Roles Of MiRNAs In The Mechanism Of Microcystin-LR-induced Male Reproductive System Toxicity

Microcystins(MCs)are a class of natural cyclic heptapeptide toxins produced by several genera of freshwater cyanobacteria.Previous studies have shown that MCs invoke hepatotoxicity and neurotoxicity,kidney impairment,and gastrointestinal disorders.In our previous study,we has showed that MCs can induce reproductive system toxicity.Chronic low dose treatment with MC-LR resulted in substantial toxicity to male reproduction,reducing serum testosterone level,causing declines in sperm quality and injury to the testis after exposure for 6 months.In addition,apoptotic cells and damaged testicular cells were observed histological after 6 months ofexposure.We also further demonstrated that spermatogo nial cell and Sertoli cell are the targets of MCs.Spermatogonial cell and Sertoli cell play an important role during spermatogenesis.It has showed that miRNA plays a critical role in regulation of spermatogenesis and the miRNAs expression are found to be modulated in the semen samples from infertile patients in previous studies.It has also indicated that MCs can alter the expression of miRNAs in liver of mice in a recent study.The main purpose of the current study was to investigate the effect of MC-LR on miRNA expression and whether this could mprovide a mechanistic explanation for declines in sperm quality.Part 1 Effects and mechanism of MC-LR on miRNA expression in GC-1(?)Effects of MC-LR on miRNA profiling expression in GC-1ObjectiveTo study the effects of MC-LR on miRNA profiling expression in GC-1 and screening the miRNA associated with spermatogenesisMethods1.GC-lcell were divided into 8 grous to detect:Control,0.5 nM,5 nM,50 nM,500 nM,1?M,10 ?M,100 ?M MC-LR goups.2.In order to choose the optimal concentration of MC-LR for miRNA array,cell viability and morphology were recorded in GC-1 cells after exposure to a range of concentrations.We observed cell morphology under microscope;We use CCK-8 to examine the MC-LR toxicology on cell viability;Fluorescent photo micrographs of cells dyed with FDA/PI were used to observe the effects of MC-LR on live or death of cells.3.We use the low density taqman microarray to detect the altertion miRNA of GC-1 cell after exposure to MC-LR.miRanda,targetscan,pictar were applied to predict the targets of alteration miRNA in order to find the miRNA associated with spermatogenesis.4.qRT-PCR was used to validate the array data.Results1.Cell viability was significantly decreased after exposure to MC-LR for 24 h at a concentration of 500 nM or higher.No significant differences were observed in groups exposed to 0.5 or 50 nM.There were no significant changes in cell morphology after treatment with up to 1000 nM MC-LR.In contrast,cells exposed to 10 or 100?C-LR displayed irreg?lar shapes and were substantially detached from the c?lture plates.FDA/PI staining was applied to confirm the toxicity of MC-LR.The changes were more pronounced when the concentrations of MC-LR were above 500 nM.2.miRNA expression profiling revealed 101 miRNAs with substantial changes in GC-1 cells.Of the 101 miRNAs with changes in their expression,46 were significantly up-regulated while 55 were evidently downregulated.Also among the changed miRNAs,25 were directly associated with spermatogenesis according to the miRNA target database.In this study,miR-96 was found to be most dramatically downregulated.miR-96 was speculated to be the candidate miRNA that targets DAZAP2 using computational prediction pro grams.3.The expression of all the selected miRNAs was consistent with the array data suggesting the reliability of the array.Conclusion1.miRNA expression profiling revealed 101 miRNAs with substantial changes in GC-1 cells.miR-96 was found to be most dramatically downregulated.2.The expression of all the selected miRNAs was consistent with the array data suggesting the reliability of the array.miRNA array is suitable for all kinds of cells.(?)Reg?lation of Microcys tin-LR-Induced Toxicity in Mouse Spermatogonia by miR-96ObjectiveTo investigate the reg?lation of microcystin-LR-induced toxicity in mouse spermatogonia by miR-96 in vitro and in vivo.Methods1.In vitro(1)The 3'-UTRs of mouse dazap2 encompassing the conserved miR-96 response elements were cloned into the pMIR-REPORT vector.These luciferase reporters were transiently transfected,together with miR-96-mimic,contro l-mimic,miR-96-inhibitor,and controlinhibitor,into 293T cells which were a model system for the validation of miRNA target genes.(2)To investigate the effects of MC-LR-induced the expression of miR-96,DAZAP2 and DAZL,the mRNA and protein levels of DAZAP2 and DAZL were detected in GC-1 cells after exposure to various concentration of MC-LR.(3)To further confirm the effects of miR-96 onDAZAP2 expression,GC-1 cells were transfected with miR-96-mimic,control-mimic,miR-96 inhibitor or control-inhibito'r followed by incubation for 24 h in the presence or absence of MC-LR.The mRNA and protein levels of DAZAP2 and DAZL were detected by qPCR and Western blot.(4)To examine the reg?latory effects of miR-96 on cellplar ffinction,GC-1 cells weretransfected with control-mimic,miR-96-mimic,control-inhibitor or miR-96-inhibitor followed by exqosure to 500 nM MCLR for 24 h.Cell viability and apoptosis were measured by CCK-8 and FDA/PI,respectively.2.In vivo(1)Male mice(n=40)were randomly divided into four groups:one control group and three experimental with 10 mice in each group.In experimental groups,mice were exposed to various concentration MC-LR(7.5?g/(kg·weight),15?g/(kg·weight),30?g/(kg·weight)).MC-LR was dissolved in physiological saline and given to rats by intraperitoneal injection(ip).Mice in control group were given the same volume of physiological saline.After two weeks treatment,blood samples were collected from carotid,respectively.The paraffin sections of testis with HE staining were made,and observed under an optical microscope.(2)miR-181a,miR-96,miR-121,miR-34 were all detected in liver,testis,epididymis and serum.(3)The mRNA and protein levels of DAZAP2 and DAZL were measured in mice testis after exposure to various concentration of MC-LR.(4)Male mice(n=30)were randomly divided into 3 groips:Mock group,Lenti-NC group and Lenti-miR-inhibitor.After 2 weeks different treatment,testis were collected.(5)The paraffin sections of testis with HE staining were made.The sperm samples were collected from testis and epididymis.Sperm quality was checked.Results1.In vitro(1)miR-96 directly recognizes and binds to the 3'-UTR of DAZAP2.(2)The expression of miR-96 was down-regulated in a dose-dependent manner,while the expression of DAZAP2 was up-regulated and the level of DAZL was down-regulated.(3)The cell viability and apoptosis were no significantly changes in GC-1 cells tranfected with miR-96-mimics followed by exposure to 500 nM MC-LR2.In vivo(1)After treatment with MC-LR for 2 weeks,the testicular structural were damaged in a dose-dependent manner,especially the lumens of spermatogenic epithelium were blockage in 15?g/kg and 30?g/kg groups.The miRNAs were alerted in testis after exposed to MC-LR.miR-96 was significantly down-regulated in 30?g/kg groups in testis and epididymis.The levels of DAZAP2 were up-regulated and the expression of DAZL was down-regulated in a dose-dependent manner.(2)The testicular structural was damaged,the viability of sperm decreased dramatically and the sperm morphology was abnormal in mice injected with miR-96-inhibitors.(3)Down-regulation of miR-96 significantly increased the level of DAZAP2,while the level of DAZL decreased.ConclusionThe expression of DAZL was negatively correlated with DAZAP2.DAZAP2 was overexpressed due to decreased levels of miR-96,resulting in the failure of DAZAL mRNA in translation.miR-96 played an important role in the regulation of cytotoxic effects of MC-LR in spermatogonia.Part 2 Roles of miRNAs and mRNA in microcystin-LR-induced Sertoli cell toxicityObjectiveTo investigate the effects of MC-LR on the reg?lation of miRNA and mRNA expression in Sertoli cells.To screening the miRNA associated with spermatogenesis.Method1.Sertoli cells were isolated from male Bal/bc mice tesits by enzymatic digestion and difference adherent.Sertoli cell were identified by FSHR immunofluorescence.2.Setoli cells were divided into 5 grous to detect:Control,0.5 nM,5 nM,50 nM,500 nM MC-LR goups.3.In order to choose the optimal concentration of MC-LR for miRNA and mRNA array,cell viability and morphology were recorded in Sertoli cells after exposure to a range of concentrations.We observed cell morphology under microscope;We use CCK-8 to examine the MC-LR toxicology on cell viability;Fluorescent photomicrographs of cells dyed with FDA/PI were used to observe the effects of MC-LR on live or death of cells.4.miRNA profiling of 768 different mouse miRNAs was acquired using a low-density miRNA Taqman array.mRNA profiling was detected by 1.0 ST GeneChip.miRNAs and genes were imported into IPA software to analyze the relationship between miRNA and mRNA.5.qRT-PCR was used to validate the array data.6.The protein levels of MAPK11,tubulin,ZO-1 and occludin were measured in Sertoli cell after exposxure to 500 nM MC-LR.Results1.Sertoli cells were isolated and purified enzymatic digestion and differential attachement,and positive rate reached to 90%through FSHR identification.2.There were no significant changes in cell morphology after treatment with various concentrations of MC-LR.There were no significant cell viability changes in cells exposed to MC-LR at 0.5 or 5 nM.However,the viability of Sertoli cells exposed to 50 or 500 nM of MC-LR for 24 h was significantly decreased compared with control cells.3.115 miRNAs were significantly altered in Sertoli cells following exposure to MC-LR for 24 h.Of the 115 miRNAs,46 were significantly up-regulated while 69 were evidently down-regulated.As indicated by mRNA profiling,2494 genes exhibited altered expression,including 1037 up-regulated genes and 1457 down-regulated genes.The expression of all the selected miRN As was consistent with the array data suggesting the reliability of the array.4.As analyzed by IPA,we found that these altered genes in Sertoli cells treated with MC-LR were involved in molecular and cellular functions,such as cell morphology,cellular growth and proliferation,cell-to-cell signaling and interaction,carbohydrate metabolism and molecular transport.In addition,predictive analysis of reproductive system disease predicted that the occurrence of sperm disorder,azoospermia,genital tumor,hypoplasia of genital organs and gonadal tumor We found that three different miRNAs(miR-374b,miR-615 and miR-362)could regulate the Sertoli cell-Sertoli cell junction pathways(ZO-1,MAPK11 and tubulin).5.Western blot results also revealed that the tight junction of Sertoli cells was damaged by MC-LR.Conclusion1.A total of 115 miRNAs and 2494 genes were altered in Sertoli cells followed by exposure to MC-LR.miR-374b-5p,miR-362-5p,miR-450a-5p,miR-193a-3p,miR-128-3p,miR-29b-3p,miR-199a-5p,and miR-miR-181a involved in both azoospermia and non-obstructive azoospermia were dysregulated.Dysregulation of miR-374b-5p,miR-193a-3p,miR-128-3p and miR-181a was also found in the semen of patients with azoospermia and asthenozoospermia.2.Three different miRNAs(miR-374b,miR-615 and miR-362)could regulate the Sertoli cell-Sertoli cell junction pathways(ZO-1,MAPK11 and tubulin).Innovative outcome obtained in this study1.This is the first study demonstrated that the alteration of miRNA expression in spermatogonial cells and Sertoli cells after exposure to MC-LR.2.This is the first study showed that the expression of miR-34,miR-122 and miR-181a was found to be modulated in the semen samples from infertile patients.3.The expression of DAZL was negatively correlated with DAZAP2.DAZAP2 was overexpressed due to decreased levels of miR-96,resulting in the failure of DAZAL mRNA in translation,damaging sperm quality.4.This is the first study demonstrated that miRNAs and mRNAs were dysregulated in Sertoli cells after exposure to MC-LR.In additional,the molecules of tight junction were affected by MC-LR in Sertoli cells.