Identification And Functional Verification Of MicroRNA Related To Early Environmental Adaptation Of Piglets

Newborn mammals are faced with a great transition from the intrauterine environment to the intrauterine environment at birth,which requires the rapid adaptive development of the body’s major systems,so as to successfully complete the transition from the fetus to the newborn.miRNAs are a class of endogenous non-coding RNAs with a length of 18-24 nt,which play an important role in degrading or inhibiting the expression of target genes through the combination of seed sequences and m RNA 3’UTR.In recent years,studies have found that miRNA plays an important role in the development of mammals,but its role and mechanism in early extra-uterine environment adaptation of newborn mammals are rarely studied.In this study,small RNA sequencing was performed on 6 tissues(heart,liver,spleen,lung,longissimus dorsi and duodenum)of piglets 113 days after gestation and6 hours after birth,aimed at mining the early postpartum adaptation-related miRNAs of newborn piglets,and verified the potential mechanism of miRNA involvement in postpartum adaptation through the in vitro miRNA overexpression and inhibition experimental.The main results of this study are as follows:(1)In this study,we conducted small RNA library sequencing on 35 samples from 6tissues of piglets before and after birth.A total of 491.16 million(M)Raw reads were generated,with an average of 14.03 M(14.03 ± 1.38 M,n = 35)for each sample.After a series of strict data filtering,a total of 467.33 M Clean reads were generated for the identification of miRNAs,and the average high-quality data obtained from each sample was 13.35 M(13.35 ± 1.6 M,n = 35).Using the miRDeep software,a total of 971 miRNAs precursors and 1511 corresponding mature miRNAs were identified.(2)A total of 881 tissue-specific miRNAs(including 141 ’Known’ miRNAs,483’Conserve’ miRNAs and 257 ’Novel’ miRNAs)were identified in 35 sequencing samples by using tissue-specific expression pattern analysis.A total of 164 non-redundant differentially expressed miRNAs(DE miRNAs)were detected in 6 tissues between pre-natal and post-natal stages,including 34 in the heart,37 in the liver,33 in the spleen,38 in the lung,21 in the longest dorsal muscle,and 60 in the duodenum(there were repeated types of DE miRNAs between tissues).Through the conservative analysis of miRNA difference patterns before and after birth in various tissues of piglets,it was found that most of difference patterns of DE miRNAs(119/164,72.56%)were not conservative between tissues(such miRNAs were only differentially expressed in a single tissue),and that DE miRNAs were differentially expressed in at most four tissues at the same time.Chi-square test showed that tissue-specific miRNAs were significantly enriched in the DE miRNAs with non-conserved DE patterns between tissues,and these tissue-specific DE miRNAs were significantly enriched in pathways related to maintenance of tissue function.However,other DE miRNAs with conservative DE patterns between tissues play an important role in tissue development and postpartum adaptation of newborn piglets.(3)Function enrichment analysis of target genes found that DE miRNAs of 6 tissue types were significantly enriched in the GO terms and pathways that highly correlated with early extra-uterine environment adaptation,including the NF kappa B signaling pathway,PI3K/AKT signaling pathway and the Hippo signaling pathway,which are responsible to the adaptation of lung to postpartum oxygen-rich environment,the adaptation of liver to postpartum hunger and hypoglycemia,and the adaptation of heart to the postpartum changes in oxygen partial pressure,respectively.(4)Through the combined miRNA and m RNA analysis,it was found that miRNAs played an important role in the regulation of postpartum adaptation of the piglets,and we found that mir-22-3p in the liver was significantly up-regulated after birth,which may be involved in PI3K/AKT signaling pathway through target gene AKT3 to promote gluconeogenesis and maintain the glucose homeostasis of the newborn.(5)After the birth of newborn piglets(0-6 h),continuous measurement of blood glucose,PAS staining of liver slices and detection of the expression level of gluconeogenesis genes showed that the blood glucose of newborn piglets continuously decreased and reached the lowest point at 3 h after birth,and then gradually increased and maintained a certain level.The liver glycogen of piglets decreased continuously from 0 to6 h after birth.The expression level of gluconeogenesis genes continued to increase,indicating that in addition to mobilize the liver glycogen reserve,newborn piglets also converted a large amount of non-carbohydrate substances into glucose by improving the gluconeogenesis of their liver,so as to maintain the glucose homeostasis of the body.(6)Dual luciferase reporter system showed that AKT3 was the target gene of mir-22-3p,and the overexpression and inhibition of mir-22-3p in Hep G2 cells showed that mir-22-3p activated the AKT/Fox O1 pathway by inhibiting the target gene AKT3,promoting gluconeogenesis and increasing glucose production,thereby maintaining the glucose homeostasis of the body.Collectively,this study found that miRNAs play an important role in early extra-uterine environment adaptation of newborn piglets.In addition,this study also found that mir-22-3p was significantly upregulated in the liver after birth.By inhibiting the expression of the target gene AKT3,it further regulated the AKT/Fox O1 pathway to promote gluconeogenesis,increase the glucose production of newborn piglets,and maintain the glucose homeostasis of the body.