MicroRNA-379-5p is associate with biochemical premature ovarian insufficiency through PARP1 and XRCC6

Abstract

Premature ovarian insufficiency (POI) imposes great challenges on women's fertility and lifelong health. POI is highly heterogeneous and encompasses occult, biochemical, and overt stages. MicroRNAs (miRNAs) are negative regulators of gene expression, whose roles in physiology and diseases like cancers and neurological disorders have been recognized, but little is known about the miRNAs profile and functional relevance in biochemical POI (bPOI). In this study, the expression of miRNAs and mRNAs in granulosa cells (GCs) of bPOI women was determined by two microarrays, respectively. MiR-379-5p, PARP1, and XRCC6 were differentially expressed in GCs of bPOI as revealed by microarrays. Subsequently, functional studies demonstrated that miR-379-5p overexpression inhibited granulosa cell proliferation and attenuated DNA repair efficiency. Furthermore, both PARP1 and XRCC6 showed lower levels in GCs from patients with bPOI and were identified as executives of miR-379-5p. Therefore, our data first uncovered potentially pathogenic miR-379-5p and two novel targets PARP1 and XRCC6 in bPOI, which corroborated the significance of DNA repair for POI, and brought up an epigenetic explanation for the disease.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1. MiR-379-5p, PARP1, and XRCC6 are differentially expressed in GCs of bPOI as revealed by microarray

a Heat map of miR-379-5p, PARP1, and XRCC6 based on microarray data. Each column represents a sample; red and green indicates up- and downregulation, respectively; r correlation coefficient. b, c, d Quantitative PCR (qPCR) analysis in GCs from women with and without bPOI shows that miR-379-5p increases (b), while PARP1 and XRCC6 decrease in bPOI patients (c, d). Two-tailed Mann–Whitney U-test

Fig. 2. PARP1 and XRCC6 are novel targets of miR-379-5p

a, b qRT-PCR and western blot analyses indicate that PARP1 and XRCC6 mRNA (a) and protein (b) levels are significantly reduced by forced expression of miR-379-5p in KGN cells. c, d Predicted target sites and relevant mutation sequences are shown. Luciferase assay demonstrates that overexpression of miR-379-5p leads to a decrease in PARP1 and XRCC6 with wild-type 3′-UTRs, while luciferase activity levels of mutated 3′-UTRs unchanged. Asterisk (*) indicates a significant difference. *P < 0.05, two-tailed Student's t test

Fig. 3. Overexpression of miR-379-5p leads to proliferation inhibition and DNA repair impairment

a CCK-8 assay reveals that miR-379-5p overexpression led to suppression in cell proliferation. b, c EdU assay shows less EdU-positive cells in miR-379-5p overexpressed group. d PCNA level is much lower in KGN cells overexpressing miR-379-5p than negative control. e After exposed to MMC for 6 h, more of γH2AX is found in KGN cells overexpressing miR-379-5p. After recovery for 2 and 4 h, miR-379-5p delays the DNA damage repair. f, g The clonogenic survival of HeLa cells suffering MMC treatment and 2 h’ recovery. The clonogenic survival percent of miR-379-5p mimics group is significantly less than that of negative control. Asterisk (*) indicates a significant difference. *P < 0.05, **P < 0.01, two-tailed Student's t test

Fig. 4. PARP1 and XRCC6 are functional targets of miR-379-5p

a An efficient decrease in PARP1 and XRCC6 protein levels is confirmed by western blot in KGN cells 48 h post transfection. b, c, d Suppressed cell proliferation in PARP1 and XRCC6 knockdown group is indicated by CCK-8 assay (b) and EdU staining (c, d). e PCNA level is much lower in PARP1 and XRCC6 silencing cells than negative control cells. f After exposed to MMC for 6 h, and recovery for 2 and 4 h, PARP1 and XRCC6 silencing delayed the DNA damage repair. g, h The percent survival relative to the control is significantly lower in PARP1 and XRCC6 depletion cells. Asterisk (*) indicates a significant difference. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Student's t test

Fig. 5. Reintroduction of PARP1 and XRCC6 abrogates the miR-379-5p-induced effects partially

a An efficient increase in PARP1 and XRCC6 protein levels is confirmed by western blot in KGN cells 48 h post co-transfection infection. b, cPARP1 and XRCC6 overexpression could promote cell proliferation. d After exposed to MMC for 6 h, more of γH2AX is found in two groups overexpressing miR-379-5p. However, after 4 h recovery, PARP1 and XRCC6 overexpression group shows similarly less γH2AX as the NC + Ad-Null group. The results uncover that PARP1 and XRCC6 is required for efficient DNA damage repair process