MicroRNA-193b-3p Regulates Hepatocyte Apoptosis In Selenium- Deficient Broilers By Targeting MAML1

Selenium(Se)is an essential trace element that plays a role in maintaining body health and maintaining homeostasis.Se deficiency affects cell proliferation,oxidative stress,and cell survival,and can cause damage to many organs,such as brain,vein,muscle,intestine,and liver.Many studies provide evidence that dietary Se deficiency can induce hepatocyte apoptosis in the body.The liver is the main organ for the body to absorb nutrients and is one of the main targets of Se deficiency.Hepatocyte apoptosis is a characteristic pathological change in Se-deficient liver tissue.As a non-coding RNA,microRNA(miRNA)can silence the target gene by targeting the transcription of the target gene and then regulating the protein expression to control different biological reactions in vivo.It plays an important role in cell proliferation,differentiation,apoptosis and tumor formation.Studies have shown that various mirnas are related to apoptotic factors and participate in the regulation of apoptosis.MiRNA is found to play a huge role in liver disease.In recent years,some studies have explained that changes in Se content can change the expression of miRNA in related organs.However,the specific mechanism of apoptosis during Se deficiency and how miRNA regulates hepatocyte apoptosis in broilers have not been perfected.The purpose of this study was to investigate whether miRNAs can regulate Se-induced hepatocyte apoptosis.In order to elucidate the research mechanism of Se deficiency on specific miRNA and its target protein in the process of hepatic cell apoptosis,we successfully replicated Se deficiency chicken model.We selected a specific miRNA,miR-193b-3p,through laboratory genomics and real-time quantitative PCR(qRT-PCR).Combined with bioinformatics prediction,dual-luciferase reporter gene detection system and qRT-PCR method were used to analyze and screen the specific target protein MAML1 of Sedeficient chicken liver apoptosis.We examined apoptosis in Se-deficient broiler livers and primary hepatocytes transfected with miR-193b-3p by ultrastructural observation,qRT-PCR,Western blot(WB),and flow cytometry.The test results are as follows:1)Based on successful replication of Se-deficient broiler models,we observed changes in hepatocytes in Se-deficient chickens.Ultrastructural observation revealed that the hepatocyte nuclei were concentrated,the nucleolus became smaller,the karyoplasm became agglomerated around the nuclear membrane,mitochondria produced vacuoles,some mitochondria ruptured,and mitochondria disappeared.It demonstrated that Se deficiency caused apoptosis of broiler hepatocytes.2)The miR-193b-3p was selected as a Se-deficient miRNA and was significantly expressed in the liver of Se-deficient chickens.Based on the model of primary cultured chicken hepatocytes,a miR-193b-3p overexpression and knockdown model was successfully constructed.The relationship between miR-193b-3p and its target gene MAML1 was predicted and successfully verified.The expression of miR-193b-3p was negatively correlated with the expression of MAML1.3)On the basis of establishment of miR-193b-3p overexpression/inhibition model in primary cultured chicken hepatocytes,miR-193b-3p overexpressing hepatocytes were treated with STAT1 inhibitor Fludarabine(Flu).The results showed that when miR-193b-3p overexpressed in hepatocytes,the cells showed early apoptosis,and the rate of late apoptosis was significantly higher than that of the control group.After knocking down miR-193b-3p,the early apoptotic cells decreased significantly compared with the control group.After adding Flu,the apoptosis rate was still lower than that of the control group.4)Through the study of the apoptosis molecular mechanism induced by Se deficiency,it was found that the apoptosis induced by miR-193b-3p overexpression may be generated through the mitochondrial pathway.qRT-PCR and WB methods detected the expression of key genes in the apoptotic pathway.The results showed that miR-193b-3p overexpression increased the release of IFN?,STAT1,IRF1,Bak,Bax,Cyt-c,Caspase9 and Caspase3,and inhibited the release of MAML1 and Bcl2.5)The expression of miR-193b-3p was up-regulated and the expression of MAML1 was down-regulated in the liver tissues of Se-deficient chickens.To further verify the hepatocytes apoptosis regulation mechanism,we detected the expression of apoptosis-related genes in the liver of Se-deficient broiler chickens.The results were consistent with miR-193b-3p transfection in primary cultured hepatocytes in vitro,suggesting that miR-193b-3p and its target gene MAML1 participate in the apoptosis of liver mitochondrial pathway caused by Se deficiency.In conclusion,our results show that Se deficiency can mediate the apoptosis of broiler chicken hepatocytes involved in the mitochondrial pathway through the regulation of miR-193b-3p expression.This provides new insights into the mechanism of hepatocellular injury induced by Se deficiency.