Transcriptome Sequencing Revealed that Knockdown of FOXL2 Affects Cell Proliferation, Cycle, and DNA Replication in Chicken pre-ovulatory Follicles Cells

Forkhead box L2 (FOXL2) is a single-exon gene encoding a forkhead transcription factor, which is mainly expressed in the ovary, eyelids and pituitary gland. FOXL2 plays an essential role in ovarian development. To reveal the effects of FOXL2 on biological process and gene expression of ovarian granulosa cells (GCs), we established stable FOXL2-knockdown GCs and then analyzed using transcriptome sequencing. It is observed that knockdown the FOXL2 affects the biological processes of cell proliferation, DNA replication, apoptosis and cell cycle. In which, the FOXL2 knockdown promoted cell proliferation and DNA replication, decreased cell apoptosis, and promoted mitosis. In addition, by comparing the transcriptome by FOXL2 knockdown, we found a serious of DEGs and their related pathways. These results indicated that, through mediating these genes and pathways, the FOXL2 might induce the cell proliferation, cycle, and DNA replication, and play a key role during ovarian development and maintenance. Sixteen mRNA profiles of primary granulosa cells which were transfected with FOXL2-specific siRNA (FOXL2-siRNA) or NC nonsense siRNA (NC-siRNA) respectively.were generated by deep sequencing, in triplicate, using Illumina Hiseq X-ten platform.Illumina Hiseq X-ten platform.

叉头框L2（FOXL2）是一种单外显子基因，编码叉头转录因子，主要在卵巢、眼睑与垂体中表达。FOXL2在卵巢发育过程中发挥关键作用。为阐明FOXL2对卵巢颗粒细胞（granulosa cells, GCs）的生物学过程及基因表达的调控效应，本研究构建了稳定敲低FOXL2的GCs，并通过转录组测序开展分析。研究发现，敲低FOXL2会影响细胞增殖、DNA复制、细胞凋亡与细胞周期等生物学过程：其中，敲低FOXL2可促进细胞增殖与DNA复制，抑制细胞凋亡，并加速有丝分裂进程。此外，通过对比FOXL2敲低组与对照组的转录组数据，本研究筛选得到一系列差异表达基因（differentially expressed genes, DEGs）及其富集的相关通路。上述结果表明，FOXL2可通过调控这些基因与通路，影响细胞增殖、细胞周期及DNA复制过程，在卵巢发育与维持中发挥关键作用。本研究利用Illumina HiSeq X Ten测序平台，对分别转染FOXL2特异性小干扰RNA（FOXL2-siRNA）与阴性对照无义小干扰RNA（NC-siRNA）的原代颗粒细胞进行深度测序，每组设置3次生物学重复，最终获得16条mRNA转录组图谱。