| MicroRNA(miRNA)is a kind of small RNA encoded by endogenous genes and not involved in protein coding,which controls gene expression after transcription through translation inhibition or mRNA degradation,and participates in the process of cell differentiation,proliferation,apoptosis and tumorigenesis,and plays a vital place in the process of metabolism regulation in vivo.In addition to providing structural support for spermatogenic cells,Sertoli cells(SCs)could also synthesize and secrete lactate,which is often regulated by insulin and other factors.Our previous research revealed that mild heat treatment(hereinafter referred to as heat treatment)could enhance the insulin receptor substrate 2(IRS2)expression,promote the activation of PI3K/Akt signal pathway,and improve insulin sensitivity,but the interactions between heat treatment and IRS2 expression is not clear,and whether miRNA participate in insulin-stimulated lactate secretion of Sertoli cells after heat treatment remains to be studied.In this experiment,the miRNA related to IRS2 expression was predicted by targetscan,mirwalk,mi RBase and other software and databases.Then,Sertoli cells were isolated from 21-day-old piglets for subsequent experiments.The cells were heat-treated at 43℃ for 30 min to extract total RNA of cells,and the level of miRNA after heat-treatment was screened by quantitative PCR.According to the screening results,miRNA mimic and miRNA inhibitor were synthesized by artificial synthesis.Mi RNA mimic and miRNA inhibitor were respectively transfected into HEK293 T with Lipofectamine 3000 as the carrier,and the dual-Luciferase kit was adopted to verify the target gene.By detecting the expression of IRS2,we found that miRNA inhibitor has asimilar effect to heat treatment.Sertoli cells were transfected with miRNA inhibitor,and treated with 100 nM insulin for 24 h after 48 h transfection,The activity of LDH and the amount of lactate production in the culture medium were detected by the kit,qRT-PCR and Western blotting was performed to analyze the phosphorylation level of PI3K/Akt pathway and the expression of MCT1,LDHA and GLUT3.These results were as followed:(1)The miRNAs that target IRS2 gene were predicted by targetscan,mirwalk and mi RBase,and then intersected with miRNAs in pigs.Finally,seven miRNAs were screened,including ssc-miR-96-5p,ssc-miR-142-3p,ssc-mi R-7-5p,ssc-miR-142-5p,ssc-miR-133a-3p,ssc-mi R-150 and ssc-miR-155-5p.(2)After heat treatment,the expression of ssc-miR-7-5p only changed significantly,which decreased by 38.2%(P<0.01)compared with the negative control group.It was found that IRS2 played a role as the functional target gene of miR-7-5p in other cells,and ssc-miR-7-5p was selected for further analysis.(3)MiR-7-5p mimic significantly reduced the luciferase activity of 3’UTR region of IRS2,which was 43.7% lower than that in the control group(P<0.01),while there was no significant effect of mimic on luciferase activity in the mutant IRS2 3’UTR region(P>0.05);transfection of miR-7-5p inhibitor significantly increased the luciferase activity of IRS2 3’UTR region,36.5% higher than the control group(P<0.05).(4)Compared with the negative control group,heat treatment and mi R-7-5p inhibitor enhanced the expression of IRS2 protein by 81.7% and 65.4%respectively(P<0.01),which indicated that the transfection of miR-7-5p inhibitor could simulate the regulation of heat treatment on the expression of IRS2 in Sertoli cells.(5)MiR-7-5p inhibitor showed a remarkable up-regulation of the PI3 K phosphorylation level,which was 52.3%(P< 0.01)higher than that of the negative control group.Compared with the single inhibitor group,the PI3 K phosphorylation level was further increased by 45.3%(P<0.05)after insulin co-treatment with inhibitor.MiR-7-5p inhibitor showed a remarkable up-regulation of the Akt phosphorylation level,which was 44.1%(P< 0.01)higher than that of the negative control group.Compared with the single inhibitor group,the Akt phosphorylation level was further increased by22.6%(P<0.05)after insulin co-treatment with inhibitor.(6)MiR-7-5p inhibitor up-regulated the expression of GLUT3,LDHA and MCT1,the activity of LDH and the synthesis of lactate in Sertoli cells(P<0.01).(7)When miR-7-5p inhibitor co-treatmented with insulin,the expression oflactate synthesis related genes,LDH activity and the amount of Lactate Synthesis in Sertoli cells were further increased(P < 0.01)than the single inhibitor group.Based on the above results,the conclusion is drawn:1.IRS2 is a functional target gene of ssc-miR-7-5p in boar Sertoli cells,and miR-7-5p regulates the expression of IRS2 by binding to the IRS2 3’UTR region.2.MiR-7-5p inhibitor simulates the effect of heat treatment on IRS2 and activates the phosphorylation level of PI3K/Akt.The activated PI3K/Akt pathway promotes the expression of GLUT3,LDHA and MCT1,enhances LDH enzyme activity,and finally promotes lactate production in SCs.3.Cooperation of mi R-7-5p inhibitor and insulin enhance the lactate secretion function of SC through IRS2/PI3K/Akt pathway.In a word,heat treatment inhibited the expression of miR-7-5p in Sertoli cells,and the decreased mir-7-5p increasd the expression of IRS2.MiR-7-5p inhibitor can simulate the promoting effect of heat treatment on the expression of IRS2,and there is a synergistic action between miR-7-5p inhibitor and insulin,which increases the phosphorylation level of PI3K/Akt,promotes the expression of GLUT3,LDHA,MCT1,improves the activity of LDH,and promotes the synthesis of lactate in Sertoli cells. |
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