MiR-19b-3p Regulates Hepatocyte Apoptosis In High Fat Diet Induced Layers By Targeting SOCS3

As a high-energy feed,oil can reduce animal stress,increase feed absorption utilization and improve animal performance,so the production will promote the growth and development of poultry by increasing the proportion of oil in feed.Liver is an important organ which could regulate lipid decomposition,and the integrity of liver cell structure and function is the premise of all kinds of life activities.Liver cells maintain dynamic balance through mitochondrial β-oxidative decomposition of fatty acids,excretion of low density lipoprotein,synthesis of triglycerides and other physiological activities.However,the excessive use of oil in feed can lead to the disruption of and the internal apoptosis of poultry liver cells.Micro RNA(miRNAs)are innate non-coding RNAs which could inhibit the expression of m RNA by binding to the 3’UTR,or control gene expression by directly degrading target m RNA.Mi RNAs play an important role in a variety of life activities,including growth and development,host-pathogen interaction,and tumorigenesis.Studies have shown that miRNAs also participate in liver cell apoptosis induced by high fat,but the specific mechanism and related pathways are unclear.The purpose of this study was to explore the molecular mechanism of miRNAs in liver disease induced by high fat diet.In this study,a high-fat diet feeding model of laying hens was established,and miR-19b-3p was screened out by preliminary test.Combined with targetscan website prediction,dual luciferase reporter gene detection and q RT-PCR analysis,the specific target gene SOCS3 was screened.HE staining,oil red O staining,transmission electron microscopy,q RT-PCR,Western blot,AO/EB staining and flow cytometry were used to observe and detect the apoptosis of liver tissues and miR-19b-3p overexpression/knockdown LMH cells.The test results are as follows:1.A fatty liver model induced by high fat diet was established.HE staining displayed that the liver lobules in the normal group were neatly arranged,while the fat vesicles in the liver tissue of the high fat group were significantly increased.Therefore,the high fat feeding model of laying hens was successfully established.TEM photos displayed that the endoplasmic reticulum of liver cells in the high fat group was expanded,the rough endoplasmic reticulum disappeared,the swelling mitochondria formed clusters and the electron density of the mitochondrial crest was reduced,and the apoptosis was obvious.2.The wild-type and mutated plasmids of SOCS3 were successfully constructed,and the negative targeting relationship between miR-19b-3p and SOCS3 was confirmed by dual luciferase reporter gene detection.In vitro,we cultured chicken liver cancer cell lines to construct knockdown and over expression models of miR-19b-3p.We found that the m RNA and protein levels of SOCS3 were significantly increased in the low-expression group of miR-19b-3p and significantly decreased in the over expression group.It has also been confirmed in vivo that high-fat diet can significantly inhibit the expression of miR-19b-3p in liver and up-regulate the expression of target gene SOCS3.3.In vitro experiments proved that miR-19b-3p induced apoptosis through JAK2/STAT3 pathway.In this study,Colivelin TFA was added with JAK2/STAT3 inducer,AO/EB staining and flow cytometry results showed that miR-19b-3p induced apoptosis through JAK2/STAT3 pathway.Colivelin TFA can reverse the increased apoptosis rate caused by the low expression of miR-19b-3p.q RT-PCR and Western blot displayed that the content of JAK2 and STAT3 was decreased in the miR-19b-3p low-expression group,and increased in the miR-19b-3p high-expression group.Moreover,Colivelin TFA can reverse the increased expression of apoptotic genes caused by the low expression of miR-19b-3p.4.In vivo and in vitro results jointly confirmed that high-fat diet mediated apoptosis of hepatocyte mitochondrial pathway through miR-19b-3p/SOCS3.The m RNA and protein levels of SOCS3 and its downstream mitochondria related apoptotic genes(Bax,Bak,Cyt-c,Caspase3,Caspase9)were significantly increased in the high-fat group,while the m RNA and protein levels of anti-apoptotic gene Bcl2 were significantly decreased in the high-fat group.The m RNA and protein levels of SOCS3 and its downstream mitochondria related apoptotic genes in LMH cells with low expression of miR-19b-3p in vitro were significantly increased,while the m RNA and protein levels of anti-apoptotic gene Bcl2 were significantly decreased.The m RNA and protein levels of SOCS3 and its downstream mitochondria related apoptotic genes in LMH cells with high expression of miR-19b-3p in vitro were significantly decreased,while the m RNA and protein levels of anti-apoptotic gene Bcl2 were significantly increased.In conclusion,high fat diet can induce reduced expression of miR-19b-3p,and miR-19b-3p can target SOCS3 to inhibit the JAK2/STAT3 pathway,and subsequently induce mitochondrial pathway apoptosis in laying hens.This provides a new insight into the role of miRNAs in liver cell apoptosis induced by high fat diet,and also provides a new idea for the development of targeted drugs for liver disease induced by high fat diet.