Effects Of MiRNA-2954 Mediated PI3K On Myocardial Apoptosis And Autophagy Induced By Selenium Deficiency In Chicken

Selenium is a trace element plays important roles in maintaining physiologic functions and homeo-stasis in human and animals.It is an important component of selenoprotein and has multiple regulatory effects on endocrine,immune response,inflammatory response and reproductive processes.Selenium deficiency can lead to a variety of diseases,including pig mulberry heart,poultry exudative quality,and injury of chicken brain,heart injury,human Keshan disease and so on.In 1930,a series of human cardiomyopathy caused by selenium deficiency was first described in China.Cardiogenic shock,cardiac hypertrophy,congestive heart failure and heart rate disorder were included above and named as Keshan disease.Multifocal necrosis and myocardial replacement by fibrous tissue are characteristic lesions of Keshan disease.In addition,selenium deficiency can lead to apoptosis,autophagy and necrosis of cardiomyocytes through increased oxidative stress and inflammation in poultry,leading to cardiac injury in chickens.In recent years,mi RNAs,a kind of highly conserved small molecule RNA has been proved paticipating in a new pathway of cell death,autophagy.In addition,evidence indicated that it plays an important role in multiple types of heart disease and various biological processes.Based on the selenium deficiency model in broilers,ultrastructure and histopathology structure of myocardial tissue were observed.mi RNAome technology was used to detect differential expressed mi RNAs between selenium deficiency group and control group heart tissue,and mi RNA-2954 exists close relationship with cardiomyopathy studies.Thus,cell transfection technique was used to establish mi RNA-2954 knockdown/over-expression model in vitro.RT-PCR,Western blot technique was used to detect expression of m RNA and protein in vivo and in vitro.Autophagy staining and flow cytometry were used to detect the apoptosis of autophagy.The results indicated that:1)In this experiment,180 broilers were randomly divided into two groups.90 broilers were fed selenium deficiency(Se 0.008 mg/kg)diet and 90 broilers were fed normal diet(Se 0.2 mg/kg)for 30 days.Results showed that there were obvious exudative diathesis and cardiac bleeding spots in broilers.Autophagy was observed in the ultrastructural observation of myocardial cells in selenium deficiency group,which confirmed that the autophagy model of myocardial cells was successfully replicated in sedeficient chicken.2)Among the 468 micro RNAs detected by mi RNAome in selenium deficiency group and control group heart tissue,11 micro RNAs were differentially expressed,among which 7 mi RNA expression was increased,4 mi RNA expression was significantly decreased,and mi RNA-2954 expression level was significantly higher than that of control group,which was proved 3.5 times higher than that of the control group by RT-PCR(P<0.05).These results suggest that selenium deficiency can change the expression profile of mi RNAs and up-regulate the expression of mi RNA-2954 in heart tissue.3)Based on the results of mi RNAome and bioinformatics,the target gene of mi RNA-2954 was predicted to be PI3 K.Knock-down/over-expression of mi RNA-2954 in cardiomyocytes were established by cell transfection technique in vitro.The results showed that the expression of mi RNA-2954 increased 75-fold in the over-expression group,and the intracellular mi RNA-2954 in the knock-down group decreased to 45% of that in the control group.The results showed that the over-expression/knock-down model of mi RNA-2954 in primary cultured cardiomyocytes was successfully established,and the results of double luciferase gene reporting,RT-PCR and Western blot confirmed that PI3 K was the target gene of mi RNA-2954.4)The m RNA levels of autophagy-associated genes IRS,RAS,MEK1,MEK2,ERK,TSC1,TSC2,ULK1,ATG13,LKB1,BECLIN1 and LC3 in heart tissue of se-deficient broilers increased significantly,while the expression levels of m TOR and AKT decreased significantly.The m RNA expression level of apoptosis-related genes Caspase3,Caspase8,Caspase9,Cytc,BAX,BAK,P53,JNK and TNF increased significantly,while the m RNA level of BCL2 decreased(P<0.05).Expression of LC3 and BECLIN1 in protein level was increased.The expression of apoptosis pathway protein BAX,Caspase3,Caspase8 increased and the expression of anti-apoptotic protein BCL2 decreased,the results showed that selenium deficiency could induce autophagy and apoptosis in myocardium.5)In vitro,the autophagy of mi RNA-2954 overexpressed cardiomyocytes was significantly increased by MDC staining,and the apoptosis rate of over-expressed cardiomyocytes was significantly higher than that of the control group by flow cytometry.The results of MDC staining and flow cytometry showed that the autophagy and apoptosis of cardiomyocytes in mi RNA-2954 inhibition group were significantly lower than those in control group.In conclusion,over-expression of mi RNA-2954 could inhibit the expression of PI3 K in chicken cardiomyocytes,leading to the increase of autophagy and apoptosis-related genes and a decrease in the expression of anti-apoptotic genes in PI3K/AKT pathway,during selenium deficiency,mi RNA-2954 was up-regulated in chicken myocardium,which induced autophagy and apoptosis via targeting PI3 K pathway,which laid a foundation for further study the mechanism and regulation pathway between mi RNA-2954 and selenium deficiency,then provide a theoretical basis for the prevention and treatment of selenium deficiency heart diseases.