Investigation Of Microcystin(-LR) Distribution On Male Reproductive System After Its Exposure And The Mechanism Of Microcystin (-LR)-induced Prostate Tumor

In recent years,with the accelerated industrialization and urbanization in the world,a large number of industrial and domestic wastewater discharged into the natural water body,serious eutrophication caused cyanobacterial blooms,the cyanotoxins has posed a serious threat to survival environment and human health.Microcystins(MCs)is a class of monocyclic heptapeptide compounds produced by freshwater algae,more than 100 kinds of its congeners have been identified so far,and microcystin-LR(MC-LR)is most widely distributed and has the strongest toxicity.Previous studies have shown that MC-LR possesses hepatotoxicity,nephrotoxicity,gastrointestinal toxicity,neurotoxicity and so on.Our study is the first to find the male reproductive toxicity of MC-LR,it can lower testosterone level in rats,impact the quality of sperm and damage the testicular structure.In order to fully illustrate the male reproductive toxicity of MC-LR and its underlying mechanisms,to study the distribution features in male reproductive system after MC-LR exposure is extremely necessary.Therefore,important male reproductive organs-the testis and the prostate were chosen as the object in this study,after its exposure in vivo and in vitro,MC-LR was analyzed in tissues and cells to observe the distribution features and determine the target cells.In addition,the prostate is the largest male accessory sex gland,which plays an important role in maintaining a healthy male reproductive function.Based on above,we took the miRNA expression profile changes in prostate epithelial cell caused by MC-LR exposure as the pointcut in this study,through in vivo and in vitro experiments and detection for clinical samples,to further explore the mechanisms of male reproductive toxicity caused by MC-LR.Part I Investigation of MC-LR distribution on important male reproductive organs and cellsObjectiveThrough in vivo and in vitro experiments,we intended to explore the distribution features and the target cells in testis and prostate after MC-LR exposure.Methods1.Male Sprague-Dawley(SD)rats(n=12,35-40g)were randomly divided into 2 groups(MC-LR treated group and the control group).For MC-LR treated group,rats were intraperitoneally injected with MC-LR at a dose of 300 ?g/kg·bw/day for 7 days;for the control group,with the same dosage of sterilized normal saline.At the next day after treatment,all rats were euthanized by cervical dislocation after weighing the body weights,testis and prostate tissues were weighed and then stored in liquid nitrogen for following study.2.Testis and prostate tissues were fixed and frozen sectioned respectively,then MC-LR was detected using immunofluorescence;total proteins were extracted from the tissues and the contents of MC-LR were analyzed using western blot;MC-LR was extracted from the tissues using chemical method,then the contents of MC-LR in tissues were detected by LC-MS.3.Using primary cell culturing system which has been established,spermatagonia,sertoli cells and Ley dig cells were separated respectively from the testes of male SD rats and then cultured in vitro.Three kinds of testicular cells were treated with MC-LR at a concentration of 500 nM,MC-LR in cells was detected respectively using immunofluorescence.4.Using cultured human prostate epithelial RWPE-1 and stromal WPMY-1 cell line,these two kinds of prostate cells were treated with 500 nM MC-LR respectively,then MC-LR in cells was detected using immunofluorescence.Results1.Compared with the control group,the bodyweights of rats in MC-LR treated group dropped significantly,the weights of testis and prostate showed no significant difference.Results from immunofluorescence detection indicated that MC-LR can enter testis and prostate tissues of rats,mainly distributed on tubal wall of seminiferous tubules and small amount in the mesenchymal in testis tissue,and mainly distributed on acinus wall and in stroma and small amount in acinus cavity in prostate tissues.Results from western blot showed that MC-LR can be detected in testis and prostate tissues.MC-LR also could be detected in extracts of testis and prostate tissues using LC-MS.2.Among three kinds of in vitro cultured testicular functional cells,after exposure to MC-LR respectively,results from immunofluorescence showed that MC-LR can enter spermatagonia and Sertoli cells,but not Leydig cells.For two kinds of in vitro cultured prostate functional cells,after exposure to MC-LR respectively,results from immunofluorescence indicated that MC-LR can enter epithelial cells and stromal cells.Conclusion1.After acute exposure to MC-LR,the body weights of rats decrease,but no significant effect can be detected on the weights of testis and prostate.2.After exposure to MC-LR,MC-LR can enter testis and prostate tissues,mainly distributed on the tubal wall of seminiferous tubules of testis,and the acinus wall of prostate.3.For three kinds of in vitro cultured testicular functional cells,after exposure to MC-LR respectively,MC-LR can enter spermatagonia and Sertoli cells,but not Ley dig cells.For two kinds of prostate functional cells,after exposure to MC-LR respectively,MC-LR can enter epithelial cells and stromal cells.Part ? Impact on miRNA expression profile of prostate epithelial cells by MC-LR and study on related mechanismsObjectiveThrough MC-LR exposure to cultured human prostate epithelial cell line RWPE-1 in vitro,we explored the impact of MC-LR on miRNA expression profile prostate epithelial cells and screened the functional miRNA and its target gene.Methods1.Human prostate epithelial cell line RWPE-1 was cultured in vitro,exposed to MC-LR for different times(6,12,24,48 h)at different concentrations(0,0.5,5,50,100,250,500 nM),then detected the cell viability using CCK-8 assay.2.Based on results of cell viability assay,prostate epithelial cell line RWPE-1 was treated with MC-LR for 48 h at different concentrations(0,10,100,500,1000 nM),then observed the cell survival rates by FDA/PI detection.3.After treated with 500 nM MC-LR for 48h,miRNAs with significantly different expression were screened using high-throughput microarray chip in prostate epithelial cells,the reliability of the results was verified using qPCR detection.4.Through databases such as miRBase and TargetScan,miRNAs that significantly different expressed after MC-LR exposure were selected and its potential target genes were retrieved.5.For the selected miRNA and its potential target gene,we built dual luciferase reporter gene plasmids including wild-type,mutant-type and empty vector sequence of target gene respectively,then transfected exogenous miRNA mimics or miRNA negative control and three kinds of reporter gene plasmids into 293T cells,to verify the associative relationship between miRNA and its target gene.6.For objective miRNA and its target gene,we built overexpression and inhibiting slow virus respectively,transfected two kinds of slow virus into prostate epithelial RWPE-1 cells for MC-LR treated group and control group respectively.Using qPCR,we validated the regulatory relationship between miRNA and its target gene,and explored the effect on miRNA regulation to its target genes by MC-LR.Results1.Prostate epithelial RWPE-1 cells were treated with different concentrations of MC-LR for different time,then CCK-8 and FDA/PI assay were employed to detect cell viability and survival rate.Results showed that treating with 500 nM MC-LR for 48 h can result in significantly declined cell viability and relatively better survival rate,therefore this treatment model was selected for screening miRNAs with significantly different expression.2.Results from high-throughput microarray detection showed that,after treated with MC-LR,30 kinds of miRNAs in prostate epithelial cells changed significantly,including 26 miRNAs up-regulated and 4 miRNAs down-regulated,and results from qPCR displayed the reliability of the chip.3.Results from bioinformatic analysis showed that,DAPK1 is a potential target gene of the most significant up-regulated miRNA-4458.The associative relationship between miRNA-4458 and its potential target gene DAPK1 was identified using dual luciferase reporter gene detection.4.After transfecting miRNA-4458 overexpression and inhibiting slow virus into RWPE-1 cells,the expression of DAPK1 gene was detected.Results showed miRNA-4458 can inhibit the expression of DAPK1,which can be can be promoted by MC-LR.Conclusion1.MC-LR can cause the miRNA expression profile changes in prostate epithelial RWPE-1 cells.2.In prostate epithelial RWPE-1 cells,miRNA-4458 has inhibitory effect on the expression of DAPK1 gene,which can be promoted by MC-LR.Part ? Study on related mechanisms of prostate tumor induced by MC-LRObjectiveThrough analyzing clinical prostate cancer tissue samples,the expression level of miRNA-4458 and its target gene DAPK1,and the correlation between their expressions and clinical pathological indicators were studied.In prostate cancer cell lines and prostate tumor burdened nude mice,after regulating the expressions of miRNA-4458 and its target gene DAPK1,tumor cell biology behaviors such as proliferation and apoptosis,migration and invasion,and related cell signaling pathways were studied.Possible mechanisms of prostate cancer induced by MC-LR were investigated.Methods1.24 pairs of clinical prostate tumor tissues and its corresponding tissues adjacent to tumor were collected.Using qPCR,immunohistochemistry and western blot,the expression of miRNA-4458 and DAPK1 were detected,and their difference and correlation were compared.Based on 24 cases clinical pathological data,the correlations between the expression level of miRNA-4458 and patients age,PSA,Gleason score,tumor staging and lymph node metastasis were respectively analyzed.2.For in vitro cultured prostate epithelial RWPE-1 cells and 4 kinds of prostate cancer cells such as PC-3,DU-145,22RV1 and LNcaP,the expression levels of miRNA-4458 were respectively detected using qPCR.3.MiRNA-4458 overexpression and inhibiting slow virus were built,and prostate cancer PC-3 and DU-145 cell lines were cultured in vitro,then slow virus were transfected into two kinds of cancer cell lines by slow virus.Using CCK-8 assay,clone formation test,effects on tumor cell proliferation ability caused by expression changes of miRNA-4458 were detected.Using cycle and apoptosis detection by flow cytometry,cell cycle and apoptosis of cancer cells caused by expression changes of miRNA-4458 were analyzed.Meanwhile,using qPCR and western blot,expression of related molecules in cell proliferation pathway such as ERK,c-Jun,cyclin D1 and CDK4 were determined.Using cell scratch assay and matrix invasion test,impact on tumor cell migration and invasion ability caused by expression changes of miRNA-4458 were analyzed.Meanwhile,using qPCR and western blot,expression of related molecules in cell invasion pathway such as ERK,c-Jun,VEGF and MMP were determined.4.Prostate cancer cell line DU-145 was cultured in vitro,and miRNA-4458 inhibiting slow virus was transfected into cancer cells through slow virus.The virus-treated cancer cells was subcutaneously injected to nude mice,mice were euthanized after 14 days,the volume of tumors were calculated to analyze the impacts on cell growth of prostate cancer caused by down-regulation of miRNA-4458.Virus-treated cancer cells were injected into nude mice from caudal vein,mice were euthanized after 42 days,pulmonary metastasis were observed to the impacts on metastasis of prostate cancer caused by down-regulation of miRNA-4458.Results1.For clinical prostate cancer tissue samples,the expression of miRNA-4458 in tumor tissues was significantly higher compared to para-carcinoma tissues,and its expression showed a negative correlation with its regulated target gene DAPK1.The expression of miRNA-4458 showed relevance with prostate cancer stages and lymph node metastasis,displaying a trend of increase with tumor progression.2.For in vitro prostate cancer cell lines such as PC-3,DU-145,22RV1 and LNcaP,the expression of miRNA-4458 was higher compared with prostate epithelial RWPE-1 cells,especially in PC-3 and DU-145 cancer cells.3.In cultured prostate cancer PC-3 and DU-145 cell lines in vitro,up-regulation of miRNA-4458 can promote cell proliferation and inhibit cell apoptosis,and down-regulation of miRNA-4458 can inhibit cell proliferation and promote cell apoptosis,whose mechanism is through the regulation of molecules such as cyclin D1 and CDK4 in cell proliferation signaling pathways.Meanwhile,up-regulation of miRNA-4458 can promote cell migration and invasion,which can be inhibited by down-regulation of miRNA-4458,whose mechanism is through the regulation of molecules such as VEGF and MMPs in cell invasion signaling pathways.4.After injecting prostate cancer cells into nude mice subcutaneously,inhibition of miRNA-4458 can inhibit tumor growth.After injecting prostate cancer cells into nude mice from caudal vein,inhibition of miRNA-4458 can inhibit tumor metastasis.Conclusion1.In clinical prostate cancer samples,compared with para-carcinoma tissues,expression of miRNA-4458 increased while the expression of DAPK1 decreased,which has the correlation with the progression and metastasis of prostate cancer.2.MiRNA-4458 can inhibit DAPK1 expression in vitro,which indirectly activates such molecules as cyclin D1 and CDK4 in cell proliferation signaling pathways,promoting proliferation of prostate cancer cell and inhibiting cancer cell apoptosis.3.MiRNA-4458 can inhibit DAPK1 expression in vitro,which in indirectly activates such moleculars as VEGF and MMPs in cell invading signaling pathways,promoting migration and invasion of prostate cancer cells.4.Inhibition of miRNA-4458 can surpress proliferation and metastasis of prostate cancer cells in vivo.