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. 2019 Apr 1;104(4):1049-1059.
doi: 10.1210/jc.2018-00544.

The HMGA2-IMP2 Pathway Promotes Granulosa Cell Proliferation in Polycystic Ovary Syndrome

Miao Li  1   2   3 , Han Zhao  1   2   3 , Shi-Gang Zhao  1   2   3 , Dai-Min Wei  1   2   3 , Yue-Ran Zhao  1   2   3 , Tao Huang  1   2   3 , Tahir Muhammad  1   2   3 , Lei Yan  1   2   3 , Fei Gao  4 , Lei Li  1   2   3   5 , Gang Lu  1   2   3   6 , Wai-Yee Chan  6 , Peter C K Leung  7 , Andrea Dunaif  8 , Hong-Bin Liu  1   2   3   6 , Zi-Jiang Chen  1   2   3
Affiliations

The HMGA2-IMP2 Pathway Promotes Granulosa Cell Proliferation in Polycystic Ovary Syndrome

Miao Li et al. J Clin Endocrinol Metab. .

Abstract

Context: The high mobility group AT hook 2 (HMGA2) gene was previously identified in a genome-wide association study as a candidate risk gene that might be related to polycystic ovary syndrome (PCOS). Whether HMGA2 contributes to promoting granulosa cell (GC) proliferation in PCOS remains unknown.

Objective: We sought to determine whether HMGA2 is involved in the ovarian dysfunction of PCOS and in the mechanism of increased GC proliferation.

Patients and cells: mRNA expression was analyzed in ovarian GCs from 96 women with PCOS and 58 healthy controls. Immortalized human GCs (KGN and SVOG cells) were used for the mechanism study.

Main outcome measures: mRNA expression in ovarian GCs was measured using quantitative RT-PCR, and KGN cells were cultured for proliferation assays after overexpression or knockdown of target genes. Protein expression analysis, luciferase assays, and RNA binding protein immunoprecipitation assays were used to confirm the mechanism study.

Results: HMGA2 and IGF2 mRNA binding protein 2 (IMP2) were highly expressed in the GCs of women with PCOS, and the HMGA2/IMP2 pathway promoted GC proliferation. Cyclin D2 and SERPINE1 mRNA binding protein 1 were regulated by IMP2 and were highly expressed in women with PCOS.

Conclusions: The HMGA2/IMP2 pathway was activated in women with PCOS and promoted the proliferation of GCs. This might provide new insights into the dysfunction of GCs in PCOS.

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Figures

Figure 1.
Figure 1.
HMGA2 expression in PCOS GCs. (A) HMGA2 protein was localized to the nuclei of mouse GCs. Left panel, staining with anti-HMGA2 antibody; middle panel, staining with 4′,6-diamidino-2-phenylindole (DAPI); right panel, merged staining. Scale bar, 25 μm. (B, C) Comparison of HMGA2 (B) mRNA and (C) protein expression in control and PCOS GCs. HMGA2 levels were higher in women with PCOS than in controls. Data were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Ctrl, control. *P < 0.05. **P < 0.01.
Figure 2.
Figure 2.
Effects of HMGA2 on GC proliferation. (A, B) CCK8 assay of control and experimental cells in which HMGA2 was downregulated or upregulated. (A) Knockdown of HMGA2 reduced cell proliferation, whereas (B) overexpression of HMGA2 increased cell proliferation. Each value represents the mean of three independent experiments. *P < 0.05. **P < 0.01. (C–E) Flow cytometry assay of GC cell cycle progression under (D) control and (E) knockdown conditions. (C) Knockdown of HMGA2 expression resulted in an increased proportion of G1-phase cells and a decreased proportion of S-phase cells. Ctrl, control; PI-A, propidium iodide-annexin V.
Figure 3.
Figure 3.
Correlation between the expression of HMGA2 and IMP2 in PCOS GCs. (A) HMGA2 and IMP2 mRNA levels were correlated in GCs from women with PCOS. R2 = 0.6773. (B, C) IMP2 mRNA and protein expression in control and PCOS GCs. IMP2 levels were higher in PCOS GCs than in control cells. Data were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). *P < 0.05. **P < 0.01. (D, E) Western blots of IMP2 expression in KGN cells. (D) Knockdown of HMGA2 reduced IMP2 levels, whereas (E) overexpression of HMGA2 increased IMP2 levels. Ctrl, control.
Figure 4.
Figure 4.
Effects of IMP2 on GC proliferation. (A) Localization of IMP2 in GCs. IMP2 protein was localized to the cytoplasm of mouse GCs. Left panel, staining with anti-IMP2 antibody; middle panel, staining with 4′,6-diamidino-2-phenylindole (DAPI); right panel, merged staining. Scale bar, 25 μm. (B, C) CCK8 assay of control and experimental cells in which IMP2 was downregulated or upregulated. (B) Knockdown of IMP2 reduced cell proliferation, (C) whereas overexpression of IMP2 increased cell proliferation. **P < 0.01. (D) Flow cytometry assay of the cell cycle phase of GCs under control and knockdown conditions. Knockdown of IMP2 expression resulted in an increased proportion of G1-phase cells and a decreased proportion of S-phase cells. Each value represents the mean of at least three independent experiments. Ctrl, control.
Figure 5.
Figure 5.
CCND2 and SERBP1 expression in the HMGA2-IMP2 pathway. (A, B) IMP2 binding sites in the CCND2 and SERBP1 genes. IMP2 bound to CCND2 and SERBP1 mRNAs. (C) RIP experiments in HEK293 cells. CCND2 and SERBP1 proteins were pulled down using anti-IMP2 antibodies. IgG and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as negative controls. (D) Knockdown of HMGA2 in KGN cells reduced the CCND2 and SERBP1 protein levels. (E) Knockdown of IMP2 reduced the CCND2 and SERBP1 protein levels in KGN cells. (F, G) Luciferase assays of IMP2 and downstream genes (F, CCND2; G, SERBP1) in KGN cells. In both cases, a dose-dependent increase in luciferase activity of CCND2 and SERBP1 was observed as IMP2 expression increased. *P < 0.05. **P < 0.01. Each value represents the mean of at least three independent experiments. Ctrl, control.
Figure 6.
Figure 6.
CCND2 and SERBP1 mRNA and protein expression in control and PCOS GCs. (A, B) CCND2 mRNA and protein expression was higher in PCOS GCs than in control cells. Data were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). **P < 0.01. (C, D) SERBP1 mRNA and protein expression was higher in PCOS GCs than in control cells. Data were normalized to GAPDH. **P < 0.01. Ctrl, control.
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