MiRNA-455-5p Regulating Adipose Cell Proliferation And Differentiation By Targeting IGF1R Gene In Pig

MicroRNA(miRNA)is a small RNA that plays critical roles in different regulatory pathways in life.It regulates life activities on post-transcriptional levels.Recently,studies have shown that miR-455-5p is involved in the regulation of occurrence and development of tumor.However,no report about the regulation of the subcutaneous adpose tissue proliferation and differentiation by this miRNA in pig had been found.In the present study,double luciferase reporter assay,QRT-PCR,Western blotting and other methods were used to explore the regulating role of miR-455-5p on adipose tissue proliferation and differentiation in pig.The main results are as follows:1.IGF1R gene is predicted as a potential target gene for miRNA-455-5p by using target predictive software.2.The measurement of miRNA-455-5p and IGF1R gene expression in subcutaneous adipose tissue from Bama miniature pig and Landrace pig on 90 d,120 d,150 d and 180 d of age showed that the expression of miR-455-5p and IGF1R increased and decreased with aging respectively.In addition,miR-455-5p was expressed widely in different tissues,which showed the highest expression in heart,relative higher expression in subcutaneous adipose tissue and spleen and the lowest expression in brain and liver.3.The pLV-miRNA-455-5p lentiviral vector was successfully constructed and over-expressed in 3T3-L1 cells.The 3’UTR of IGF1R with binding site or with no binding site of miRNA-455-5p were amplified and cloned into psi-CHECKTM-2 vector to construct normal vector IGF1R-WT and mutant vector IGF1R-MUT.The constructed vectors were transfected into 293T cell lines.The results showed that the luciferase activity was significantly lower in miWT group compared with that in miMUT or miNC or inWT group(P<0.01)and no significant difference in luciferase activity was observed among miMUT,miNC and inWT group(P>0.05).The luciferase activity of WT group was significantly lower compared with MUT group and NC group(P<0.01),and there was no significant difference in luciferase activity between MUT group and NC group(P>0.05).The results confirmed that miRNA-455-5p interacted with IGF1R gene by binding to the specific site of IGF1R3’UTR.IGF1R was the target gene for miRNA-455-5p and the target site was the mutant site in IGF1R 3’UTR.4.The change of expression of miRNA-455-5p,IGF1R,PPARγ and C/EBPa during the 3T3-L1 cells differentiation were measured.The results showed that miRNA-455-5p,PPARγ and C/EBPα expressions were significantly up-regulated and IGF1R was significantly down-regulated during the 3T3-L1 cells differentiation(P<0.01).The miR-455-5p mimic or inhibitor was transfected into 3T3-L1 cell for 48 hours and the cells were collected for qRT-PCR.The qRT-PCR results showed that the miR-455-5p expression was successfully up-regulated and down-regulated in 3T3-L1 cells transfected with mimic and inhibitor respectively(P<0.05).The over-expression and inhibited-expression of miR-455-5p in 3T3-L1 cells led to the significant down-regulated and up-regulated exgression of PPARγ and C/EBPα,respectively(P<0.05),but had no significant effect on the expression of IGF1R mRNA(P>0.05).The Western blotting results showed that the expression of IGF1R was down-regulated by miR-455-5p over-expression and up-regulated by miR-455-5p inhibited-expression(P<0.01).The results indicated that IGF1R gene expression regulated by miRNA-455-5p at translated level other than transcriptional level.In conclusion,miRNA-455-5p may involve in the negative regulation of adipose tissue proliferation and differentiation by inhibiting the expression of its target gene IGF1R.