| MicroRNA(miRNA)is a class of non-coding single-stranded RNA molecules,which consist of 22 nucleotides in length encoded by an endogenous gene.miRNA can primarily decrease or inhibit the expression of target gene mRNA by complementary binding to the non-coding region of their 3’-UTR.Involved in regulation of post-transcriptional gene expression,miRNA plays an important role in a series of important physiological activities such as body’s early development,cell proliferation,cell differentiation,apoptosis and lipid metabolism.Sertoli cells(SCs)are somatic cells located in the testis.The lactate secreted by SCs is not only the preferred energy substrate for the development of spermatogenic cells,but also has an anti-apoptotic effect in spermatogenesis process.The studies of miRNA expression in SCs are important for testicular development and spermatogenesis regulation.In this study,3~4 week-old testis were used as experimental materials.SCs were treated at 43° C for 30 min to induce heat stress.Then extracted RNA of control group and heat-stress group.The miRNAs expression differentially were identified by high-throughput sequencing and analyzed by GO and KEGG.Compared with miRBase database,the novel miRNAs related to AMPK signaling pathway were selected and the target genes were predicted.On this basis,miRNA mimics and miRNA inhibitors were artificially synthesized,which were transfected into Sertoli cells with the vectors of RNAiMAX.The medium were collected for measurement of lactate secretion by lactate detection kit,and measurement of LDH activity by LDH detection kit.The expression of miRNA in diffierent treatments and the expression of genes related to lactate secretion were detected by RT-PCR.The phosphorylation level of AMPK and the proteins expression of GLUT3,LDHA and MCT1 were detected by western blot.These results were as followed:(1)Compared with the control group,16 miRNAs are differentially expressed in heat stress group,including 10 known miRNAs(ssc-miR-27b-5p,ssc-miR-215 and ssc-miR-4332 and so on),and 6 novel miRNAs(ssc-novel-miR-8-3p,ssc-novel-miR-11-3p,ssc-novel-miR-62-5p and so on).(2)Compared with the control group,miR-8-3p mimic decreased the phosphorylation level of AMPK significantly(P < 0.01)whileras miR-8-3p inhibitor increased the level of phosphorylated AMPK in Sertoli cells(P < 0.05).(3)Protein and mRNA expression of GLUT3 in miR-8-3p mimic group were increased by 35.06% and 166.25% respectively,compared with the control group.Protein and mRNA expression of GLUT3 also showed an upward trend after heat stress.MiR-8-3p inhibitor resulted in a decrease in GLUT3 protein expression by 5.32% and mRNA expression by 16.38%.(P < 0.01)(4)Compared with the control group,miR-8-3p mimic increased the protein and mRNA expression of LDHA by 14.72% and 16.74%,respectively.MiR-8-3p inhibitor resulted in a decrease of 9.56%(P < 0.05)and 22.44%(P < 0.01)of LDHA protein and mRNA expression in Sertoli cells.(5)Heat stress and miR-8-3p mimic both increased LDH activity significantly.Compared with heat stress group,heat stress after transfected miR-8-3p inhibitor induced the activity of LDH decreased by 5.21%,but increased by 19.53% compared with the control group.(P <0.01)(6)MiR-8-3p mimic increased the protein and mRNA expression of MCT1 by 15.87% and 71.13% respectively.After transfected miR-8-3p inhibitor,the protein eand mRNA expression of LDHA decreased by 9.14% and 42.43%.(P < 0.01)(7)MiR-8-3p mimic induced an increase of lactate production by 26.09%,which was 5.04% higher than that of heat stress group.MiR-8-3p inhibitor resulted in a decrease by 57.14% in SCs lactate secretion.(P < 0.01)From the analysis of the above results,the following conclusions are drawn:1.After heat stress,the most abundant target genes predicted for differential miRNAs are PI3K-Akt signaling pathway,including COL4A4(ssc-miR-29c),COL4A2(ssc-novel-miR-11-3p),ITGB1(ssc-miR-27b-5p)and other 14 target genes;followed by the Calcium signaling pathway,including CYSLTR2(ssc-novel-miR-113-3p),ITPKA(ssc-novel-miR-6-3p),P2X7R(ssc-novel-miR-8-3p)and so on.2.Ssc-novel-miR-8-3p expressed differentially,which related to response to ATP,G protein coupled receptor,glucosyltransferase,protein tyrosine/serine/threonine phosphatase activity,protein Serine/threonine phosphatase complexes,phosphatase activity,and other protein functions.3.Ssc-novel-miR-8-3p is associated with the AMPK signaling pathway,hypoxia-inducible factor-1 signaling pathway,insulin signaling pathway,and adipocytokine signaling pathway.And miR-8-3p affected the activity of AMPK phosphorylation by regulating the activity of PP2 A.4.MiR-8-3p mimic promoted the expression of GLUT3(SLC2A3),LDHA(LDHA),MCT1(SLC16A1)and the activity of LDH,also increased the lactate production significantly.MiR-8-3p inhibitor reduced the expression of GLUT3(SLC2A3),LDHA(LDHA),MCT1(SLC16A1)and the activity of LDH,also reduced the lactate production of SCs.In summary,heat stress induces an alterations of miRNA expression in SCs.As a novel miRNA,ssc-novel-miR-8-3p closely related to AMPK signaling pathway and promoted the lactate production in SCs.MiR-8-3p mimic reduced the activity of AMPK phosphorylation.It up-regulated the expression of genes which related to lactate production,and the proteins expression of GLUT3,LDHA,MCT1,and promoted the LDH activity and lactate production.Therefore,ssc-novel-miR-8-3p is a positive regulator that induces an increase of lactate production in SCs and it can be a candidate molecular for anti-apoptosis of spermatogenic cells. |
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