| As a miniture pig,it has been widely studied in recent years that the Bama mini pig has formed a closed pig population unique in China under the influence of long-term inbreeding and local feeding conditions.As a dwarf breed,it has developed many unique genetic markers,such as morphological markers-body dwarfs and molecular markers-DNA level genetic polymorphisms,and so on.microRNA(miRNA)is a highly conserved non-coding RNA with a sequence length of about 22 nt.It has tissue and timing specificity and plays an important role in the growth and development of animals.In recent years,with further miRNA research,it has been found that miRNAs are closely related to the growth and development of animals.Among them,miR-140,which is located in the intron of the WWP-2 gene,plays a role in mammalian cartilage development and cancer treatment.However,the study of miR-140 for pig has not been fully reported.In this study,Large White(LW)and Bama mini pigs(BM)were used as experimental subjects.The difference in the amount of miR-140 expression in the cells of the embryonic stage,the 1-3 day old and the adult stage of the six tissues(heart,liver,lung,kidney,muscle,and cartilage)was examined,respectively.It was found that miR-140 in porcine fetal fibroblasts(PFFs)of LW was significantly higher than that in PFFs of BM(P<0.05).In addition,the expression of mi R-140 in hearts,muscles,lungs,and kidneys of the BM with 1-3 days age of non-weaning was greater than that of the LW.In contrast,LW expressed more miR-140 in cartilage and liver than BM.And the expression levels of miR-140 in the liver and kidney were extremely different(P<0.0001),in lung was significantly different(P<0.01).For adulthood stage,miR-140 expression levels in all tissues in LW was higher than that in BM.The difference of miR-140 expression level in heart,muscle,lung,and liver was extremely significant(P<0.0001),in cartilage was significant(P<0.05).To further examine the effect of miR-140 on cell function,flow cytometry wasused to detect the effect of overexpression miR-140 mimics on the apoptosis and cycle of PFFs in LW.For apoptosis,the amount of early apoptosis,late apoptosis,and total apoptosis were lower in the experimental group than in the control group after transfecting miR-140 mimics for 24 h,of which only late apoptosis was significantly different(P<0.05).After transfecting for 48 h,the proportion of apoptosis cells at each stage of the experimental group was lower than that of the control group,and the proportion of total and late apoptosis were significantly different(P<0.05),and the difference in early apoptosis was extremely significant(P<0.01).At the same time,the cell cycle experimental results showed that the apoptosis proportion of the two groups after 48 hours of transfection was significantly different at the S phase(P<0.05),and the trend in the proportion of three phases(G1,S and G2)is the same as 24 hours after transfection.According to the characteristics of miRNAs that bind to the mRNA of target genes and thus affect biological function,71 potential 3’UTR and miR-140 complementary target pairs were predicted using bioinformatics websites and software.It was further predicted that 500 genes co-expressed with the target genes,of which 200 were transcription factors.And through analyzing network diagram of co-expression genes,9 key genes(hub genes)that regulated the apoptotic pathway and 12 cell cycle-related genes were screened out.The results of qPCR showed that in the PFFs of LW and BM,7 predicted target genes were negatively correlated with the expression of miR-140,which can be speculated that those genes are the candidate target genes of miR-140.Based on the above experimental results,it was concluded that miR-140 may be one of the functional mi RNAs affecting somatotype traits in pigs,but its target genes and their interaction mechanisms need further confirmed.This experiment provided a molecular theoretical basis for studying the effect of miR-140 on pig’s somatic traits,which laid the foundation for further research on the mechanism of growth and development of miniture pigs. |
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