Characterization of Long Non-Coding RNA Profiles in Porcine Granulosa Cells of Healthy and Atretic Antral Follicles: Implications for a Potential Role in Apoptosis

Li Meng,Zhenfang Wu,Kun Zhao,Katja Teerds,Jian Tao,Shouquan Zhang,Chi Chiu Wang

doi:10.3390/ijms22052677

Abstract

Long non-coding RNAs (lncRNAs) play important roles in multiple biological processes including ovarian follicular development. Here we aimed to gain novel information regarding lncRNAs transcriptome profiles in porcine granulosa cells of advanced atretic antral (AA) and healthy antral (HA) follicles using RNA-seq. A total of 11,321 lncRNAs including 10,813 novel and 508 annotated lncRNAs were identified, of which 173 lncRNAs were differentially expressed (DE-lncRNAs); ten of these were confirmed by qRT-PCR. Gene Ontology indicated that DE-lncRNAs associated with developmental processes were highly enriched. Pathway analysis demonstrated predicted cis- and trans-targets of DE-lncRNAs. Potential mRNA targets of up-regulated DE-lncRNAs were mainly enriched in apoptosis related pathways, while targeted genes of downregulated DE-lncRNAs were primarily enriched in metabolism and ovarian steroidogenesis pathways. Linear regression analyses showed that expression of upregulated DE-lncRNAs was significantly associated with apoptosis related genes. NOVEL_00001850 is the most-downregulated DE-lncRNA (FDR = 0.04, FC = −6.53), of which miRNA binding sites were predicted. KEGG analysis of its downregulated target genes revealed that ovarian steroidogenesis was the second most highlighted pathway. qRT-PCR and linear regression analysis confirmed the expression and correlation of its potential targeted gene, CYP19A1, a key gene involved in estradiol synthesis. Our results indicate that lncRNAs may participate in granulosa cells apoptosis and thus antral follicular atresia.

Highlights

The main function of the ovary is the production of steroid hormones necessary for the development of female secondary sex characteristics, and to provide an optimal environment for oocyte maturation and release [1]
In agreement with a previous study [10], our data showed that the identified porcine granulosa cell long noncoding RNAs (lncRNAs) had a shorter total length than coding mRNA transcripts (Figure 1A) as well as reduced length of the open reading frame (Figure 1B), and possessed fewer exons (Figure 1C)
Given that our present study shows that lncRNAs are possibly involved in granulosa cells apoptosis and antral follicular atresia, we hypothesized these lncRNAs may contribute to follicle section during the follicular phase

Summary

Introduction

The main function of the ovary is the production of steroid hormones necessary for the development of female secondary sex characteristics, and to provide an optimal environment for oocyte maturation and release [1]. Ovarian follicular development is regulated by various factors including gonadotropins and growth factors all involved in the process of selecting the best follicles to ovulate [2]. Atresia can be considered a physiologically normal process, which ensures that only oocytes from the best quality follicles will ovulate, dysregulated atresia can lead to reproductive diseases such as polycystic ovary syndrome (PCOS) and premature menopause in women at reproductive age [2]. Non-protein-coding transcripts have been detected by means of next-generation sequencing, a technique with unprecedented resolution [3]. It has come as quite a surprise that less than 2% of the human genome encodes proteins; the majority of nucleotides are not transcribed into proteins, representing noncoding transcripts [4]. Among the various types of noncoding transcripts, such as miRNAs and circular RNAs (circRNAs), a class referred to as long noncoding RNAs (lncRNAs) is attracting increasing attention

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