Several retrospective studies reported PCOS as one of the most common comorbidities of COVID-19 26. However, why PCOS patients are susceptible to COVID-19 remains uncertain. Therefore, understanding the molecular mechanisms, early diagnosis and intervention is of great clinical importance. In this work, we performed bioinformatics analyses on two independent gene chip databases containing SARS-CoV-2 infected NHBE and PCOS granulosa cells and identified 27 common DEGs.
According to GO, KEGG and GSEA enrichment analysis, the DEGs were enriched primarily in immune response, NF-kappaB transcription factor activity regulation, Toll-like receptor (TLR) binding, virus infection, TNF signaling pathway, IL-17 signaling pathway and NOD-like receptor signaling pathway. These findings suggest that COVID-19 and PCOS have DEGs associated with inflammation and immune response. It has also been reported that severe COVID-19 triggers an exaggerated inflammatory response that can lead to acute respiratory distress syndrome, a life-threatening condition associated with multi-organ failure and high mortality 27. By Pearson correlation analysis, we found that 7 hub genes were significantly positively correlated with immune scores, implying that they are associated with immune infiltration. Hub gene expression heatmaps showed that hub gene expression was low in innate immune cells such as monocytes, activated NK cells, and neutrophils. Most of the hub genes were lower expressed in M2 macrophages, whereas highly expressed in M1 macrophages. As is well known, M2 macrophages suppress inflammation while M1 macrophages promote it 28. The differential expression of hub genes in macrophages may be a common pathogenic cause of PCOS and SRAS-Cov-2. Furthermore, in the training set, validation set, animal models and clinical samples, the expression of hub genes was significantly higher in the PCOS and SARS-Cov-2 groups than in the control group. This suggests that high expression of these genes is associated with the development of inflammation. Recent findings have indicated that the periovulatory follicles of women with PCOS contain elevated inflammatory mediators and that this low-grade inflammation might serve as a precursor to ovarian dysfunction in PCOS 29. This underlying proinflammatory predisposition may put women with PCOS at increased risk of severe COVID-19 30.
Based on the MCODE and seven algorithms of CytoHubba, we screened seven hub genes, including S100A9, TLR2, CD86, ICAM, THBD, MMP9, and ITGB2. TLR2 was a key hub gene among the hub genes. During a viral infection, the host uses pattern recognition receptors, especially TLRs, to sense the virus and activate the innate immune system 31. TLR2 was thought to be one of the most significant members of the TLR family and is responsible for sustaining airway inflammation 32. In line with this, GO and KEGG Pathway identified significant enrichment in signaling pathways involving toll-like receptors. In addition, COVID-19 severity was associated with TLR2 expression. Coronavirus infection caused proinflammatory cytokines to be produced independently of viral entry via TLR2-dependent signaling. TLR2 sensed the SARS-CoV-2 envelope protein as its ligand 33,34.
Besides, there was evidence that TLRs are located in ovary granulosa cells, cumulus cells, and theca cells 35. The abnormal expression of TLRs did not result in good oocyte quality and insufficient fertility 36. In another study, TLR2 was found to be highly expressed in granulosa cells from PCOS patients and could mediate inflammation and oxidative stress caused by LPS 37. These results showed that TLR2 was a key molecule mediating inflammation in COVID-19 and PCOS. We speculate that the state of chronic inflammation in PCOS primarily mediates the susceptibility of affected patients to COVID-19. Other hub genes were also associated with inflammatory pathways or metabolic pathways, which were involved in PCOS and became potential risk factors for susceptibility to COVID-19 38–40.
This study also revealed the interaction of hub genes with ovary-specific genes. GO and KEGG Pathway analyses showed significant enrichment in inflammatory pathway, ovarian follicle development and insulin signaling pathway. According to our findings in the PPI network, TLR2 interacted with MYD88, TLR1, IRF3, and other key players in the inflammatory response signaling pathway 41,42. S100A9 could potentially interact with S100A8, and its complex was believed to facilitate cyst migration in PCOS development 43. S100A9 could increase the production of inflammatory cytokines and disturb the steroidogenesis of PCOS 44. Researchers have reported that S100A8 and S100A9 have diagnostic biomarker values and can be used to identify COVID-19 patients admitted to intensive care units 45. Hence, S100A8 and S100A9 appeared to have important roles in causing COVID-19, as well as immune responses.
In addition, we also found that EGFR proteins had direct or indirect interactions with S100A9, TLR2, ITGB and ICAM1. As a critical factor in cell growth, differentiation, implantation, and decidualization, EGFR was vital for reproduction 46. It is believed that the EGFR was responsible for facilitating proliferation and inhibiting apoptosis of granulosa cells by activating the MAPK/ERK signaling pathway and inducing transcription factor AP1 expression 47. According to other studies, EGFR was expressed by cells of the lungs after SARS-CoV-2 infection. An elevated level of EGFR expression can further exacerbate pulmonary disease and cause fibrosis. Nimotuzumab can block the EGFR, which could be a novel treatment strategy for COVID-19 48.
We have identified micro-RNA species has-miR-21-5p, has-miR-335-5p, has-miR-146a-5p and has-miR-143-5p shared by the hub genes. These micro-RNAs have been linked to the development of PCOS 49–51. Among them, has-miR-335-5p and has-miR-146a-5p were demonstrated to be involved in the DEGs-miRNA network regulation in COVID-19 52. In particular, our previous study displayed that miR-335-5p may function as a mediator in the etiopathogenesis of PCOS and has the potential as both a novel diagnostic biomarker and therapeutic target for PCOS 11. Establishing hub gene-miRNA network might provide new insights into the pathogenesis of PCOS and COVID-19.
We also identified TFs closely associated with the hub genes. For instance, we identified STAT3 that participated in both TLR2 and S100A9 transcription. STAT3 has been shown to regulate the expression of TLR2 and S100A9. Given the roles of TLR2 and SA100A9 discussed above, we speculated that STAT3 might have the same function in PCOS and COVID-19 53. Additionally, our previous study found that STAT3 could act directly on the miR-27a-3p promoter to induce granulosa cell apoptosis during the development of PCOS 24.
The drugs and chemical compounds found in our analysis include resveratrol, lipopolysaccharides, methotrexate, nickel, tretinoin, Captopril and Glucosamine. For instance, tretinoin has been stipulated to regulate steroid biosynthesis in human ovarian theca cells 54. In addition, resveratrol was used to regulate inflammation and oxidative stress of granulosa cells in PCOS via targeting TLR2, which was consistent with our above finding of TLR2 as a hub gene 55.