Integrative Analyses of Transcriptome Sequencing Identify Functional miRNAs in the Chicken Embryo Fibroblasts Cells Infected With Reticuloendotheliosis Virus

Abstract

In this study, we found a much higher proportion of reticuloendotheliosis virus (REV) infected chicken embryo fibroblasts (CEF) were in active cell division phase than that of control cells which indicated that REV can affect the fate of CEF. So, we performed high-throughput sequencing and transcriptomic analysis to identify functional miRNAs, in order to figure out the possible mechanism in the interaction of REV with CEF. In total, 50 differentially expressed miRNAs (DEmiRNAs) were identified. Then target genes of DEmiRNAs were predicted and identified by transcriptome profile results. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were conducted to analyze the identified target genes of miRNAs which showed that metabolism, cell cycle, and apoptosis were the most related pathways involved in infection of REV. We analyzed the genes related to cell cycle which indicated that CyclinD1-CDK6 complex played an important role in regulating the transition of the cell cycle from G1 phase to S phase during REV infection. Fluorescence microscope identification showed that REV inhibited the apoptosis of CEF which was in accordance with transcriptome results. A novel miRNA, named novel-72 was found, KEGG analysis was conducted to predict the biological function of its target genes which showed that those target genes were significantly enriched in mTOR signaling pathway and functioned to promote cell cycle and cell growth during the REV infection. In conclusion, REV could induce the up-regulation of cell metabolism, cell cycle and mTOR signaling pathway while inhibit apoptosis of the cell.

Integrated Analysis of miRNA and mRNA Expression Profiles in Spleen of Specific Pathogen-Free Chicken Infected with Avian Reticuloendotheliosis Virus Strain SNV

“…Zhiqiang Yu et al identified the effect of REV infections on miRNA expression profile changes in the bursa of SPF chickens, and found that miRNAs are not only involved in up-regulating pro-apoptotic, proto-oncogene, and cancer cell-associated genes, but also down-regulating genes that are associated with anti-apoptotic cytokines [ 31 ]. Jie Zhai et al identified functional miRNAs in REV-infected CEF cells using transcriptome sequencing [ 32 ]. Defang Zhou et al demonstrated that REV and ALV-J synergistically increased the accumulation of exosomal miRNAs, shedding light on the synergistic molecular mechanism of ALV-J and REV [ 33 ].…”

The Role of MicroRNA in DNA Damage Response

“… Zhao et al (2019) found that under the infection of herpes simplex virus 2, miR-H4-5p bound to 3′-UTR of CDK inhibitor 2A and cyclin-dependent kinase-like 2 for negative regulation, to promote HeLa cells of S phase to the ratio of 41.8%, which was significantly higher than that in HeLa cells in control. In another study, transcriptomic sequencing of reticuloendotheliosis virus infected chicken embryo fibroblasts found that cyclin D1-CDK6 complex played a key role in the transition process from G1 phase to S phase ( Zhai et al, 2018 ). KEGG enrichment analysis showed that target genes of a new miRNA, novel-72 was found to promote cell cycle and cell proliferation in this virus-infected cells ( Zhai et al, 2018 ).…”

“…In another study, transcriptomic sequencing of reticuloendotheliosis virus infected chicken embryo fibroblasts found that cyclin D1-CDK6 complex played a key role in the transition process from G1 phase to S phase ( Zhai et al, 2018 ). KEGG enrichment analysis showed that target genes of a new miRNA, novel-72 was found to promote cell cycle and cell proliferation in this virus-infected cells ( Zhai et al, 2018 ). From the reports so far, miRNAs might promote the transition from G1 phase to S phase in cells under the infection by virus, but more studies are needed to further elucidate that if the conjecture can be applied to more viruses.…”

Epigenetic Regulation by Non-Coding RNAs in the Avian Immune System

“…High-throughput sequencing has been conducted in REV-infected and non-infected tissues or cells and the results identified many candidate DEmiRNAs involved in REV infection. These DEmiRNAs could target immune-related genes and participate in immune-related pathways [138][139][140]. Among them, miR-2945, miR-106-3p/5p, miR-29b/3p/, miR-7b, miR-16c-5p/, miR-122-5p, miR-155, miR-18a-5p, miR-147, miR-184-3p, miR-222b-5p/3p, miR-145b-5p, miR-20b-5p, and miR-1b-3p expressions were verified by qRT-PCR.…”

“…For novel-72, it showed a significantly lower expression in REV-infected cells. Four predicated target genes of novel-72, PDPK1 (phosphoinositide dependent protein kinase-1), mTOR (mammalian target of rapamycin), S6K1 (S6 Kinase 1), and eIF4E (Eukaryotic Initiation Factor 4E) of mTOR signaling pathway were related to cell proliferation and survival, indicating that novel-72 could be involved in mTOR signaling pathway to mediate the cell fate during REV infection [139].…”