The Role And Mechanism Of MiR-128-3p In Spermatogenic Apoptosis Induced By Testicular Heat

Part 1 Differential miRNA analysis and validation of sequencing of mouse testis before and after heat[Purpose] Bioinformatics analysis and verification of miRNA sequencing results before and after testicular heat treatment in mice to identify key miRNAs and important pathways in spermatogenic damage after testis heated.[Methods] Based on the previous results of the sequencing,bioinformatics analysis of the obtained differential miRNAs(multiple ? 2)was carried out,including prediction of differential miRNAs target genes by miRanda software,and GO enrichment and KEGG pathway analysis of predicted target genes.The mouse testis was then water bathed at43 °C,and the mouse testis tissue was obtained.The RNA of the testis tissue of the mice before and after heat shock was extracted,and then the miRNA sequencing results were verified by stem-loop primer RT-qPCR.[Results] A total of 5,392 target genes of differentially expressed miRNAs were predicted,and it was found that they were enriched in biological processes,cell components and molecular functions through GO analysis.KEGG pathway analysis revealed that they were mainly involved in several pathways,including ribosomal pathway,hypoxia inducible factor 1(HIF-1)signaling pathway,mitogen-activated protein kinase(MAPK)pathway and oxidative phosphorylation.RT-qPCR of these differentially expressed miRNAs showed that seven down-regulated miRNAs were consistent with the sequencing results,while only mir-21a-3p of the four up-regulated miRNAs was consistent with the sequencing results.[Conclusions] From the results we can see that the eight miRNAs of miR-449a-3p,miR-298-5p,miR-92a-1-5p,miR-423-3p,miR-423-5p,miR-128-3p,miR-340-3p and miR-21a-3p are identical in accord with sequencing results,and these identified miRNAs are involved in different pathways(such as ribosome,HIF-1 and MAPK),which may bethe key to testicular injury caused by heat stress.Part 2 Explore the role and mechanism of miR-128-3p through MAPK14 in heat-induced spermatogenic apoptosis in vitro[Purpose] On the basis of the first part,it was found that miR-128-3p was down-regulated after testis heated.The aim of this study was to investigate the expression of MAPK14 activation-related proteins in mice before and after testis heated,and to verify the relationship between miR-128-3p and MAPK14.The effect of miR-128-3p on the proliferation and apoptosis of spermatogenic cells by regulating MAPK14 activation was explored in the mouse spermatocyte cell line(GC2-spd).[Methods] The expression of MAPK14 and phosphorylated MAPK14 was detected by Western blot after testis heated 3,6 and 12 hours.The several target gene prediction software of miRNA TargetScan,DIANA microT,miRDB and miRPath,etc.,were used to predict the target relationship between miR-128-3p and MAPK14.Then,it was verified by the dual luciferase gene reporter system.Based on the spermatocyte heated model established in the our previous group,optimize the heat shock conditions and construct a new heat shock model.firstly,the cells were cultured to a suitable density in a 37°C 5%CO2 incubator and moved to 43°C in a 5% CO2 incubator for one hour before returning to the original incubator for further culture.The expression of miR-128-3p was detected by RT-qPCR in GC2-spd cells before heat treatment and 3,6,9,and 12 hours after heat treatment,and the expression of MAPK14 and phosphorylated MAPK14(p-MAPK14)were detected by Western blot,and CCK8 cell proliferation assay method for detecting cell proliferation activity.The inhibitors and mimics of miR-128-3p were used to interfere with the spermatocytes before and after heat treatment,and then the corresponding expression changes of MAPK14 and p-MAPK14 were detected by Western blot,and the cellproliferation activity were detected by CCK8 cell proliferation assay and AnnexinV-FITC/PI apoptosis assay was used to detect cell apoptosis.[Results] Compared with the control group,phosphorylated MAPK14(p-MAPK14)was significantly increased at 3 and 6 hours after testis heated in mice(p<0.001),and there was no statistical significance at 12 hours after testis heated(p=0.149).Among the six groups co-transfected,only the luciferase activity of the co-transfected wild-type vector and the miR-128-3p mimics group was significantly lower than that of the other groups,and there was no statistical difference between the other groups.After GC2-spd cells were heated for one hour at 43°C in a 5% CO2 incubator,no large number cell death occurred when observed under the microscope.Compared with the control group,the morphology was only slightly rounded.Compared with pre-heat treatment,miR-128-3p was significantly decreased(p<0.01)in GC2-spd cell after heat treatment 3 and 6 hours,and recovered after heat treatment 9 hours and increased after heat treatment 12 hours(p<0.01);The p-MAPK14 which is activated form of MAPK was found increased significantly at 3and 6 hours after heat treatment(p<0.0001,p=0.004)and recovered at 9 and 12 hours(p=0.03,p=0.735);After GC2-spd cells heated 3,6,and 9 hours,the proliferation activity of GC2-spd cells was decreased(p=0.002,p=0.005,p<0.001),and recovered at 12 hours after heat treatment(p=0.444).In the miR-128-3p interference assay,the expression of p-MAPK14 in the miR-128-3p inhibitor(antagomir)group increased and reached the heat treatment group level,while the p-MAPK14 in the group combined heat treatment with transfecting miR-128-3p mimic(agomir)was significantly lower than that in the only heat treatment group and reached the level of the control group(no heat treatment).The cell proliferation activity of each group was detected.Compared with the control group,the proliferation activity between the group transfected with miR-128-3p antagomir and heat treatment group was not statistically significant,but lower than that of the control group(p<0.01),while the cell proliferation activity of the group combine heat treatment with miR-128-3p agomir was significantly reversed(p<0.001)compared with heat treatmentonly group.There was not statistically significant compared with the control group.In the apoptosis assay,the cell apoptosis rate of miR-128-3p antagomir group and heat treatment group was not statistically significant,but significantly elevated(p<0.001)compared with the control group.The cell apoptosis rate of group heated coupled with transfected with miR-128-3p mimics was significantly reversed compared with heat treatment only group(p=0.004),but not fully recovered,still higher than the control group(p=0.007).[Conclusions] At 3 and 6 hours after testis heated in mice,MAPK14 was activated and p-MAPK14 expression was elevated.And MAPK14 is a target gene of miR-128-3p.miR-128-3p was down-regulated and p-MAPK14 was up-regulated at 3 and 6 hours after GC2-spd heat treatment in vitro.miRNA interference experiments in vitro showed that the activation of MAPK pathway may be mediated by the down-regulation of mir-128-3p after GC2-spd heat treatment,and miR-128-3p down-regulated plays an important role in the proliferation inhibition and apoptosis induction after GC2-spd heat treatment.