Preliminary Study Of MiR-148a And Dnmt1 Interaction,the Effects On Development Of Pig Nuclear Transfer Embryos

The efficiency of somatic cell nuclear transfer is still low,the main reason is considered as incomplete epigenetic reprogramming of somatic cell nucleus,which closely associated with the abnormal epigenetic modifications.The epigenetic modification includes DNA methylation and histone modification etc.DNMT1 maintain the methylation,caused abnormal DNA methylation level.MicroRNA(miRNA)is a group of small and non-coding RNAs that regulate expression of protein by repressing translation or promote degradation of the target mRNA.Many studies showed that the expression of miR-148a was restricted due to abnormal methylation,and DNMT1 was one of the target genes of miR-148a,which was regulated by miR-148a.In order to understand whether they have interaction in normal somatic cells,and the function and mechanism of endogenous miR-148a associated with epigenetic modification in the SCNT embryo development,we verified that pig DNMT1 was one of target genes of miR-148a by bioinformatics and dual luciferase experiments.We explored the regulation relationship of miR-148a and DNMT1 in pig fibroblasts.Then we studied the effects and the mechanisms of miR-148a on development of somatic cell nuclear transfer embryos.The main results of the study are as follows.1.Bioinformatics analysis of miR-148 and the confirmation of miR-148a targeting on DNMT1 geneThe target genes of miR-148a were predicted by using software and dual luciferase experiment.Multiple sequence alignment results found that 1 to 8 and 11 to 19 site matured sequences of miR-148/152 gene family were highly conservative,which demonstrated that miR-148/152 gene family was highly conserved among species.Evolutionary tree analysis results showed that miR-148 gene family was divided into two types,mammals miR-152,fish miR-152 and miR-148a/b.The GO and KEGG of target genes predicted were analyzed.The results showed that the target genes enriched in gene expression,regulation of transcriptional activity,embryonic development process.We verified that the pig DNMT1 was one of target genes by dual luciferase experiments.2.The regulation relationship of miR-148a and DNMT1 in pig fibroblastsWe cloned and analyzed the miR-148a promoter sequences.The results showed that high methylation site and two promoter region had in the 2043 bp sequence,which were priority to SP1 and AP2 transcription factors.The fragments were inserted into the pGL3-Basic vector.Compared with blank group,the luciferase ratio had no significant difference in cells of pGL-148a-0.6K and pGL3-148a-1K transfection group(P>0.05),increased significantly in cells of pGL3-148a-2K and pGL3-promoter transfection group(P<0.05),which illustrated that 2043bp sequence had promoter efficiency.Adding 5 and 10 ng/mL bFGF to the pig fibroblasts transfected by pGL3-148a-2K and pRL-TK plasmids,the results showed that dual luciferase expression level in bFGF treatment group deacreased significantly compared with the untreated group(P<0.05).Adding 5 and 10 ng/mL bFGF to the pig fibroblasts,the expression of miR-148a reduced significantly,DNMT1 gradually increased significantly(P<0.05).Adding 0.5 and 1 μM 5-aza-dC to the pig fibroblasts,DNMT1 expression gradually reduced,miR-148a expression gradually increased with 5-aza-dC concentration increasing(P<0.05).3.Establishment of the porcine fibroblast line overexpressing miR-148aThe three plasmid package system was used to prepare high title lentivius.Compared with untreated and miR-control groups,the expression of miR-148a increased five times(P<0.05),and the expression of target gene DNMTl significant suppressed(P<0.05),while the expression of DNMT3a has no significant change(P>0.05).DNMT1 protein level was significantly reduced by western blot analysis(P<0.05).The change of genome-wide methylation was detected by immunifluorescence analysis technology,we found methylation level of overexpressing miR-148a group was significantly lower than that of untreated and miR-control groups(P<0.05).The growth of pig fibroblasts was significantly inhibited(P<0.05),and the G0/G1 cells increased significantly(P<0.05).4.The effects of miR-148a on porcine SCNT embryonic development and related gene expressionWe analyzed the expression pattern of miR-148a in pig embryos during maturation,parthenogenetic and SCNT embryos.Further we stuied the effects of overexpressing miR-148a on development of nuclear transfer embryos,expression of the DNA methylation,histone modification and related genes.The results showed that,miR-148a had lower expression during oocyte maturation.In early parthenogenetic embryos,the expression of miR-148a firstly increased,then decreased.Excepting to the blastocyst,the expression of miR-148a in IVF and PA embryos was significantly higher than that of the SCNT embryos(P<0.05).Compared with blank and miR-control,more SCNT embryos developed to blastocyt stage(20.79%vs 14.36%、14.9%,P<0.05),and more total cell numbers in blastocytsts(39.15 ± 6.36 vs 33.25 ±7.14、34.38±8.50,P<0.05)in overexpressing miR-148a group.Overexpressing miR-148a could significantly decreased DNA methylation levels in 2-,4-cell embryos(P<0.05).The expression of DNMT1 gene at 2-,4-and blastocyst stages was decreased(P<0.05),and the DNMT3a,TET1 and TET3 gene expression at 2-and 4-cell stages were significant increased(P<0.05),and TET2 had the same expression at 2-cell and blastocyst stage.Overexpression of miR-148a could significantly improve H3K4me3 levels in 2-and 4-cell embryos(P<0.05),significantly decrease H3K9me3 levels in 4-cell and blastocyst embryos(P<0.05),and significantly increase the H3K9ac levels in 2-cell embryos(P<0.05).The expression profiles of HDAC1、HDAC2、KDM5A、HAT1 genes were significantly increased(P<0.05),the expression of HDAC2 gene was decreased(P<0.05).MiR-148a could significantly increase the expression of eIF3A and TFIIA in SCNT 2-and 4-cell embryos(P<0.05),and also significantly increase the expression of Oct4,Nanog in blastocyst.In conclusion,the above results demonstrated that pig DNMT1 gene was one of the target genes of miR-148a,miR-148a and DNMT1 had regulation relationship in pig fibroblasts.Overexpressing miR-148a could decrease the expression of DNMT1,and increase the expression of reprogramming related genes,and improve the development ability of early nuclear transfer embryos.