# LncRNA RWDD3 Facilitates Leydig Cell Steroidogenesis by Regulating the miR-1388-5p/NPY1R/cAMP Pathway in Yanshan Cashmere Goats

## TL;DR

This study identifies a new long non-coding RNA, lncRWDD3, that helps regulate testosterone production in cashmere goats by interacting with miR-1388-5p and the NPY1R/cAMP pathway.

## Contribution

The discovery of lncRWDD3 as a ceRNA that regulates the miR-1388-5p/NPY1R/cAMP pathway in Leydig cells of cashmere goats.

## Key findings

- LncRWDD3 acts as a ceRNA by sponging miR-1388-5p to regulate NPY1R expression.
- Prolactin at 200 ng/mL maximizes testosterone secretion in Leydig cells.
- Overexpression of lncRWDD3 or NPY1R increases cAMP levels and testosterone production.

## Abstract

The underlying mechanisms of prolactin-regulated testosterone secretion and the roles of long non-coding RNAs (lncRNAs) in this process remain poorly understood. We performed a comprehensive analysis of the testicular tissues of cashmere goats with different prolactin levels via RNA sequencing and constructed an lncRNA–miRNA–mRNA interaction network. We identified a novel lncRNA named lncRWDD3 and investigated its effects on the testosterone secretion of goat Leydig cells. We found that 200 ng/mL of prolactin achieved the highest testosterone secretion and that lncRWDD3 acts on miR-1388-5p as a competing endogenous RNA (ceRNA). In addition, neuropeptide Y receptor Y1 (NPY1R) was proven to be a target of miR-1388-5p. Our study shows that prolactin can regulate testicular function through the ceRNA network and that the novel lncRWDD3 acts as a sponge for chi-miR-1388-5p to activate the NPY1R/cAMP pathway, thereby facilitating Leydig cell steroidogenesis.

Prolactin is a polypeptide hormone that plays a critical role in male reproduction. However, the underlying mechanisms of prolactin-regulated testosterone secretion and the roles of lncRNAs in this process remain unclear. We performed a comprehensive analysis of the testicular tissues of cashmere goats with different prolactin levels by means of RNA-sequencing. Then, we constructed a lncRNA–miRNA–mRNA interaction network by integrating previously submitted testicular mRNA sequencing data. We identified a novel lncRNA named lncRWDD3 and investigated its effects on testosterone synthesis in the Leydig cells of cashmere goat. The primary Leydig cells were used to explore the biological function of lncRWDD3/miR-1388-5p/NPY1R in vitro. This study found that 200 ng/mL of prolactin achieved the highest testosterone secretion in Leydig cells. LncRWDD3 or NPY1R overexpression promoted cAMP levels, testosterone secretion, and related gene expression, while lncRWDD3 or NPY1R interference had the opposite effect. It was found that lncRWDD3 acts on miR-1388-5p as a ceRNA, and neuropeptide Y receptor Y1 (NPY1R) was confirmed to be a target of chi-miR-1388-5p. Our research shows that prolactin regulates the testicular function of cashmere goats via the lncRNA–miRNA–mRNA ceRNA network, and lncRWDD3 acts as a ceRNA to activate NPY1R/cAMP signaling via the sponging of miR-1388-5p in order to govern testosterone synthesis in the Leydig cells of cashmere goats. Our results provide insights for future studies on the molecular mechanism of the prolactin regulation of testicular function in goats.