DNA Double-Strand Break-Related Competitive Endogenous RNA Network of Noncoding RNA in Bovine Cumulus Cells

Abstract

(1) Background: DNA double strand breaks (DSBs) are the most serious form of DNA damage that affects oocyte maturation and the physiological state of follicles and ovaries. Non-coding RNAs (ncRNAs) play a crucial role in DNA damage and repair. This study aims to analyze and establish the network of ncRNAs when DSB occurs and provide new ideas for next research on the mechanism of cumulus DSB. (2) Methods: Bovine cumulus cells (CCs) were treated with bleomycin (BLM) to construct a DSB model. We detected the changes of the cell cycle, cell viability, and apoptosis to determine the effect of DSBs on cell biology, and further evaluated the relationship between the transcriptome and competitive endogenous RNA (ceRNA) network and DSBs. (3) Results: BLM increased γH2AX positivity in CCs, disrupted the G1/S phase, and decreased cell viability. Totals of 848 mRNAs, 75 long noncoding RNAs (lncRNAs), 68 circular RNAs (circRNAs), and 71 microRNAs (miRNAs) in 78 groups of lncRNA–miRNA–mRNA regulatory networks, 275 groups of circRNA–miRNA–mRNA regulatory networks, and five groups of lncRNA/circRNA–miRNA–mRNA co-expression regulatory networks were related to DSBs. Most differentially expressed ncRNAs were annotated to cell cycle, p53, PI3K-AKT, and WNT signaling pathways. (4) Conclusions: The ceRNA network helps to understand the effects of DNA DSBs activation and remission on the biological function of CCs.

miR-302d Targeting of CDKN1A Regulates DNA Damage and Steroid Hormone Secretion in Bovine Cumulus Cells

“…The dual luciferase reporter assay showed that miR-302d mimics remarkably inhibited the expression of CDKN1A WT (Figure 3e). opposite to the trend of CDKN1A expression (Figure 1b) and was also in agreement with the results of earlier studies [24]. Transfection of bovine cumulus cells with miR-302d mimics enhanced miR-302d expression and significantly diminished CDKN1A expression (p < 0.01, Figure 3c,d).…”

“…TargetScan database predictions illustrate that miR-302d targets binding to the CDKN1A 3 UTR at positions 614 to 620 (Figure 3a). BLM treatment of bovine cumulus cells resulted in a significant down regulation of miR-302d expression (p < 0.01, Figure 3b), which was opposite to the trend of CDKN1A expression (Figure 1b) and was also in agreement with the results of earlier studies [24]. Transfection of bovine cumulus cells with miR-302d mimics enhanced miR-302d expression and significantly diminished CDKN1A expression (p < 0.01, Figure 3c,d).…”

“…Collection and culture of bovine CCs were performed in the same way as previously described [24]. In short, the ovaries were taken from healthy cows from Changchun Haoyue Halal Meat Products Co., Ltd.* (Changchun, Jilin, China) and transferred to the laboratory within 1 h. Follicular fluid containing COCs in follicles 3-8 mm in diameter was obtained using a 10 mL syringe.…”

“…Mammalian cell cycle progression is regulated by CDKs and the regulatory subunit cyclins, cell cycle progression is triggered by partial phosphorylation of Rb by CDK-Cyclin, and CDKN1A disrupts this interaction and affects cell cycle progression and cell proliferation [22,23]. Earlier studies showed that miR-302d and CDKN1A were abnormally expressed during DNA damage in bovine cumulus cells [24]. This indicated that CDKN1A and miR-302d may be potential markers of DNA damage.…”

Knockdown of KDM5B Leads to DNA Damage and Cell Cycle Arrest in Granulosa Cells via MTF1

“…It has been reported that reactive oxygen species (ROS) causes adverse changes, such as proliferation arrest, aging, apoptosis and necrosis [ 17 ]. Recently, there were research studies pointing out that chloroquine increased ROS in ovarian cancer cell lines, causing DSBs [ 18 , 19 ]. The primary sources of ROS were mitochondria and plasma membrane [ 20 ].…”