Microrna-144Inhibites The Expression Of Abca1in RAW264.7Macrophages

Object：In this research, the eukaryotic expression plasmid of miR-144has been constructed in order to detect whether ABCA1expressioncould be regulated by miR-144in RAW264.7macrophages.Method： According to the published documents and severalbioimformatic tools, the miR-144was selected due to a high probabilitybinding to ABCA1mRNA3’UTR. The DNA sequences of miR-144andABCA1mRNA3’UTR amplified by PCR with technology of molecularcloning were recovered from gel extraction kit, which were after theninserted into plasmids of pcDNA3.1（+） and pcDNA3.1（+）-luciferase respectively. After the sequencing of plasmids, The dualluciferase reporter gene system was applied to determine whether thereexisted a direct targeting relationship between miR-144and ABCA1mRNA3’UTR. Secondly, the plasmids of empty pcDNA3.1（+）andpcDNA3.1（+）-miR-144were transfected to RAW264.7macrophagesrespectively by means of LipofectamineTM2000. The expression of ABCA1at mRNA and protein standard was determined by qRT-PCR and Westernblot.Result： The DNA sequences of miR-144and ABCA1mRNA3’UTR amplified by PCR were successfully inserted to plasmids ofpcDNA3.1（+） and pcDNA3.1（+）-luciferase respectively. The dualluciferase reporter gene system divovered that, compared to control grouptransfeting with empty pcDNA3.1（+）, pcDNA3.1（+）-luciferase- ABCA13’ UTR and PRL-SV40, the treatment group transfected withpcDNA3.1（+）-miR144, pcDNA3.1（+）-luciferase-ABCA13’ UTRand PRL-SV40indicated a significant decrease of firefly luciferase activitywith P＜0.05, showing that miR-144could regulate ABCA1expressiondirectly by targeting to ABCA1mRNA3’ UTR. The qRT-PCR elucidatedthat the miR-144expression was signicantly increased in RAW264.7macrophages after transfection of pcDNA3.1（+）-miR-144with P＜0.01,compared to the group transfected of pcDNA3.1（+）with nearly noalteration and P＞0.05. The qRT-PCR also elucidated that the expression ofABCA1mRNA was not obviously changed after transfection of pcDNA3.1（+）and pcDNA3.1（+）-microRNA-144respectively, with P＞0.05.But the Western blot showed that overexpression of miR-144bytransfection of pcDNA3.1（+）-microRNA-144could significantlydecrease the expression of ABCA1at protein standard compared to thecontrol group, with P＜0.01.Conclusion：miR-144can inhibit ABCA1expression in RAW264.7macrophages at posttranscriptional level.