A GHR NAT Promotes Chicken Fat Deposition And Muscle Cell Proliferation By Inhibiting Its MRNA Expression

Natural antisense transcripts(NATs)are transcribed from the opposite DNA strands of encoding or non-coding proteins genes in a natural state,and are a class of RNA molecules about 200 to several thousand nucleotides(nt)in size.They can directly or indirectly regulate target genes expression.In animals,some NATs can affect the expression of target genes through epigenetic mechanisms,as well as form double-stranded RNA(ds RNA)by complementary with target m RNAs to protect m RNA from degradation or promoting protein synthesis at the post-transcriptional level,thereby affecting cells differentiation,proliferation and apoptosis,and play pivotal regulatory roles in life activities.The GHR protein is a single-channel transmembrane receptor,which could deliver the GH that it binds into the cell,and then transmits the signal to downstream factors,thereby causing a series of physiological reactions in the body.That will stimulate cells proliferation and differentiation,regulate the metabolism of three major nutrients in the body,and promote the transcription of related genes involved in these processes,ultimately affect individuals' growth and development.The expression of GHR gene is strictly controlled,and the NAT generated by chicken GHR gene,such as GHR-AS,plays an important role in regulation.However,this gene can lauch more than one kind of NAT,whose biological function and machnism needs to be further studied.Therefore,here we cloned another GHR NAT,analyzes its tissue expression profile,sequence features and its effect on the regulation of GHR,to explain the regulation and molecular mechanism of GHR expression by NAT.Explore the role of GHR-AS-EST in cell growth and development in hepatocellular carcinoma cell line(LMH)and primary myoblasts.In this experiment,six 16-embryonic old of White Recessive Rock chickens(WRR)were selected for tissue sample collection to extract RNA.The full length of GHR-AS-EST transcript was obtained by RACE and Nothern blot.Several 11.5-embryonic age of WRR were selected for isolating primary myoblasts.The hepatocellular carcinoma cell line(LMH)and chicken primary myoblasts were transfected by various constructing vectors,and then analyzed the effect of GHR-AS-EST on GHR gene expression,as well as the regulatory effect of cell growth and development.The main results are as follows,(1)A novel GHR NAT,GHR-AS-EST,was successfully cloned from normal WRR liver tissues,is 688 nt in length and is transcribed from the opposite sequence of exon 6,exon 5 to 5'-UTR,with the ability to encode proteins that encoding a protein of approximately 17 k Da.(2)Chicken GHR-AS-EST overlaps with the sequence of 5'-UTR and exon 1-6 of GHR sense transcripts,and its sequence is less than 60% similar to other species' potential GHR NAT,but closer to that of dogs and rats.(3)GHR-AS-EST distributs widely in different tissues of chicken embryos,and is with higher expression level in liver and muscle tissues than other tissues.It can inhibit the expression of GHR m RNA and protein,and promote 0.7-kb GHR and GHBP at both RNA and protein levels.(4)GHR-AS-EST can reduce the transcriptional activity of GHR V1 promoter through repressing GHR-AS,and promoting both Sp1 and Sp3 expression to inhibite GHR m RNA expression,and prolonging the half-life of GHBP by forming a length of 537 bp ds RNA with GHBP in exon 2-6 region,for maintaining GHBP stability.(5)In LMH cells,GHR-AS-EST can promote cell proliferation by increasing the expression of genes involved in JAK2/STAT pathway,and can also affect fat deposition by inhibiting the expression of SOCS3 and LEPR,increasing m RNA expression of adipocyte differentiation markers genes PLIN,GOS2,C/EBP?,FAS,PPAR?,and A-FABP.GHR-AS-EST can affect the cell cycle of chicken primary myoblasts,inhibits the expression levels of muscle cell differentiation marker genes Myo D,Myo G,Myf5 and MRF4,and promotes the expression of both My HC and Myomaker.In summary,chicken GHR-AS-EST is a new NAT of GHR,which negatively regulates GHR m RNA expression,and affects the growth and development of individuals by promoting cell proliferation and the expression levels of genes involved in fat synthesis,as well as inhibiting the expression levels of myogenic regulatory factors.These findings have accumulated data for studying the expression of GHR gene regulated by NAT and provide a reference for exploring whether such NAT affects cell growth,development and metabolism.