Preliminary Study On The Expression Of Zearalenone On MicroRNA In TM3 Cells And Its Apoptosis Mechanism

Background: Zearalenone(ZEA)is an estrogen-active mycotoxin that acts primarily on the reproductive system of animals and can cause reproductive failure in livestock,causing severe damage to agriculture and animal husbandry;micro RNAs(mi RNAs)are eukaryotic A18-26 bp non-coding nucleotide fragment in the organism,which is closely related to the developmental and physiological processes of the organism.Objective: This study was to elucidate whether mi RNA is involved in the regulation of ZEA in germ cell toxicology and to verify whether ZEA causes germ cell apoptosis through the PI3K/AKT pathway.Methods: The optimal concentration of ZEA on mouse Leydig cells was determined by CCK-8 kit and the median lethal concentration was calculated.The cell-induced model of ZEA on mouse Leydig cells was established.The apoptosis of mouse Leydig cells was detected by TUNEL by TUNEL.RNAs from the blank group and the ZEA-treated group were extracted,and mi RNA sequencing was performed to screen differentially expressed mi RNAs after 3 replicates in each group.The mi RNA sequencing results were verified by RT-PCR.The PI3K/AKT signaling pathway screened by mi RNA sequencing confirmed whether ZEA promoted apoptosis of mouse Leydig cells via PI3K/AKT pathway by constructing adenoviral vector to interfere with PTEN gene.Flow cytometry confirmed the apoptosis of mouse testicular cells.Western-blot confirmed the changes of PTEN,AKT,p-AKT,Bax and Bcl-2proteins in PI3K/AKT pathway.Result:1.The CCK-8 kit showed that the growth of mouse Leydig cells was inhibited with the increase of ZEA concentration.The IC50 value of ZEA on mouse Leydig cells was IC50=53.33?mol/L.The results of TUNEL showed that when the mouse Leydig cells were treated with 53.33 ?mol/L ZEA for 24 hours,the mouse Leydig cells showed obvious apoptosis.2.mi RNA sequencing results showed that there were 197 mi RNAs significantly changed in the ZEA-treated group compared with the control group(p<0.05),of which 86 were up-regulated and 111 were down-regulated.Target gene prediction was performed on differentially expressed mi RNAs by bioinformatics and GO and KEGG analysis were performed.Sixteen zebra-related mi RNAs were screened and mi RNA-target gene-signal pathway interaction maps were mapped.The mi RNA-target gene-signal pathway interaction map indicates that the Ras signaling pathway,Rap1 signaling pathway,PI3K-AKT signalingpathway,Foxo signaling pathway,AMPK signaling pathway and MAPK signaling pathway are closely related to ZEA toxicology.The mi RNA and target genes were verified by RT-PCR,and the results were consistent with the sequencing results.3.After constructing the adenoviral vector,the transfection fluorescence microscope was used to observe the transfection of adenovirus carrying the GFP fluorescence enhanced label,and the optimal multiplicity of infection(MOI)was 50 MOI.Western-blot results showed that adenovirus effectively interfered with the expression of PTEN protein.The results of cytometry showed that PTEN protein silencing in mouse Leydig cells inhibited ZEA-induced apoptosis.The PTEN and Bax proteins in the ZEA group were significantly up-regulated compared with the blank group(p<0.01).The AKT,p-AKT,and Bcl-2 proteins in the ZEA group were significantly down-regulated compared with the blank group(p<0.05).The expression of the NC+ZEA group and the ZEA histone were basically the same.The PTEN and Bax proteins in the sh RNA+ZEA group were significantly down-regulated compared with the ZEA group(p<0.05),and the AKT,p-AKT,Bcl-2 proteins in the sh RNA+ZEA group were significantly down-regulated compared with the ZEA group(p<0.05).Conclusion:1.ZEA can induce apoptosis of mouse Leydig cells.The half-inhibitory concentration of ZEA on mouse Leydig cells is IC50=53.33?mol/L2.Sixteen micro RNAs were associated with ZEA toxicity in TM3 Leydig cells after ZEA exposure.GO enrichment analysis and pathway interaction analysis showed that micro RNAs and target genes were closely related to the toxicological mechanism of ZEN through Ras signaling pathway,Rap1 signaling pathway,PI3K-AKT signaling pathway,Foxo signaling pathway,AMPK signaling pathway and MAPK signaling pathway.3.ZEA may induce apoptosis of mouse Leydig cells through PI3K/AKT signaling pathway.PTEN protein may be an important target of ZEA in PI3K/AKT signaling pathway.