The Mechanism About MC-LR-induced The Suppression Of Gonadotropin-releasing Hormone Synthesis In GT1-7 Cells

Microcystins(MCs),which have strong toxicity on liver,nerve,kidney and gastrointestinal,are natural toxins composed of seven amino acids and secreted by freshwater cyanobacteria in the process of blooming.In previous studies of our laboratory,it was discovered that MC-LR has strong male reproductive toxicity for the first time.Previously,we have found that MC-LR(the most abundant and the most toxic isoform of microcystins)can cause the significant reduction of serum testosterone by 60%to 80%according to both acute and chronic experiments.Leydig cells are the main place for testosterone biosynthesis in male.MC-LR cannot enter into the leydig cells without damaging to them by both in vitro and in vivo experiments.The testosterone synthesis in leydig cells are affected by the hypothalamic-pituitary-gonad axis.The GnRH neurons in hypothalamus regulate the synthesis of testosterone indirectly.Based on previous work,MC-LR can get into the hypothalamus and reduce male animals'testosterone synthesis and secretion.Due to the cellular complexity of the brain and the absence of cultured model systems representing differentiated central nervous system neurons,we use the GT1-7 cells for further molecular mechanisms study.GT1-7 cells are neuronal in physiology and morphology and secrete GnRH steadily in a pulsatile fashion,as it does in the intact hypothalamus.Organic anion transporting polypeptides(Oatps)constitute the specific transport protein that can mediate the transportation of MCs from the blood into the cell.Oatp1a5 is the pivotal protein that transport MC-LR into GT1-7 cells based on our previous study.Our research will clarify the mechanism about MC-LR-induced the suppression of Gonadotropin-releasing hormone synthesis in GT1-7 cells.The thesis is divided into three parts:Part I:The effects of MC-LR on miRNA profiling expression in GT1-7ObjectiveTo study the effects of MC-LR on miRNA profiling expression in GT1-7 cells and screen the miRNA associated with the synthesis of GnRH.Methods1.After exposure to MC-LR for different concentrations and time,CCK-8 method was used to determine the viability of GT1-7 cells.2.In order to choose the optimal concentration of MC-LR for miRNA array,cell viability and morphology were recorded in GT1-7 cells after exposure to a range of concentrations.We observed cell morphology under the microscope;We use CCK-8 method to examine the MC-LR toxicology on cell viability.3.We use the Hybrid chip microarray to detect the altertion miRNA of GT1-7 cells after exposure to 500 nM MC-LR for 48h.miRanda?Targetscan and microRNA.org were applied to predict the targets of the alteration miRNA in order to find the miRNA associated with the suppression of GnRH.qRT-PCR was used to validate the array data.4.The prediction and variation of the targets of miR-329-3p1)Using bioinformatics software,such as miRanda?Targetscan and microRNA.org,we can predict the target gene of the significantly changed miRNA and filter out the miRNAs associated with the synthesis of GnRH.Taqman probe is used to test miR-329-3p again.2)The 3'-UTRs of mouse Prkarla and Prkacb encompassing the conserved miR-329-3p response elements were cloned into the pMIR-REPORT vector.These luciferase reporters were transiently transfected,together with miR-329-3p-mimic,control-mimic,miR-329-3p-inhibitor,and control inhibitor into 293T cells which were a model system for the validation of miRNA target genes.5.The Promega kit is used to test the change of PKA enzyme activity in GT1-7 cells treated by MC-LR.Results1.In CCK-8 tests,Cell viability was decreased after exposure to MC-LR in does-dependent and time-dependent manner.Cell viability was significantly decreased after exposure to MC-LR for 48 h at a concentration of 500 nM or higher.2.GT1-7 cells were treated by 500 nM MC-LR for 48h,comparing with the control group,miRNA expression profiling revealed 101 miRNAs with substantial changes in GT1-7 cells.Of the 101 miRNAs with changes in their expression,42 were significantly up-regulated while 59 were evidently downregulated.The expression of all the selected miRNAs was consistent with the array data suggesting the reliability of the array.3.The prediction and variation of the targets of miR-329-3p.1)miR-329-3p was speculated to be the candidate miRNA that targets Prkar1a and Prkacb using computational prediction programs.2)Compared with transfection negative controls,the plasmid group and transfection miR-329-3p after plasmid can significantly reduce the loading of wild type Prkar1a and Prkacb sequence of fluorescein enzyme activity,but the negative control luciferin enzyme plasmid and load mutant Prkarla and Prkacb sequence of fluorescein had no effect on enzyme activity.4.Comparing with the control group,GTl-7 cells treated by MC-LR evoke the PKA enzyme.Conclusions1.The Prkar1a?Prkacb are the target genes of miR-329-3p confirmed by luciferase reporters.2.GT1-7 cells treated by MC-LR evoke the PKA enzyme.Part II:To study the effect and mechanism of the activation of PKA enzyme in GT1-7 cells by MC-LR.ObjectiveStudy the effect and mechanism of the activation of PKA enzyme in GT1-7 cells by MC-LR.Methods1.After exposure to MC-LR for different concentrations and time,GnRH mRNA level was determined using qPCR and GnRH concentration in supernatant was tested by ELISA.2.GT1-7 cells were exposed to 500 nM MC-LR for 0,0.25,0.5,1,3,6 h.Expression levels of Prkarla?Prkacb.c-Jun?c-Fos were determined by qPCR.cAMP and was tested by ELISA.The protein expression of Prkar1a?Prkacb?c-Jun?c-Fos?creb?p-creb were determined by Western blot.3.After exposure to the PKA inhibitor H-89 2HCl(10?M)for 1h individually,GT1-7 cells were dealt with 500 nM MC-LR for 24h,GnRH mRNA level was determined using qPCR.The protein expression of creb?p-creb were determined by Western blot.4.After exposure to 500 nM MC-LR for 6h,CHIP assays were performed to assess the interaction of p-CREB with c-Fos and c-Jun promoter,the interaction of c-Fos and c-Jun with GnRH promoter as well as enhancer.Results1.Q-PCR indicated GnRH transcription level decreased significantly as MC-LR concentration and exposure time increased.After exposure of MC-LR to GT1-7,we found as MC-LR concentration increased,GnRH level in the cell culture medium exhibited an increase-followed-by-decrease trend.2.After exposure of GT1-7 to MC-LR,the qPCR results showed that the expression of Prkar1a?Prkacb?c-Jun?c-Fos were upregulated indicating that PKA pathway was evoked;the Westernblot showed that the protein expression of Prkarl a?Prkacb?c-Jun?c-Fos?p-CREB were upregulated further confirming that PKA pathway was evoked.3.Pretreatment with PKA inhibitors abolished the suppression of GnRH induced by MC-LR.4.ChIP assay studies demonstrated that MC-LR increased p-CREB/c-Jun,p-CREB/c-Fos interaction,and c-Jun/GnRH promoter and enhancer interaction,and c-Fos/GnRH promoter and enhancer interaction.Conclusions1.MC-LR showed significant toxicity on GT1-7 cells.Cell viability was decreased and GnRH synthesis was suppressed after exposure to MC-LR.2.MC-LR evokes the PKA pathway in GT1-7 cells.Suppressing the pathway can abolished suppression of GnRH induced by MC-LR.Part III:The effect of regulating miR-329-3p on MC-LR-induced the suppression of GnRHObjectiveRegulate the expression of miR-329-3p to study the effects of MC-LR-induced the suppression of GnRH.Methods1.miR-329-3p affect the synthesis of GnRH by Prkarla and PrkacbTransfecting miR-329-3p-mimic,control-mimic,miR-329-3p-inhibitor,and control inhibitor into GT1-7 cells.miR-329-3p,Prkar1a?Prkacb and GnRH mRNA level was determined using qPCR.The protein expression of Prkar1a?Prkacb were determined by Western blot.2.Overexpression miR-329-3p can reverse the suppression of GnRH synthesis induced by MC-LRGT1-7 cells were transfected with miR-329-3p-mimic,control-mimic,miR-329-3p inhibitor or control-inhibitor followed by incubation for 24 h in the presence of MC-LR.miR-329-3p,Prkar1a?Prkacb and GnRH mRNA level was determined using qPCR.The protein expression of Prkar1a?Prkacb?c-Jun?c-Fos?creb?p-creb were determined by Western blot.Results1.miR-329-3p affect the synthesis of GnRH by Prkar1a and PrkacbCompared with the control group,transfection miR-329-3p mimics can significantly increase the amount of miR-329-3p,reduce the mRNA level of Prkar1?Prkacb and GnRH.Transfection miR-329-3p inhibitors causes the country results.2.Overexpression miR-329-3p can reverse the suppression of GnRH synthesis induced by MC-LRCompared with the control group,transfection miR-329-3p and MC-LR can abolish the increase of Prkar1a?Prkacb?c-Jun?c-Fos?p-CREB induced by MC-LR,and reverse thesuppression of GnRH caused by MC-LR.Conclusions1.miR-329-3p affect Prkar1a?Prkacb in transcription and translation level inhibits the expression of Prkarla?Prkacb.2.Overexpression of miR-329-3p can suppress the expression of Prkar1a?Prkacb,thus suppress the activation of PKA pathway,and abolish the suppression of GnRH caused by MC-LR.