Expression Regulatory Mechanism And Function Of MicroRNA-122 In Chicken Liver

Micro RNAs are a class of tiny, single-stranded noncoding RNAs that can target m RNA via base pairing and lead to translational repression or m RNA degradation at the level of gene posttranscription. They are involved in organ development, cell differentiation, proliferation, apoptosis and other biological process, closely related with many diseases. mi R-122 is highly expressed in liver, which plays an important role in the growth and development, lipid metabolism and other processes of liver, its abnormal expression is related to the development of liver disease. The objectives of this study were to clarify the expression regulatory mechanism and function of mi R-122 in chicken.The main results were as follows:(1) We used bioinformatics analysis and 5’RACE method confirm the 3294 bp site(chr Z:757760) upstream of mi R-122 precursor(pre-mi R-122) as the transcription start site, and sequence deletion technique showed 3.8~3.2 kb region upstream of pre-mi R-122 as the core promoter region of mi R-122.(2) In order to study the expression regulatory mechanism of mi R-122 in chicken, we predicted the promoter region of mi R-122 contains the binding site of transcription factor HNF3β, mutant of this site dramatically decreased the mi R-122 promoter activity. Moreover, EMSA showed HNF3β directly interacted with mi R-122 promoter in vitro, suggesting that HNF3β binding to mi R-122 promoter can activate the transcription of mi R-122.(3) Further study on the fuction of mi R-122, bioinformatics prediction and dual luciferase report system proved that VNN1 and BZW2 were target genes of mi R-122, target site mutation assays showed their complementary sites of 3’UTR with mi R-122 seeds region were target sites of mi R-122.(4) After overexpression of mi R-122 in LMH cells, BZW2 m RNA expression leveldecreased obviously, while VNN1 m RNA expression level did not change significantly. Using LNA-antimi R-122 knockdown the expression of mi R-122 in primary chicken hepatocytes, the target genes m RNA expression level both increased significantly. These data suggested that mi R-122 negatively regulates the expression of BZW2 m RNA, and mi R-122 does not regulate VNN1 in m RNA level.(5) The characteristics of target gene VNN1 expression in various tissues was showed consistent with the expression of mi R-122 features, they were both highly expressed in liver, followed by lung, trace expressed in spleen, kidney, adipose tissue, and extremely low expressed in other organization. These data showed there was tightly correlate between VNN1 and mi R-122, and VNN1 may be play an important role in liver. While BZW2 m RNA was widely expressed in chickens, with the highest level in spleen and low expressed in liver.(6) In the different growth period of chicken liver, the expression of VNN1 has a certain amount of volatility, there were differences between different individuals at the same time. The expression of BZW2 was first descent again rise and then downward trend. These data suggested that VNN1 and BZW2 m RNA level was not related to mi R-122 in the developmental period of chicken liver.