lnc-MAP3K13-7:1 Inhibits Ovarian GC Proliferation in PCOS via DNMT1 Downregulation-Mediated CDKN1A Promoter Hypomethylation

doi:10.1016/j.ymthe.2020.11.018

. 2021 Mar 3;29(3):1279-1293.

doi: 10.1016/j.ymthe.2020.11.018. Epub 2020 Nov 17.

lnc-MAP3K13-7:1 Inhibits Ovarian GC Proliferation in PCOS via DNMT1 Downregulation-Mediated CDKN1A Promoter Hypomethylation

Xueying Geng¹, Jun Zhao¹, Jiayu Huang¹, Shang Li¹, Weiwei Chu¹, Wang-Sheng Wang¹, Zi-Jiang Chen², Yanzhi Du³

Affiliations

¹ Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200135, China; Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai 200135, China.
² Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200135, China; Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai 200135, China; Center for Reproductive Medicine, Cheeloo College of Medicine, Shandong University, Jinan, Shandong 250012, China; National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, Shandong 250012, China; Key Laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, Shandong 250012, China.
³ Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200135, China; Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai 200135, China. Electronic address: duyz@sjtu.edu.cn.

PMID: 33212300
PMCID: PMC7934583
DOI: 10.1016/j.ymthe.2020.11.018

lnc-MAP3K13-7:1 Inhibits Ovarian GC Proliferation in PCOS via DNMT1 Downregulation-Mediated CDKN1A Promoter Hypomethylation

Xueying Geng et al. Mol Ther. 2021.

. 2021 Mar 3;29(3):1279-1293.

doi: 10.1016/j.ymthe.2020.11.018. Epub 2020 Nov 17.

Authors

Xueying Geng¹, Jun Zhao¹, Jiayu Huang¹, Shang Li¹, Weiwei Chu¹, Wang-Sheng Wang¹, Zi-Jiang Chen², Yanzhi Du³

Affiliations

¹ Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200135, China; Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai 200135, China.
² Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200135, China; Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai 200135, China; Center for Reproductive Medicine, Cheeloo College of Medicine, Shandong University, Jinan, Shandong 250012, China; National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, Shandong 250012, China; Key Laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, Shandong 250012, China.
³ Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200135, China; Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai 200135, China. Electronic address: duyz@sjtu.edu.cn.

PMID: 33212300
PMCID: PMC7934583
DOI: 10.1016/j.ymthe.2020.11.018

Abstract

Polycystic ovary syndrome (PCOS) is an endocrine-related disease and global cause of infertility that is associated with abnormal folliculogenesis. Inhibited granulosa cell (GC) proliferation is recognized as a key factor that underlies aberrant follicle maturation. Many epigenetic landscape modifications have been characterized in PCOS patients. However, the epigenetic regulation pathways in follicular dysplasia are not completely understood. In this study, we reported a novel mechanism of DNA hypomethylation induced by long non-coding RNAs (lncRNAs) and its function in cell cycle progression. We observed that lnc-MAP3K13-7:1 was highly expressed in GCs from patients with PCOS, with concomitant global DNA hypomethylation, decreased DNA methyltransferase 1 (DNMT1) expression, and increased cyclin-dependent kinase inhibitor 1A (CDKN1A, p21) expression. In KGN cells, lnc-MAP3K13-7:1 overexpression resulted in cell cycle arrest in the G₀/G₁ phase, as well as the molecular inhibition and genetic silencing of DNMT1. Mechanistically, lnc-MAP3K13-7:1 inhibited DNMT1 expression by acting as a protein-binding scaffold and inducing ubiquitin-mediated DNMT1 protein degradation. Moreover, DNMT1-dependent CDKN1A promoter hypomethylation increased CDKN1A transcription, resulting in attenuated GC growth. Our work uncovered a novel and essential mechanism through which lnc-MAP3K13-7:1-dependent DNMT1 inhibition regulates CDKN1A/p21 expression and inhibits GC proliferation.

Keywords: DNA methylation; DNMT1; PCOS; epigenetics; granulosa cell proliferation; lncRNA.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Figure 1**
lnc-MAP3K13-7:1 Is Upregulated in Polycystic Ovary Syndrome (PCOS) and Associated with DNA Methyltransferase 1 (DNMT1) Levels (A) Quantitative real-time PCR detection of lnc-MAP3K13-7:1 expression levels in ovarian granulosa cells (GCs) from 42 patients with PCOS and 42 normal controls (p = 0.0002). (B) Global DNA methylation of genomic DNA sequences from women with PCOS and normal controls. For each group, the whiskers indicate the 10th and 90th percentiles, the central solid line indicates the median, and the mean for each group is noted by “+” in each box (p < 0.0001). (C and D) Quantitative real-time PCR detection of *DNMT1* (C) and *DNMT3A* (D) expression in the same cohort described in (A) (*DNMT1*, p = 0.0018; *DNMT3A*, p = 0.0173). (E) Western blot analysis of DNMT1 protein expression in GCs from 18 patients with PCOS and 18 normal controls (p = 0.0171). The 36 samples were derived from the same experiment. In (A)–(E), data are presented as the mean ± SD. In (A), (C), and (E), ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (Student’s t test). In B, ∗∗∗p < 0.001 (Mann-Whitney U test).

**Figure 2**
lnc-MAP3K13-7:1 Upregulation Decreased DNMT1 Protein Levels and Inhibited GC Proliferation *In Vitro* (A) Quantitative real-time PCR analysis of lnc-MAP3K13-7:1 overexpression using lentiviral vectors in KGN cells. Expression of lnc-MAP3K13-7:1 in stably transfected cells in comparison with that in empty vector-transfected cells (left, n = 9, p < 0.0001). Quantitative real-time PCR analysis of *DNMT1* and *DNMT3A* expression after lnc-MAP3K13-7:1 overexpression (middle and right, *DNMT1*, n = 9, p = 0.614; *DNMT3A*, n = 3, p = 0.433). (B) Global DNA methylation of KGN cells after lnc-MAP3K13-7:1 overexpression compared with that in empty vector-transfected cells (n = 3, p = 0.0864, Mann-Whitney U test). (C) Western blot analysis of DNMT1 and DNMT3A protein expression in KGN cells after lnc-MAP3K13-7:1 overexpression. The quantification of protein levels is presented at the bottom (n = 3 for each group; DNMT1, p < 0.0001; DNMT3A, p = 0.682). (D) Quantitative real-time PCR detection of cyclin-dependent kinase inhibitor 1A (*CDKN1A*) expression in GCs from 42 patients with PCOS and 42 normal controls (p = 0.0004). (E and F) Effects of lnc-MAP3K13-7:1 overexpression on KGN cell growth measured using the Cell Counting Kit-8 assay (E, n = 3, p < 0.0001) and cell cycle distribution determined using flow cytometry (F, n = 4, p < 0.0001 for each group). (G) Quantitative real-time PCR analysis of changes in proliferation-related marker expression (proliferating cell nuclear antigen [*PCNA*] as a positive regulator and *CDKN1A* and *CDKN1B* as negative regulators) 72 h after lnc-MAP3K13-7:1 lentivirus transfection (n = 6; *PCNA*, p = 0.019; *CDKN1A*, p = 0.004; *CDKN1B*, p = 0.339). (H) Western blot analysis of changes in proliferation-related marker expression (PCNA, p21, cyclin E1, CDK2, p-CDK2 [Thr160]), retinoblastoma protein [Rb], and p-Rb [Ser807/Ser811]) normalized to β-actin protein expression in lnc-MAP3K13-7:1-overexpressing KGN cells. The quantification of protein levels is presented on the right (n = 3; Rb, p = 0.156; p-Rb, p = 0.003; cyclin E1, p = 0.923; PCNA, p = 0.003; CDK2, p = 0.663; p-CDK2, p < 0.0001; p21, p = 0.007). In (A)–(C) and (E)–(H), mean ± SD. In (A) and (C)–(H), ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (Student’s t test).

**Figure 3**
The DNMT Inhibitor 5-Aza-2′-Deoxycytidine (5-Aza) Concentration-Dependently Inhibited KGN Cell Proliferation Similarly as DNMT1 Silencing (A) Western blot analysis of DNMT1 levels following 24 h of treatment with different concentrations of 5-Aza. Different letters (a, b, c) indicate significant differences (n = 4, p ≤ 0.05). (B and C) Effects of 5-Aza treatment on the cell cycle distribution of KGN cells as measured using flow cytometry (B, n = 3; G₀/G₁, p = 0.0068; S, p = 0.0004; G₂/M, p = 0.0009) and growth curves calculated using the Cell Counting Kit-8 assay (C, n = 3, p < 0.0001). (D) Western blot analysis of changes in proliferation-related marker levels (PCNA, p21, CDK2, p-CDK2 [Thr160], Rb, and p-Rb [Ser807/Ser811]) normalized to β-actin protein levels in 5-Aza-treated KGN cells (n = 3). See also Figure S5A. (E) Western blot analysis of DNMT1 silencing using three different siRNAs in KGN cells. The quantification of protein levels is presented on the right (n = 3; si-NC versus si-DNMT1-1, p = 0.005; si-NC versus si-DNMT1-2, p = 0.007; si-NC versus si-DNMT1-3, p = 0.21). (F and G) Effects of *DNMT1* silencing on the cell cycle distribution of KGN cells as measured using flow cytometry (F, n = 4) and growth curves as calculated using the Cell Counting Kit-8 assay (G, n = 3, p < 0.0001 for each group). In (G), cell viability was significantly changed by two different siRNAs compared with that in cells transfected with si-negative control (NC). (H) Western blot analysis of changes in proliferation-related marker levels (PCNA, p21, CDK2, p-CDK-2, Rb, and p-Rb) normalized to β-actin levels after DNMT1 silencing in KGN cells (n = 3). See also Figure S5B. In (A)–(H), mean ± SD. ∗∗p < 0.01, ∗∗∗p < 0.001 (Student’s t test). In (A), p < 0.05 (one-way ANOVA).

**Figure 4**
Expression of Exogenous DNMT1 Rescued lnc-MAP3K13-7:1-Induced Cell Cycle Arrest (A) Western blot analysis of DNMT1 and proliferation-related marker expression (PCNA, p21, CDK2, p-CDK2, Rb, and p-Rb) normalized to β-actin protein levels after DNMT1 overexpression in KGN cells (n = 3; DNMT1, p = 0.0002; p-Rb, p = 0.002; Rb, p = 0.7602; PCNA, p = 0.0266; p-CDK2, p = 0.0076; CDK2, p = 0.187; p21, p = 0.0207). (B and C) Cell cycle distribution (B) and cell growth curves (C) after exogenous *DNMT1* expression in lnc-MAP3K13-7:1-overexpressing cells. The analysis was performed using Duncan’s multiple range test. Different letters (a, b, c, d) in the same column indicate significant differences (p < 0.05; B, n = 3; C, n = 8). (D) Effects of exogenous *DNMT1* expression on protein expression in lnc-MAP3K13-7:1-overexpressing cells. DNMT1 overexpression rescued lnc-MAP3K13-7:1 overexpression-induced protein downregulation. The analysis was performed using Duncan’s multiple range test. Different letters (a, b, c, d) in the same column indicate significant differences (p < 0.05, n = 3). In (A)–(D), data are representative of three independent experiments. Mean ± SD. In (A), ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (Student’s t test). In (B)–(D), p < 0.05 (one-way ANOVA).

**Figure 5**
DNMT1 Silencing or 5-Aza Treatment Upregulated *CDKN1A* Expression by Decreasing Its Promoter Methylation Levels (A and B) Quantitative real-time PCR analysis of *DNMT1* and negative proliferation marker levels (*CDKN1A*, *CDKN1B*, *TP53*) after *DNMT1* silencing using two different siRNAs (A, n = 6 for each group; *DNMT1*, p < 0.0001; *CDKN1A*: si-NC versus si-DNMT1-1, p = 0.0004; si-NC versus si-DNMT1-2, p = 0.003; *CDKN1B*: si-NC versus si-DNMT1-2, p = 0.0012) or after 5-Aza treatment (5 μM) for different durations (B, n = 3 for each group; *DNMT1*: 12 h, p = 0.0022, 24 h, p = 0.0057; *CDKN1A*: 24 h, p = 0.0035, 48 h, p < 0.0001; *CDKN1B*: 24 h, p = 0.0145; *TP53*: 24 h, p = 0.0324, 48 h, p = 0.0179). (C) The selected pyrosequencing sequence was located upstream of *CDKN1A* exon 1 and had 16 CpG sites named pos1–pos16. CpG island enrichment in the *CDKN1A* promoter region was predicted by the MethPrimer website, which was located in the promoter and first exon regions of *CDKN1A*. (D and E) Methylation status of the *CDKN1A* promoter CpG island after DNMT1 silencing using two different siRNAs (D, n = 6, pos2, p = 0.0073) or after 5-Aza treatment (5 μM) for different durations (E, n = 5, pos9, p = 0.0166). Pos, CpG site in the *CDKN1A* promoter; Meth, methylation level. Mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (Student’s t test).

**Figure 6**
lnc-MAP3K13-7:1 Directly Bound DNMT1 and Facilitated DNMT1 Ubiquitination and Translocation (A) Quantitative real-time PCR analysis of nuclear and cytoplasmic *DNMT1* expression in lnc-MAP3K13-7:1-overexpressing KGN cells using an RNA cellular fractionation assay. *ACTIN* and U6 were used as cytoplasmic and nuclear controls, respectively (n = 4). (B) Representative images of DNMT1 protein expression (red) in lnc-MAP3K13-7:1-overexpressing KGN cells and control cells using immunofluorescence staining. Images are representative of three independent experiments. Nuclei were counterstained with DAPI. Scale bars, 100 μm. (C) Western blot analysis of DNMT1 protein expression in different subcellular fractionations in lnc-MAP3K13-7:1-overexpressing KGN cells. Glyceraldehyde-3-phosphate dehydrogenase and lamin A/C served as cytoplasmic and nuclear markers, respectively. The quantification of protein expression is presented on the right. Different letters (a, b, c) in the same column indicate significant differences (p < 0.05, n = 3). (D) DNMT1 protein degradation was observed after 8 h of exposure to high concentrations (100 μM) of the proteasome inhibitor MG132 but not after treatment with low concentrations (1 and 10 μM) (n = 4; DMSO, p = 0.003; MG132, 1 μm, p = 0.004; MG132, 10 μm, p = 0.0008). (E) Stabilization of DNMT1 protein expression following treatment with cycloheximide (CHX). KGN cells were transfected with vector (control) or lnc-MAP3K13-7:1 overexpression plasmids and treated with 100 μM CHX for the indicated times. β-Actin was used as a loading control. DNMT1 levels were quantified and expressed as percentages relative to the control (n = 3, 10 h, p = 0.0406). (F) Immunoprecipitation assay of DNMT1-ubiquitin in KGN cells. KGN cells were transfected with lnc-MAP3K13-7:1 lentiviral vectors and treated with 100 μM MG132. Whole-cell lysates were prepared and subjected to immunoprecipitation using anti-DNMT1 antibodies. Immunoprecipitates were probed to detect the polyubiquitination of DNMT1 using anti-ubiquitin antibodies. Approximately 5% of the cell lysates used for co-immunoprecipitation were loaded as the inputs. Immunoglobulin G (IgG) was used as a control (n = 4, p = 0.0028). (G) Quantitative real-time PCR analysis of lnc-MAP3K13-7:1 enrichment in DNMT1 RNA immunoprecipitates in lnc-MAP3K13-7:1-overexpressing KGN cells. IgG was used as a negative control, and EZH2 was used as a positive control. Nuclear paraspeckle assembly transcript 1 was used as a negative control, and uridine-rich 1 small nuclear RNA was used as positive controls for quantitative real-time PCR (n = 6 for DNMT1 primers, n = 3 for controls). In (A) and (D)–(G), data are presented as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (Student’s t test). In (C), data are presented as the mean ± SD. p < 0.05 (one-way ANOVA).

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References

1. Bozdag G., Mumusoglu S., Zengin D., Karabulut E., Yildiz B.O. The prevalence and phenotypic features of polycystic ovary syndrome: a systematic review and meta-analysis. Hum. Reprod. 2016;31:2841–2855. - PubMed
1. Skiba M.A., Islam R.M., Bell R.J., Davis S.R. Understanding variation in prevalence estimates of polycystic ovary syndrome: a systematic review and meta-analysis. Hum. Reprod. Update. 2018;24:694–709. - PubMed
1. Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Fertil. Steril. 2004;81:19–25. - PubMed
1. Franks S., Stark J., Hardy K. Follicle dynamics and anovulation in polycystic ovary syndrome. Hum. Reprod. Update. 2008;14:367–378. - PubMed
1. Webber L.J., Stubbs S., Stark J., Trew G.H., Margara R., Hardy K., Franks S. Formation and early development of follicles in the polycystic ovary. Lancet. 2003;362:1017–1021. - PubMed

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[1] Bozdag G., Mumusoglu S., Zengin D., Karabulut E., Yildiz B.O. The prevalence and phenotypic features of polycystic ovary syndrome: a systematic review and meta-analysis. Hum. Reprod. 2016;31:2841–2855. - PubMed

[2] Bozdag G., Mumusoglu S., Zengin D., Karabulut E., Yildiz B.O. The prevalence and phenotypic features of polycystic ovary syndrome: a systematic review and meta-analysis. Hum. Reprod. 2016;31:2841–2855. - PubMed

[3] Skiba M.A., Islam R.M., Bell R.J., Davis S.R. Understanding variation in prevalence estimates of polycystic ovary syndrome: a systematic review and meta-analysis. Hum. Reprod. Update. 2018;24:694–709. - PubMed

[4] Skiba M.A., Islam R.M., Bell R.J., Davis S.R. Understanding variation in prevalence estimates of polycystic ovary syndrome: a systematic review and meta-analysis. Hum. Reprod. Update. 2018;24:694–709. - PubMed

[5] Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Fertil. Steril. 2004;81:19–25. - PubMed

[6] Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Fertil. Steril. 2004;81:19–25. - PubMed

[7] Franks S., Stark J., Hardy K. Follicle dynamics and anovulation in polycystic ovary syndrome. Hum. Reprod. Update. 2008;14:367–378. - PubMed

[8] Franks S., Stark J., Hardy K. Follicle dynamics and anovulation in polycystic ovary syndrome. Hum. Reprod. Update. 2008;14:367–378. - PubMed

[9] Webber L.J., Stubbs S., Stark J., Trew G.H., Margara R., Hardy K., Franks S. Formation and early development of follicles in the polycystic ovary. Lancet. 2003;362:1017–1021. - PubMed

[10] Webber L.J., Stubbs S., Stark J., Trew G.H., Margara R., Hardy K., Franks S. Formation and early development of follicles in the polycystic ovary. Lancet. 2003;362:1017–1021. - PubMed

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lnc-MAP3K13-7:1 Inhibits Ovarian GC Proliferation in PCOS via DNMT1 Downregulation-Mediated CDKN1A Promoter Hypomethylation

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lnc-MAP3K13-7:1 Inhibits Ovarian GC Proliferation in PCOS via DNMT1 Downregulation-Mediated CDKN1A Promoter Hypomethylation

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