: Lihua ZHOU E-mail:historical1990@163.com

Abstract: Objective

To investigate the expression of miR-27a in villus tissue of the patients with recurrent abortion (RM), and to clarify its effects on trophoblast cell proliferation and apoptosis and their mechanisms.

Methods

The villus tissues of 17 RM patients (RM group) and normal pregnant women (healthy control group) were collected. The expression levels of miR-27a in villus tissue of the subjects were detected by real-time fluorescence quantitative PCR (RT-qPCR). The human chorionic trophoblasts HTR-8/SVneo were cultured in vitro. The miR-27a mimic or inhibitor or negative control sequence (miR-NC) were transfected into the trophoblasts by liposome transient transfection;at the same time,control group(without transfection) was set up. After RT-qPCR method was used to detect the transfection efficiency, CCK-8 method and EdU experiment were used to detect the proliferation activities of the cells and the rates of EdU positive cells in various groups, and flow cytometry was used to detect the percentages of trophoblasts at different cell cycles in various groups; Annexin Ⅴ-FITC/PI method was used to detect the apoptotic rates of cells in various groups. The target genes of miR-27a were analyzed by bioinformatics, Cyclin D1 and insulin-like growth factor 1 (IGF1) were screened for verification, and luciferase target gene reporting experiment, RT-qPCR method and Western blotting method were used to detect the regulatory relationship between miR-27a and Cyclin D1 IGF1; RT-qPCR method and Immunohistochemistry were used to detect the expressions of Cyclin D1 and IGF1 mRNA and proteins in villi tissue of subjects in RM group and healthy control group. Pearson’s correlation analysis was used to analyze the correlation between miR-27a and the expression levels of Cyclin D1 and IGF1 mRNA.

Results

Compared with healthy control group, the expression level of miR-27a in villus tissue of the patients in RM group was increased significantly (P<0.01).Compared with control group, the expression level of miR-27a in the cells in miR-27a mimic group was significantly increased (P<0.01), while that in the miR-27a inhibitor group was significantly decreased (P<0.01),and the expression level of miR-27a in the cells in miR-NC group had no significant difference(P>0.05).Compared with control group, the proliferation activity and percentage of cells in S phase in miR-27a mimic group were decreased significantly(P<0.05), while the percentage of cells in G0/G1 phase and apoptosis rate were increased significantly (P<0.05);the proliferation activity and percentage of cells in S phase in miR-27a inhibitor group were significantly increased(P<0.05), and the percentage of cells in G0/G1 phase and apoptotic rate were significantly decreased (P<0.05),and the above indicators in miR-NC group had no significant differences(P<0.05). The results of luciferase target gene reporter assay, RT-qPCR and Western blotting confirmed that miR-27a could targetedly inhibit the expressions of Cyclin D1 and IGF1. Compared with healthy control group, the expression levels of Cyclin D1 and IGF1 mRNA and proteins in villi tissue of the patients in RM group were significantly reduced (P<0.05), and there were significantly negative correlations between the mRNA expression levels of Cyclin D1 and IGF1 and the expression level of miR-27a in villus tissue of the RM patients (r=-0.214, P<0.05; r=-0.307, P<0.05).

Conclusion

Abnormally elevated miR-27a in villus tissue of the RM patients may block the cycle progression of trophoblasts, inhibit their proliferation, and promote apoptosis by targeted regulating the expression of Cyclin D1 and IGF1.

Key words: Recurrent miscarriage, MicroRNA-27a, Cyclin D1, Insulin-like growth factor 1, Trophoblasts

CLC Number: 

  • R714.21