Long non-coding RNA TCONS_00814106 regulates porcine granulosa cell proliferation and apoptosis by sponging miR-1343

https://doi.org/10.1016/j.mce.2020.111064Get rights and content

Highlights

  • The long non-coding RNA, TCONS_00814106, regulates reproductive processes in pigs.

  • TCONS_00814106 regulates cell function by sponging miR-1343.

  • TCONS_00814106 serves as a competitive endogenous RNA for miR-1343.

Abstract

Recent evidence shows that long non-coding RNAs (lncRNAs), a class of non-coding RNAs, are involved in the regulation of reproductive processes. In this study, we identified a lncRNA, TCONS_00814106, that was upregulated in high-fecundity sow ovarian tissues and influenced by reproductive hormones. Bioinformatics analyses and luciferase reporter assays showed that TCONS_00814106 is a miR-1343 target. Cell counting kit (CCK)-8 and apoptosis assays showed that TCONS_00814106 promotes proliferation and inhibits apoptosis in porcine granulosa cells (GCs), and that this could be reversed by miR-1343. Also, we observed that transforming growth factor-β receptor type I (TGFBR1) is a functional target of miR-1343 in GCs. TCONS_00814106 serves as a competing endogenous RNA to regulate TGFBR1 expression by sponging miR-1343, thereby exerting regulatory functions in GCs. Overall, these results provide new insights into the biological function of the lncRNA TCONS_00814106.

Introduction

Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs that are over 200 nucleotides in length and ubiquitous in various mammalian tissues (Ponting et al., 2009; Volders et al., 2013; Mas-Ponte et al., 2017; Hu et al., 2018). As epigenetic regulators, lncRNAs possess numerous functions (Bánfai et al., 2012). Moreover, mounting evidence indicates that lncRNAs may serve as competing endogenous RNAs (ceRNAs) for miRNAs, thus regulating target gene expression. Tao et al. (2017) reported that the lncRNA GAS5 functions as a ceRNA for miR-21, which controls cardiac fibroblast activation and fibrosis via the PTEN/MMP-2 signaling pathway. Song et al. (2017) have demonstrated that lncRNA-KRTAP5-AS1 and lncRNA-TUBB2A serve as ceRNAs to regulate claudin-4 expression by sponging miR-3620–3p in gastric cancer. Also, lncRNA-MEG3 has been reported to enhance bovine skeletal muscle differentiation by sponging miR-135 and regulating MEF2C function (Liu et al., 2019). Taken together, these reports suggest that some lncRNAs function as miRNA sponges to regulate protein-coding transcripts indirectly.

In recent years, increased attention has been focused on lncRNAs. This is because lncRNAs play crucial roles in multiple biological processes (Song et al., 2017; Liang et al., 2018; Liu et al., 2018, 2019; Liu et al., 2018a, Liu et al., 2018b; Tao et al., 2017; Li et al., 2018). Notably, several articles have confirmed, using RNA sequencing, that lncRNAs are present in the ovarian tissues of pigs (Tang et al., 2017; Liu et al., 2018a, Liu et al., 2018b) and sheep (Miao et al., 2016). Differentially expressed lncRNAs have been identified in the ovaries of Duroc pigs at different follicular stages (Liu et al., 2018a, Liu et al., 2018b). Additionally, differentially expressed lncRNAs have also been identified in high-fecundity Han sheep and low-fecundity Dorset sheep, and these deregulated lncRNAs may play an important role in sheep prolificacy (Miao et al., 2016). These studies suggest that lncRNAs may be involved in reproductive processes. However, the biological functions and regulatory mechanisms of most of these lncRNAs remain relatively unknown.

In our previous studies, we obtained ovarian lncRNA expression profiles (GEO: GSE134001) of Canadian Large White sows with high (total number of piglets born, TNB > 15.73; n = 4) and low fecundity (TNB < 11.11; n = 4) in the luteal phase of the estrous cycle, using Ribo-Zero RNA-seq technology (Hu et al., 2020a, Hu et al., 2020b). Notably, the differentially expressed lncRNA TCONS_00814106 located at chromosome 7: 127,717,970–127,731,679, according to the reference genome (Sscrofa10.2), was of particular interest. The expression of lncRNA TCONS_00814106 (fold-change = 5.633) was upregulated in the high-fecundity group. In the present study, we aimed to characterize the functions and regulatory mechanisms of TCONS_00814106 in porcine follicular GCs. Our findings may help to explain the potential role of TCONS_00814106 in reproductive processes in pigs.

Section snippets

Cell culture and treatment

The animal experiments were performed in accordance with the guidelines for the care and use of experimental animals established by the Ministry of Science and Technology of the People's Republic of China (Approval Number, 2006-398), and the work was approved by Hebei Agricultural University, Baoding, China.

Fresh ovaries of cyclic sows were obtained from a local slaughterhouse and rapidly immersed in normal saline (NS) containing 1% penicillin-streptomycin at 37 °C. The ovaries were reached the

The expression of the lncRNA TCONS_00814106 is affected by FSH or hCG

To explore the effect of reproductive hormones on TCONS_00814106 in vitro, cultured GCs were treated with FSH (10 IU/mL) or hCG (5 IU/mL) for 24 h. RT-qPCR results showed that the expression levels of TCONS_00814106 were significantly increased in response to treatment with FSH or hCG (Fig. 1). These results show that TCONS_00814106 expression was influenced by FSH or hCG stimulation.

The lncRNA TCONS_00814106 functions as a miR-1343 sponge

To further elucidate the regulatory mechanism of the lncRNA TCONS_00814106, we first determined the

Discussion

In this study, we reported a differentially expressed lncRNA, TCONS_00814106, which was identified in the ovarian tissues of sows with both high and low fecundity. We found that TCONS_00814106 was upregulated in the high-fecundity sows and associated with reproductive processes. Given the significant effects of the lncRNA TCONS_00814106 on fecundity, its functions and regulatory mechanisms warrant further exploration.

In our current work, we validated the functions of TCONS_00814106 in promoting

Funding

This work was supported by the Modern Agriculture Industry Technology System Foundation of Hebei Province [grant # HBCT2018110201].

CRediT authorship contribution statement

Huiyan Hu: Conceptualization, Methodology, Software, Writing - original draft. Yanfang Fu: Data curation. Bo Zhou: Software, Validation. Zhiqiang Li: Visualization, Investigation. Zhongwu Liu: Visualization, Investigation. Qing Jia: Supervision, Writing - review & editing.

Declaration of competing interest

None.

Acknowledgments

We thank Charlesworth Author Services (www.cwauthors.com) for providing linguistic assistance during the preparation of this manuscript.

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