MicroRNA-31 Negatively Regulates Inter Leukin-34 Expression In Vitro

Background & Objective: Interleukin-34(IL-34)is a recently discovered interleukin family member that has two receptors,CSF-1R and RPTP-?.IL-34 is involved in the inflammatory and immune responses in various diseases,it has a variety of biological functions,for example,enhancing monocyte cell viability,accelerating osteoclast formation and so on.IL-34 is produced by a variety of cells and is expressed in many tissues of the body.Micro RNA-31 is an important gene regulatory molecule that is involved in the proliferation and differentiation of tumor cells,migration and metastasis.By using on-line Target Scan and Mi Randa software,we predicted the presence of the mi R-31 binding site in the 3 'non-coding region(3' UTR)of IL-34.We hypothesized that mi R-31 may play an important role in the regulation of IL-34 expression.First,we examined the expression levels of IL-34 and mi R-31 in various cell lines.Next,we investigated whether mi R-31 bound to the 3'UTR of IL-34 and the regulatory role of mi R-31 on IL-34 expression.This study aims to explore whether mi R-31 regulates IL-34 gene expression.Methods:1.Western blotting was used to detect the expression of IL-34 in various cells(Hep G2,GES-1,SGC-7901,HGC-27,MGC-803,HT-29,KYSE-450,MKN-45,U87MG).2.QRT-PCR was used to detect the expression levels of micro RNA-31 and IL-34 m RNA in various cells(Hep G2,GES-1,SGC-7901,HGC-27,MGC-803,HT-29,KYSE-450,MKN-45,U87MG).3.The psi-check2 plasmid containing IL-34 3'UTR was constructed by the common method of molecular cloning.The mi R-31 binding site in IL-34 3'UTR was depletely mutated by the plasmid mutation kit4.The psi-check2 plasmid containing IL-34 3'UTR and its mutant(The mi R-31 binding site in IL-34 3'UTR was depletely mutated)were co-transfected into MGC-803 cell with mi R-31 mimic,NC or mi R-31 inhibitor,inhibitor nc,48 hours lates,luciferase activity was analyzed using dual-luciferase reporter assay system.5.MGC-803 cell were transfected with mi R-31 mimic,NC,the q RT-PCR method was used to detect the expression of mi R-31 and IL-34 m RNA by co-immunoprecipitation of RNA-binding protein after 48 hours.6.IL-34 and Mi R-31 expression levels were detected by Western blotting and q RT-PCR respectively after transfection of with mi R-31 mimic,NC,mi R-31 inhibitor and inhibitor nc in HT-29 and KYSE-450 cells.Results:1.IL-34 was expressed at different levelsin various cells.2.The expression level of Mi R-31 is different in different cells,and its expression level is negatively correlated with the level of IL-34 expression.3.Mi R-31 binds directly to IL-34 3'UTR.4.Mi R-31 negatively regulates the post-transcriptional expression of IL-34.Conclusion:MiR-31 binds directly to IL-34 3'UTR and negatively regulates post-transcriptional expression of IL-34.