MiR-144-3p Targets STC1 Activate PI3K/AKT Pathway To Induce Bursa Cell Apoptosis And Cell Cycle Arrest In Selenium Deficient Broilers

Selenium is an essential trace element in the animal body.It participates in various physiological processes in the form of selenoprotein in the body.In recent years,studies have found that selenium has the functions of preventing cardiovascular and cerebrovascular diseases,reducing the risk of cancer,and enhancing immunity.Selenium deficiency can cause damage to the animal body.At present,it is known that selenium deficiency can cause damage to the animal body.However,the specific mechanism of selenium deficiency on the immune system of broilers,especially the damage to the bursa of Fabricius,remains to be further studied.With the in-depth research on micro RNA(miRNA)in recent years,it has been discovered that miRNA plays an important role in the development of the body.Selenium deficiency can regulate the expression of miRNAs,and changes in the expression of these miRNAs can participate in a variety of damage processes in tissues.Therefore,it is very important to study the damage of selenium deficiency to the organs and tissues of broilers and the role of miRNAs in the prevention and treatment of selenium deficiency diseases in broilers.MiRNAs are endogenous m RNAs found in eukaryotes,about 20-25 nucleotides in length,and have certain regulatory functions in the body.MiRNAs can control the expression of the target gene by inhibiting the transcription of the target gene RNA.MiR-144-3p has been proven to induce apoptosis and inhibit cell proliferation.The bursa of Fabricius is a unique central immune organ of poultry.It is located above the cloaca.The cyst wall is filled with lymphocytes and is the main place for the development of B lymphocytes in young birds.After B cells mature,they produce specific antibodies in the form of humoral immunity to complete the immune response.In this experiment,a selenium-deficient broiler model was first constructed,and miRNA-144-3p with significantly different expression in the bursa of fabric was screened out.Through the observation of the microstructure of the bursa tissue,the prediction of biological information,the dual luciferase reporter gene method,qRT-PCR,Western Blot,and flow cytometry,the broiler bursa of fabric was detected and knocked down or overexpressed miRNAs.-144-3p DF1 cells,changes in apoptosis and cell cycle,and changes in the expression of related genes.The main research results are as follows:(1)In this experiment,after collecting the bursa tissue of selenium-deficient broilers,the microstructure of the bursa tissue was observed by fixing and staining and other steps.The results showed that in the selenium-deficient group,the inside of the bursa tissue The boundaries of the cortex and medulla become blurred,the number of lymphocytes is significantly reduced,and the connective tissue that connects the vesicles is broken.It shows that selenium deficiency can cause obvious damage to broiler’s bursa tissue.(2)The expression of miR-144-3p was significantly increased in the bursa tissue of selenium-deficient broilers.Target gene prediction was carried out through miRDB,mirbase and other websites,and the potential miR-144-3p target gene STC1 was screened out,and the target gene was verified by the method of dual luciferase reporter gene.The knockdown and overexpression model of miR-144-3p was established by DF1 cells.Western Blot and qRT-PCR were used to detect the expression of STC1 gene and protein.The results showed that the m RNA and protein levels of STC1 in the overexpression group were significant.Decreased,but significantly increased in the knockdown group.Combined with the dual luciferase gene verification results,it jointly proved the targeting relationship between miR-144-3p and STC1.(3)After knocking down miR-144-3p to increase the expression of STC1,the expression of PI3 K and AKT was tested by qRT-PCR and Western Blot,and it was found that the expression of PI3 K and AKT was significantly reduced when STC1 was increased.,While in the overexpression group,when the expression of STC1 decreased,the expression of PI3 K and AKT increased significantly.At the same time,by detecting the gene expression downstream of the PI3K/AKT pathway,it was found that when the expression of STC1 decreased,while the expression of PI3 K and AKT increased,the expression of the downstream genes of the PI3K/AKT pathway increased at the same time,and when the expression of STC1 increased,The expression of downstream genes in the PI3K/AKT pathway was also reduced accordingly.Therefore,the results of this experiment indicate that the expression changes of STC1 are involved in the regulation of the PI3K/AKT pathway.(4)TUNEL was used to detect cell apoptosis in the bursa of Fabricius,and it was found that compared with the control group,the number of apoptotic cells in the selenium-deficient group increased significantly.QRT-PCR and Western Blot were used to detect the expression of genes caspase9,BAD,CREB,MCL-1 and BCL-2 downstream of the PI3K/AKT pathway in the bursa tissue and DF1 cells,which are involved in regulating cell apoptosis and cell apoptosis.Finally,the cells in the knockdown and overexpression groups were tested for apoptosis by flow cytometry.The results showed that compared with the control group,in the selenium deficiency and miR-144-3p overexpression groups,the expression of pro-apoptotic genes increased,and the proportion of apoptotic cells increased significantly.On the contrary,the expression of apoptotic genes in the knockdown group decreased,and the proportion of apoptotic cells decreased.The results show that selenium deficiency can activate the PI3K/AKT signaling pathway through miR-144-3p,promote the expression of apoptosis-related genes downstream of this pathway,and induce cell apoptosis in the bursa of fabric.(5)In this experiment,qRT-PCR and Western Blot were used to detect the expression of PI3K/AKT downstream cell cycle-related genes in the bursa tissue and DF1 cells,and it was found that the selenium deficiency group and the overexpression group DF1 cells,The expression of GSK-3β,P21,P27 was significantly reduced,and the expression of CDK2,CDK6,Cyclin B1,Cyclin D1,and Cyclin E1 were significantly increased.The cell cycle distribution changes were detected by flow cytometry and it was found that compared with the control group,the proportion of cells in the G1 and S phases in the overexpression group was significantly increased,and the proportion of cells in the G2 phase was significantly reduced;while in the knockdown group,the G1 phase The ratio of cells in the S and S phase decreased,and the ratio of cells in the G2 phase increased significantly.It proves that selenium deficiency can target STC1 through miR-144-3p to participate in cell cycle regulation.In summary,selenium deficiency can cause significant damage to the bursa of fabric in broilers.The expression of miR-144-3p is significantly increased when selenium is deficient,miR-144-3p can activate PI3K/AKT pathway by targeting STC1,induce apoptosis and cell cycle arrest,and ultimately lead to the damage of the bursa tissue structure.This study further supplements the mechanism of the effect of selenium deficiency on the immune organs of the bursa of fabric.By studying miRNA methods,it provides new data support for the prevention and treatment of broiler immune damage caused by selenium deficiency in production.