The Mechanism Of Multiple MicroRNAs Modulate FABP1 Expression

Objective: Human liver type fatty acid binding protein(human liver fatty acid binding protein, FABP1) is a molecular weight of about 14~15KD, and it can be combined with long chain fatty acids of high conserved protein. It’s one of fatty acid binding protein family. Based on the previous studies in our lab showed that hepatocyte nuclear factor 3 beta(hepatocyte nuclear factor 3 beta, HNF3 beta) and CCAAT/enhancer binding protein alpha(CCAAT/enhancer binding protein, alpha, C/EBPA) act on FABP1 gene promoter regulateing its expression, but 3’ untranslated region(3’ untranslational region, 3’ UTR) control mechanism is still not clear. This study was designed to investigate the effect of mi RNA on expression in FABP1-3’ UTR to explore the mechanism of hepatic steatosis, and it contribute to provide a new direction for diagnosis and treatment of fatty liver.Methods: 1. using Target Scan, Pic Tar and other software to carry out bioinformatics analysis of FABP1 related mi RNAs, screening the mi RNAs which are target of FABP1-3’ UTR.2. Chemical synthesis FABP1-3’ UTR, inserted into pmir Glo vector to construct a recombinant vector pmir GLO-FABP1-3’ UTR. Transfect mi RNA mimics or NC(Negative Control, NC) and pmir GLO-FABP1-3’ UTR recombinant vector into Hep G2 cells respectively mediated by liposome. Use dual luciferase reporter gene assay system to detecte the luciferase activity 48 hours later to select the potential mi RNAs act on FABP1-3’ UTR compared with the control group.3. Find out the paired bases between initial screened mi RNAs with FABP1-3’ UTR, than mutate the corresponding paired bases, chemical synthesis of pmir GLO-FABP1-3’ UTRmuts. pmir GLO-FABP1-3’ UTRmuts and the corresponding mi RNA mimics were co transfected into Hep G2 cells by liposome mediated. Use dual luciferase reporter gene assay system to detecte the luciferase activity 48 hours later to rescreened. 4. the effect of mi RNAs on the expression of m RNA and protein to FABP1 was detected by fluorescence quantitative PCR and Western-blot.Results: Through the prediction of software and the analysis of database, screening out 68 possible mi RNAs targeting FABP1-3’ UTR.The selected mi RNA mimics and pmir GLO-FABP1-3’UTR recombinant vector were co transfected into Hep G2 cells. luciferase activity assay showed that 16 mi RNAs can significantly reduce the luciferase values(t analysis, P<0.05) compared with the control group(NC) 48 h later. And we found there are 6 FABP1-3 ’UTR muts still exists fluctuation of downregulation. therefore, we determine 6 mi RNAs can down regulate FABP1-3’ UTR’s expression finally. They are mi R-323a-3p、mi R-362-3p、mi R-4672、i R-466、mi R-3941、mi R-4517. the experimental results show that part of mi RNAs level does not affect or even rise in m RNA level using GAPDH as realtime rt-pcr standard, and partly can down regulate the expression of FABP1: mi R-323a-3p、mi R-362-3p、mi R-4672. and the results also show that mi RNAs are able to down regulate the protein expression level of FABP1 in different level using beta actin as western blot’ s standard.Conclusion: 6 mi RNAs can down regulate the expression of the FABP1’ s 3’ UTR after transcription, and it can affect FABP1’ s normal expression resulting in a series of physiological and pathological changes.