Whole Genome Transcriptomic Analysis of Ovary Granulosa Cells Revealed an Anti-Apoptosis Regulatory Gene DLGAP5 in Polycystic Ovary Syndrome - PubMed (original) (raw)

doi: 10.3389/fendo.2022.781149. eCollection 2022.

Hu Li 2 3 4, Yi Song 1, Juan Cen 5, Yuying Zhang 6, Yi Sui 7, Dexuan Cui 8, Tin Chiu Li 1, Yan Xu 3, Chi Chiu Wang 1, Pui Wah Jacqueline Chung 1, Tao Tang 1 3

Affiliations

Whole Genome Transcriptomic Analysis of Ovary Granulosa Cells Revealed an Anti-Apoptosis Regulatory Gene DLGAP5 in Polycystic Ovary Syndrome

Yan Deng et al. Front Endocrinol (Lausanne). 2022.

Abstract

The mechanisms underlining pathogenesis of polycystic ovary syndrome (PCOS) remain largely unknown. Dysfunction of ovarian granulosa cells plays an important role. The present study performed the lncRNA and mRNA profiling by whole genome transcriptomic sequencing of ovary granulosa cells from women with PCOS and investigated the potential role of differentially expressed gens (DEGs) in the pathomechanism of PCOS. In total, 1,936 DEGs (30 upregulated and 1,906 downregulated mRNAs and lncRNAs) were identified in the ovary granulosa cells between control and PCOS group. Functional enrichment analysis showed that DEGs were mainly associated with cytokine-cytokine receptor interaction, neuroactive ligand-receptor interaction, and olfactory transduction. qRT-PCR validated the upregulation of DLGAP5 mRNA in ovary from PCOS group when compared to control group. Immunostaining and TUNEL assays showed that DLGAP5 protein level was increased while apoptosis was decreased in follicles of ovary in PCOS group. In vitro functional assays showed that DLGPA5 knockdown repressed viability and proliferation, but enhanced apoptosis and disrupted cell cycle in granulosa cells; while DLGAP5 overexpression had the opposite effects in granulosa cells. In conclusion, the study showed differentially expressed lncRNA and mRNA profile in the granulosa cells in ovaries of PCOS. Functional results demonstrated that DLGAP5 is a dysregulated candidate gene in the pathogenesis of PCOS, especially granulosa cell apoptosis and proliferation.

Keywords: DLGAP5; PCOS; apoptosis; ovary granulosa cells; transcriptomic analysis.

Copyright © 2022 Deng, Li, Song, Cen, Zhang, Sui, Cui, Li, Xu, Wang, Chung and Tang.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1

Figure 1

RNA-seq analysis of differentially expressed genes between PCOS and control group. (A) Boxplots of abundance of transcripts in each sample. (B) RPKM density distribution in each sample. (C) PCA of averaged, rank-normalized read counts from RNA-seq. (D) Heatmap analysis of differentially expressed genes between control group and PCOS group. (E) Volcano plots showing the significance and log2 fold-change (logFC) for all gene transcripts reliably detected in the RNA-seq analysis between control group and PCOS group.

Figure 2

Figure 2

Functional enrichment analysis of differentially expressed genes. (A) GO enrichment analysis of DEGs in biological process. (B) GO enrichment analysis of DEGs in cellular component. (C) GO enrichment analysis of DEGs in molecular function. (D) KEGG enrichment analysis of DEGs. DEGs, differentially expressed genes.

Figure 3

Figure 3

Alternative splicing analysis. (A) Alternative splicing types. A3SS, alternative 3’ splice site; A5SS, alternative 5’ splice site; MXE, mutually exclusive exons; RI, Retained Intron; SE, skipped exon. (B) Alternative splicing number. (C) GO enrichment analysis of alternative spliced genes in cellular component. (D) GO enrichment analysis of alternative spliced genes in molecular function. (E) GO enrichment analysis of alternative spliced genes in molecular function. (F) KEGG enrichment analysis of alternative spliced genes.

Figure 4

Figure 4

Validation analysis of selected differentially expressed candidate genes between control group and PCOS group. The mRNA expression levels of (A) LOC105379507, (B) CXCL8, (C) AREG, (D) LOC107986562, (E) CXCL2, (F) DLGAP5, (G) Lin001778, (H) LOC105379355, (I) PADI6, and (J) SNRA9 in the ovarian tissues between control group and PCOS group. N = 8–11. *P < 0.05 and **P < 0.01.

Figure 5

Figure 5

Immunostaining analysis of DLGAP5 in the ovarian tissues. (A) Immunostaining of DLGAP5 in control group. Scale bar = 200 µm. (B) Immunostaining of DLGAP5 in PCOS group. Scale bar = 200 µm. (C) Immunostaining of DLGAP5 in control group. Scale bar = 200 µm. (D) Immunostaining of DLGAP5 in PCOS group. Scale bar = 200 µm. Blue arrows indicate primary or antral follicles.

Figure 6

Figure 6

TUNEL analysis of ovarian tissues in the normal group and PCOS group. (A, C) TUNEL staining of ovarian tissues in control group. Scale bar = 200 µm. (B, D) TUNEL staining of ovarian tissues in PCOS group. Scale bar = 200 µm. Scale bar = 200 µm. Red arrows indicate primary, secondary or antral follicles.

Figure 7

Figure 7

Effects of DLGAP5 knockdown on the granulosa cell proliferation and apoptosis. Granulosa cells were transfected with si-NC or si-DLGAP5, and after siRNA transfection, (A) the mRNA expression level of DLGAP5 in the granulosa cells was determined by qRT-PCR; (B) the protein expression level of DLGAP5 in the granulosa cells was determined by western blot; (C) the cell viability of granulosa cells was determined by CCK-8 assay; (D) the cell proliferation of granulosa cells was determined by EdU assay, scale bar = 50 µm; (E) the cell apoptosis of granulosa cells was determined by TUNEL assay; scale bar = 50 µm. N = 3. **P < 0.01.

Figure 8

Figure 8

Effects of DLGAP5 knockdown on the granulosa cell apoptosis and cell cycle. Granulosa cells were transfected with si-NC or si-DLGAP5, and after siRNA transfection, (A) the cell apoptotic rates and (B) cell cycle of granulosa cells were determined by flow cytometry; (C) the protein levels of cleaved caspase-3, cleaved caspase-9, CDK2, cleaved caspase-8, cyclin C and cyclin D in granulosa cells were determined by Western blot assay. N = 3. **P < 0.01 and ***P < 0.001.

Figure 9

Figure 9

Effects of DLGAP5 overexpression on the granulosa cell proliferation and apoptosis. Granulosa cells were transfected with LV-control or LV-DLGAP5, and after transfection, (A) the mRNA expression level of DLGAP5 in the granulosa cells was determined by qRT-PCR; (B) the protein expression level of DLGAP5 in the granulosa cells was determined by western blot; (C) the cell viability of granulosa cells was determined by CCK-8 assay; (D) the cell proliferation of granulosa cells was determined by EdU assay, scale bar = 100 µm; (E) the cell apoptosis of granulosa cells was determined by TUNEL assay, scale bar = 100 µm. *P < 0.05 and **P < 0.01.

Figure 10

Figure 10

Effects of DLGAP5 overexpression on the granulosa cell apoptosis and cell cycle. Granulosa cells were transfected with LV-control or LV-DLGAP5, and after transfection, (A) the cell apoptotic rates and (B) cell cycle of granulosa cells were determined by flow cytometry; (C) the protein levels of cleaved caspase-3, cleaved caspase-9, CDK2, cleaved caspase-8, cyclin C and cyclin D in granulosa cells were determined by Western blot assay. *P < 0.05.

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References

    1. Chung PW, Chan SS, Yiu KW, Lao TT, Chung TK. Menstrual Disorders in a Paediatric and Adolescent Gynaecology Clinic: Patient Presentations and Longitudinal Outcomes. Hong Kong Med J = Xianggang Yi Xue Za Zhi (2011) 17:391–7. - PubMed
    1. Ovesen PG, Moller N, Greisen S, Ingerslev HJ. [Polycystic Ovary Syndrome Ii. Endocrinol Metabolism] Ugeskrift laeger (1998) 160:265–9. - PubMed
    1. Homburg R. Polycystic Ovary Syndrome - From Gynaecological Curiosity to Multisystem Endocrinopathy. Hum Reprod (Oxford England) (1996) 11:29–39. doi: 10.1093/oxfordjournals.humrep.a019031 - DOI - PubMed
    1. Kong GW, Cheung LP, Lok IH. Effects of Laparoscopic Ovarian Drilling in Treating Infertile Anovulatory Polycystic Ovarian Syndrome Patients With and Without Metabolic Syndrome. Hong Kong Med J = Xianggang Yi Xue Za Zhi (2011) 17:5–10. - PubMed
    1. Norman RJ, Dewailly D, Legro RS, Hickey TE. Polycystic Ovary Syndrome. Lancet (London England) (2007) 370:685–97. doi: 10.1016/S0140-6736(07)61345-2 - DOI - PubMed

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