Potential Contributions Of MiRNAs And Their Target Genes Interaction In The STAT5B Signaling Pathway On Sex-Size Dimorphism In Mauremys Reevesii

Sexual size dimorphism(SSD)is a phenomenon in which size differences occur between male and female individuals of the same age in the species.Many species of turtles have significant sexual size dimorphism in which adult female turtles(Mauremys reevesii)have significantly larger than males that indicates typical female-biased SSD type.Upto date,most studies on SSD in turtles have focused on sexual selection-driven evolution and the influence of environmental conditions but few studies have been reported on its endogenous mechanisms.Studies in humans have shown that the GH/IGF-1 axis plays an important role in regulating somatic growth and metabolism that knocking out the Signal transducer and activator of transcription 5B(STAT5B)gene from mice and fish which leads disappearance of SSD,showing that STAT5 B signaling pathway play an important role in the endogenous mechanism of SSD formation.In this study,we analyzed the m RNA expression profile in the liver tissues of adult tortoise using transcriptome sequencing technology,furthermore,we analyzed and validated the epigenetic regulation of STAT5 B signaling pathway on GH/IGF-1 axis in order to reveal the endogenous mechanism of forming male and female size dimorphism in tortoises.The specific findings are as follows.1.In this study,we first performed transcriptome analysis of liver tissues from 3? and3? adult tortoises individuals(all 5-year-old adults)using high-throughput sequencing technology.A total of 659019 transcripts were obtained by assembly splicing,and 20836 differentially expressed genes(DEGs)were screened by edge R software,in which 7343 DEGs were up-regulated.GO annotation and KEGG enrichment analysis were performed on these DEGs and a total of 7 pathways related to STAT5 B were enriched,five significant genes were selected for validation in combination with the transcriptome sequencing results of DEGs.The results showed that the qRT-PCR assay was consistent with the results of transcriptome sequencing,in which the expression levels of GHR and IGF-1 were higher in the liver of female turtles than in males(P < 0.05 and P < 0.001)while the expression levels of JAK2 and SOCS2 were higher in males than in females(P < 0.001 and P < 0.001).An interesting finding was that the expression of STAT5 B gene was not different between female and male livers(P > 0.05).Accordingly,it is hypothesized that there might be epigenetic regulation of STAT5 B gene expression to play its role in turtle SSD formation which requires further experiments to follow.2.Using immunoflorescence(IF)and protein immunoblotting(Western Blot,WB)techniques,the expression of STAT5 B upto the level of protein were analyzed.The results showed that STAT5 B protein was distributed in the nucleoplasm of nucleated cells of liver tissue,activated by phosphorylation in the cytoplasm,and entered the nucleus to promote the transcription of its target genes(e.g.IGF-1).WB assay showed that the protein expression level of STAT5 B in the liver of female turtles was significantly higher than that of males(P < 0.05).This result suggests that there might be post-transcriptional regulation of STAT5 B gene expression in the liver of male turtles which inhibits protein translation and micro RNA(miRNA)is one of the most common forms of regulation.3.To explore the above speculation,this study used the bioinformatics software Mi Randa and Target Scan to predict the miRNAs that regulate the STAT5 B signaling pathway.The predicted miRNAs/m RNAs pairs were: miR-129-5p/ STAT5 B,miR-128-3p/IGF-1,miR-148a-3p/IGF-1 and let-7a-5p /GHR.To explore the optimal target sites for binding energy between miRNAs and target genes,the RNAhybrid software was further used and calculated the binding energies of miRNAs/m RNAs where the binding energies of miR-129-5p/STAT5 B was-34.5 kcal/mol,let-7a-5p/GHR was-21.9 kcal/mol,miR-128-3p/IGF-1 and miR-148a-3p/IGF-1 was-22.9 kcal/mol,respectively.Therefore,the next qRT-PCR experiments of miRNAs were performed.4.Based on the predicted results,the expression levels of the miRNAs were examined by qRT-PCR.The results showed that the expression levels of miR-129-5p,miR-128-3p,let-7a-5p and miR-148a-3p were higher in adult male liver cells than in females(P < 0.001,P < 0.01,P < 0.001 and P < 0.05),and it was hypothesized that miR-129-5p might be act on the target gene STAT5 B,which inhibits the expression of this protein posttranscriptionally in male liver cells by preventing translation;let-7a-5p might be act on the target gene GHR,miR-128-3p and miR-148a-3p both might be act on the target gene IGF-1 while GHR and IGF-1 are expressed at higher levels in the liver of adult female turtles than in males,Speculating that miRNA might be involved in the degradation of m RNAs in the liver cells of male individuals which in turn showed the differences in expression between female and male turtles.In this study,the endogenous mechanism of SSD production in turtles was explored for the first time from an epigenetic perspective.The miRNAs potentially regulating the relevant genes were predicted by bioinformatics software.qRT-PCR was used to detect the expression of miRNAs and by comparison of its expression with its target m RNAs.It was hypothesized that several miRNAs were involved in the regulation of STAT5 B signaling pathway,which in turn had an impact on the formation of SSD in turtles.This study fullfils a gap in the turtle-related field and provides a new idea to further reveal the endogenous mechanism of SSD formation in vertebrates.