| In this paper,we study the miR-378-2 sequence mutations of miR-378 in Chinese local porcine with Sanger sequencing,in order to analyze the genetic effect of mutations and explore the effect of mutations on expression and function of miR-378.The results are shown as follows:1.The results showed that miR-378-2 gene of Rongchang pig and Neijiang pig has the same two mutations,which were in miR-378-2 precursor sequence +49A>G(miR-378 seed sequences+5A>G)and +68A>G mutation,respectively.Both of these mutations could affect the expression of miR-378,+49A>G mutation also result in changes in functions of miR-378.2.The secondary structure and free energy of the miR-378-2 sequence with two mutations showed that the miR-378-2 secondary structure and free energy of the mutant were all changed significantly,stem structure of the convex ring became small,free energy entropy decreased by 17.2%.The results showed that miR-378-2 secondary structure sequence mutations making it more stable,more conducive to the identification and processing enzymes.3.We constructed the expression vector of miR-378-2W/M,and overexpressed in HEK293 cells.The results showed that mature miR-378-2M expression level was about two times more than that in miR-378-2W(P<0.01).The expression of pri-miR-378-2 and pre-miR-378-2 was about 1.6 times more than that in miR-378-2W(P<0.01).Compared with miR-378-2W,expression level of pri-miR-378-2 of miR-378-2M had no significant difference(P>0.05),indicating that miR-378-2 sequence mutation could increased the expression of pre-miR-378-2 and miR-378.4.The prediction of target genes of wild type miR-378 and mutant miR-378 showed that the mutations of miR-378 seed sequence(+5A>G mutation)leads to change a large number of target genes.The total number of target genes increased by 27,58 target genes were gained,31 target genes were deleted,13 target genes were not affected.The GO and Pathway analysis of predicted target genes showed that miR-378 seed sequence +5A>G mutation changed miR-378 regulatory pathway.All these results showed that miR-378 seed sequence +5A>G mutation did not only affected the recognition of target gene,but also affected the biological function of miR-378.5.We selected GDF6,Runxlt1,Galnt3 and RAB10 predicted target genes by random sampling.Then,using Dual Luciferase Reporter Assay and RNA-pull down experiments certified the target validation on the predicted target genes.The results showed GDF6 was the target gene of miR-378 mutation,RAB10 was the target gene for miR-378 mutation deletion,while Runxlt1 and Galnt3 were target genes that were not affected by miR-378 mutation.RNAhybrid software was used to predict the secondary structure and minimum free energy of target gene binding to its miRNA.It is proved that miR-378 seed sequence+5A<G mutation changed the secondary structure and minimal free energy of miR-378 after binding to the target gene,resulting in the loss or gain of miR-378 partial target genes.6.By transfecting the control mimics,miR-378W mimic and miR-378M mimic into the pre-differentiation 3T3-L1 cell,respectively.The results showed that compared with miR-378W group,the miR-378M group of lipid droplets were significantly lower,the expression level of PPAR-y,PGC-1? and PGC-1? were downregulation,and the expression level of HSL,ATGL and CGI-58 were upregulation.The results showed that miR-378 seed sequence +5A>G mutation affected adipocyte differentiation and lipolytic metabolism-related gene expression,which led to the loss of adipocyte differentiation and lipid deposition.These results provided bases for studying the acquisition or deletion of miR-378 seed sequence mutation functions,and gave some new ideas of miRNA target gene recognition,identification and functional validation of miRNA mutations. |
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