Effects of Circular RNA of Chicken Growth Hormone Receptor Gene on Cell Proliferation.

Animal growth and development are regulated by neural and endocrine growth axes, in which cell proliferation plays key roles. Recently, many research showed that circular RNAs were involved in hepatocyte and myoblast proliferation. Previously, we identified a circular RNA derived from the chicken GHR gene, named circGHR. However, the function of circGHR is unclear. The objective of this study was to investigate circGHR expression pattern and its roles in cell proliferation. Results indicated that circGHR was a closed-loop structure molecule, and it was richer in the nucleus of hepatocytes and myoblast. Real-time PCR showed that circGHR was increased from E13 to the 7th week in the liver but decreased in the thigh and breast muscle. The CCK-8 assay displayed that circGHR promoted cell proliferation. Simultaneously, the biomarker genes PCNA, CCND1, and CDK2 and the linear transcripts GHR and GHBP were upregulated when circGHR was overexpressed. Altogether, these data exhibited that circGHR could promote cell proliferation possibly by regulating GHR mRNA and GHBP expression.

[Cloning and expression analysis of chicken circular transcript of insulin degrading enzyme gene].

Insulin-degrading enzyme (IDE) is a highly conserved metallopeptidase that functions in the catabolism of bioactive peptides. In our previous study, we identified a putative circular transcript in that chicken insulin-degrading enzyme (IDE) gene through analyzing a high throughput sequencing result. Here we set to confirm the circular transcript of IDE (circIDE) and explore its expression regularity in normal barred Plymouth chicken. The circIDE was confirmed by PCR amplification and sequencing. The circular structure of circIDE was determined by RNase R processing and reverse transcription experiments. Then we analyzed the spatiotemporal expression pattern of circIDE and IDE mRNA and compared the differential expression of circIDE and IDE mRNA in the normal barred Plymouth chicken and the dwarf ones. The results showed that the full length of chicken circIDE was 1332 nt, divided form exon 2-11 of the IDE gene. RNase R tolerance analysis showed that chicken circIDE had the general characteristics of circular molecule, and was highly resistant to RNase R. The random primers had higher transcription efficiency than the oligo-d(T)18 primers, confirming that circIDE is a circular structured molecule without poly(A). circIDE was highly expressed in the liver and heart tissues but less in the muscle tissues of leg and breast in normal chickens at the age of 1 and 12 weeks. The expression profile of circIDE in liver tissue showed that circIDE level was lower in1 to 6 weeks and then became higher after 8 weeks of age. The expression of circIDE in liver tissue was significantly higher in normal chicken than that in dwarf barred Plymouth chicken (P<0.05). This study confirmed a circIDE strucutre in chicken IDE gene and uncovered its expression regularity. We demonstrated that the expression level of circIDE in the liver tissue was higher in normal barred Plymouth chicken compared to dwarf species. This study paves the way for further understanding the biological function of chicken circIDE, including its roles in regulating chicken growth and development.

Identification and characterization of circular RNAs in chicken hepatocytes.

CircRNAs play important roles in chicken's growth. In this context, we studied the expression profiles of circRNAs between the GHR antisense transcript overexpressed and the control groups of chicken hepatocytes by using the deep RNA-sequencing technique to identify the key circRNAs involved in chicken's growth. A total of 4772 circRNAs were detected in both groups and 92 circRNAs displayed significant differential expression between two groups. Reactome pathway analysis on the parental genes of differential circRNAs indicated that most enriched and meaningful 9 pathways were related to "Mitotic G1-G1/S-M phases", and "FGFR2c ligand binding and activation". Additionally, Five exonic circRNAs were confirmed including circGHR separated from GHR. CircGHR developed in HuaiXiang chicken from d 1 to 5 w of age and was expressed at a higher level in the nucleus than in the cytoplasm. Moreover, our results specify 5 circRNAs target sites for microRNA Let-7b that is expected to target GHR mRNA, signifying their potential role in regulating GHR gene expression in chicken.

Chicken GHR antisense transcript regulates its sense transcript in hepatocytes.

An increasing number of evidences indicated that long noncoding RNAs (LncRNAs) regulate a variety of biological progresses via different mechanisms. Our previous study had identified a chicken growth hormone receptor (GHR) antisense transcript (GHR-AS) which regulated GHR sense transcript (GHR-S) in LMH cells. In the present study, roles of GHR-AS and its regulatory mechanism were analyzed in chicken hepatocytes. The expression patterns of liver GHR-S, GHR-AS and Let-7b ascended with the development of chicken. The hepatocytes proliferation was promoted and more cells entered into DNA synthesis (S) phase when GHR-AS was overexpressed while the cell proliferation was slowed and fewer cells were in S phase when GHR-AS was interfered. Meanwhile, the GHR-S increased when we overexpressed GHR-AS while it reduced when GHR-AS was inhibited. The S1 Nuclease protection assay indicated that GHR-S and GHR-AS formed RNA duplex via GHR-S 3' untranslation regon (3'UTR). In hepatocytes or LMH cells, the half-time of GHR-S showed a delayed trend when GHR-AS or GHR-AS 5' untranslation regon (5'UTR) was overexpressed. Furthermore, the level of GHR-S can be decreased by Let-7b mimics whereas it was partially rescued when co-transfected pGHR-AS or pGHR-AS 5'UTR with Let-7b mimics. Based on our findings, GHR-AS affected hepatocytes proliferation and improved GHR-S stability possibly by forming RNA duplex between GHR-S and GHR-AS, competing with Let-7b.