Chicken GHR-AS And Let-7b Regulate The GHR-S In Hepatocytes

Gene can transcribe the natural antisense transcripts(NATs)which are in opposite direction as compared with m RNAs.Both NATs and micro RNA can regulate the expression of gene m RNAs in a dynamic balance.Growth hormone receptor(GHR)is a critical gene in animal growth axis.Previous studies indicated that chicken GHR m RNA(GHR-S)can be regulated by GHR antisense transcript(GHR-AS)and the mi RNA Let-7b.However,the interacting mechanism among GHR-AS,Let-7b and GHR-S in chicken embryo hepatocytes remain unknown.In this study,we analyze the relationships of the above mentioned three molecules by S1 Nuclease protection assay,RNA stability assay and the other technologies in Huai Xiang chicken embryo hepatocytes.The following contents were detected:(1)The express characteristics of GHR-S,GHR-AS and Let-7b in chicken liver and hepatocytes;(2)Effects of GHR-AS on chicken embryo hepatocytes proliferation;(3)The interaction among GHR-S,GHR-AS and Let-7b.The temporal expression profile results indicated that the liver GHR-S,GHR-AS and Let-7b exhibited similarly increased trends in the development of chicken.In chicken hepatocytes,GHR-S was highly expressed in cytoplasm and the half-life was about 2.5 h while GHR-AS was highly expressed in nucleus and the half-life was more than 4 h.In chicken LMH cells,the half-life of GHR-AS was about 6 h while the half-life of GHR-S was less than 4 h.The results suggested GHR-AS was more stable than GHR-S.We overexpressed GHR-AS in chicken hepatocytes,the results showed that the expression of both mature RNAs and pre-RNAs of GHR-S increased.Also,the cell proliferation was raised and more cells were found in phase S.When we knockdown the GHR-AS,the expression of both mature RNAs and pre-RNAs of GHR-S decreased.Also,the cell proliferation was slowed and more cells were found in phase G1.The sequences of GHR-S and GHR-AS were partially complementary.S1 Nucleaseprotection assay results indicated GHR-AS and GHR-S can form RNA duplex via the3?UTR of GHR-S.The half-time of GHR-S was prolonged when p GHR-AS or p GHR-AS 5?UTR was transfected in hepatocytes and LMH cells.Furthermore,Let-7b mimics can decrease the level of GHR-S.Whereas the levels of GHR-S can be partially rescued when p GHR-AS or p GHR-AS 5?UTR was co-transfected with Let-7b mimics.The above results suggested that GHR-AS could protect GHR-S from being degraded by let-7b.In general,GHR-AS promoted the chicken embryo hepatocytes proliferation,GHR-AS and GHR-S showed a correlated expression pattern in chicken liver.GHR-AS can enhance the stability of GHR-S possibly by forming the RNA duplex in GHR-S3?UTR.