Effects Of MiR-302s/367 Cluster On Gene Expression And Epigenetic Modification Of Sheep Fetal Fibroblasts

MicroRNAs(miRNAs),a type of small,non-coding RNAs,~22 nt in length,can bind to 3' untranslated region(3'-UTR),even coding sequence and promoters of target mRNAs,inhibit mRNAs translation and/or cause mRNAs degradation.It is well-known that miRNAs have the ability to influence many cellular processes,including reprogramming,proliferation,differentiation and apoptosis.miR-302s/367 family have the ability to reprogram mouse and human somatic cells into pluripotent stem cells(iPSCs),inhibit the proliferation of several types of cancer cells,even cause cancer cells apoptosis.However,the functions of miR-302s/367 family in other mammals have not been explored.In this study,a synthetic precursor miR-302s/367 cluster(pre-miR-302s/367 cluster)was inserted into the entry vector pcDNA6.2-GW/EmGFP-miR,and then the cluster was recombined into the destination vector pT-RE-DEST30 by gateway technology and the recombinant vector was identified.The recombinant vectors pT-REx-DEST30 and pcDNA6/TR vectors were introduced into sheep fetal fibroblast cells(SFFs)using Lipofectamine3000 transfection method.Transgenic SFFs were screened by G418 and Blasticidin,cultured in medium with(Tet-on)or without(Tet-on)1 ?g/mL tetracycline.Tetracycline could induce the expression of pre-miR-302s/367 cluster and EmGFP in transgenic SFFs.The expressions of miR-302s/367 cluster and EmGFP in transgenic SFFs were detected by fluorescence inverted microscope and stem-loop RT qPCR.The proliferation and apoptosis of(Tet-on)/(Tet-off)transgenic SFFs were detected by CCK and flow cytometry,respectively.RNA from(Tet-on)/(Tet-off)transgenic SFFs was extracted and highthroughput RNA-seq technology was used to detect RNA expression in cells.Genomic DNA and histones of(Tet-on)/(Tet-off)transgenic SFFs were extracted and genomic DNA methylation and histone H3 acetylation levels were detected by ELISA.The purpose of this study was to explore the effect of miR-302s/367 cluster on the gene expression and the epigenetic modification of SFFs,and the results were showed as following:1.Recombinant destination vector pT-REx-DEST30 was successfully constructed by gateway recombination technology.2.The transgenic SFFs were successfully obtained.The expression of EmGFP and miR-302s/367 cluster in(Tet-on)transgenic SFFs was successfully induced by thetetracycline at a final concentration of 1 ?g/mL in the culture medium.The levels of miR-302 s cluster and miR-367 in(Tet-on)transgenic SFFs were as high as 21 times and 4 times of U6 siRNA(which was used as reference siRNA).3.After 3 days of miR-302s/367 expression,which was induced by Tetracycline in the culture medium,the proliferation rate of(Tet-on)transgenic SFFs was significantly lower than that of(Tet-off)transgenic SFFs;and the apoptosis rate of(Tet-on)transgenic SFFs(4.76 ± 0.31%)was also significantly lower than that of(Tet-off)transgenic SFFs(14.21 ± 0.41%).After miR-302s/367 cluster was induced to express for 7 days,the(Teton)transgenic SFFs transformed to epithelial-like cells.4.A total of 42.57 G clean data was obtained from High-throughput RNA-seq of 6samples.Of them,the minimum value of Q20,Q30 and clean reads ratio was 96.23%,88.51% and 98.06% respectively,which conformed to the quality standard of Highthroughput RNA-seq(Q20?95% and Q30?85%).Some of genes in the PI3 K and cell cycle pathway were differentially expressed in(Tet-on)/(Tet-off)transgenic SFFs.RT qPCR were used to confirm the change in the level of these genes.The results showed that PI3 K,c-Myc,CDK6 and cyclin D1 were significantly up-regulated,while,CDK2,cyclin E1,cyclin E2,E2F1 and E2F2 were significantly down-regulated in(Tet-on)transgenic SFFs,only the change in the level of P15INK4 B was not significant.5.After tetracycline induced miR-302s/367 cluster to express for 3 days,the level of DNA methylation in(Tet-on)transgenic SFFs(0.157%)was significantly lower than that of(Tet-off)transgenic SFFs(0.324%).However,the level of histone H3 acetylation in the(Tet-on)transgenic SFFs(0.84%)was significantly higher than that of(Tet-off)transgenic SFFs(0.55%).RT qPCR was used to examine the level of DNA methylation-and histone H3 acetylation-related genes.The results showed that both the levels of DNA methylationrelated genes,DNMT1,DNMT3 a,DNMT3b,AOF1 and AOF2,and histone H3 acetylationrelated genes,HDAC1 and HDAC2,were significantly lower in(Tet-on)transgenic SFFs than that in(Tet-off)transgenic SFFs.