| Microcystins(MCs)is a cyclic heptapeptide compounds which produced by cyanobacteria blooms,with liver and kidney toxicity,neurotoxicity,gastrointestinal toxicity,pulmonary toxicity and male reproductive toxicity.The first study in our laboratory has found that MC-LR(one of the most widely distributed and the most toxie MCs isomers in water)has the female reproductive toxicity.The research showed that MC-LR could induce the apoptosis of mouse ovarian granulosa cells(GCs),follicular atresia acceleration,significantly reduce the number of follicles in different stages,estrus cycle disorder and decrease the reproductive ability of mice.Apoptosis of GCs and accelerated follicular atresia was acknowledged as the key link of female reproductive damage induced by MC-LR,and GCs is essential in the process of follicular development,so the damage of GCs will directly affect the normal function of ovary.Latest recent research has shown that microRNAs(miRNAs)could influence gene expression of GCs by regulating mitogen-activated protein kinases(MAPKs)signaling pathway.Furthermore,increasing evidence has proven that miRNAs played crucial role in embryonic development and cancer progression which induced by MC-LR.Therefore,on the basis of previous work,this study was to screen the changed miRNA and mRNA by chip technology in vitro experiment,and through bioinformatics to analyze main signaling pathway and biological processes which aberrant miRNA involved in.Then,from the perspective of miRNA to investigate the mechanism of mouse ovarian granulosa cells(mGCs)injury induced by MC-LR which could provide a mechanistic explanation for the toxicity of MC-LR on mGCs.Part 1 The effect of MC-LR on the expression of miRNA and mRNA in mGCsObjectiveTo investigate the effects of MC-LR on the regulation of miRNA and mRNA expression in mGCs.To screening out aberrant miRNA and mRNA which correlated with normal function of mGCs by bioinformatics.Methods1.mGCs were isolated from 3-week old female Balb/c mice by mechanical separation.mGCs were identified by FSHR immunofluorescence and flow cytometry.2.Immunofluorescence and Western Blot assay were used to determine whether MC-LR could enter into mGCs.3.In order to choose the optimal concentration of MC-LR for miRNA and mRNA array,cell viability,morphology and flow cytometry were recorded in mGCs after exposure to a range of concentrations.CCK-8 was used to examine the MC-LR toxicology on cell viability.Flow cytometry was used to observe the effects of MC-LR on cell apoptsis rate.The morphology of cells was observed under light microscope.4.The expression of miRNA after MC-LR exposure was screened by hybridization miRNA microarray.The expression of mRNA was screened by MTA 1.0 gene chip.5.RT-PCR was used to verify the accuracy of miRNA and mRNA chips.6.Bioinformatics analysis was used to analyze the signaling pathways and biological processes involved in the differential expression of miRNA and mRNA.7.The interaction of miRNA and mRNA network constructed by Cytoscape software.miRanda and TargetScan were used to predict the target genes of differentially expressed miRNAs.The predicted target genes were compared with the mRNA microarray results.If genes with an expression pattern that negatively correlated with its regulatory miRNAs were included.Results1.The mGCs were isolated by mechanical separation.The positive rate reached to 94%through FSHR identification and flow cytometry.2.Immunofluorescence and Western Blot assay proved that MC-LR could enter into mGCs.3.There were no significant changes in cell morphology after treatment with 0-5 μM MC-LR.However,when concentration of MC-LR reached to 20 μM,We observed some cell dead.There were no significant cell viability changes in cells exposed to MC-LR at 0.04,0.2 or 1 However,the viability of mGCs exposed to 5 or 20 μM of MC-LR for 48 h was significantly decreased compared with control cells.The flow cytometry experiments showed that 25 μM MC-LR significantly increased the apoptosis rate of mGCs.4.miRNA microassay showed that there are 125 miRNAs significantly changed in mGCs after 48 h exposure of MC-LR at a concentration of 5 μM.Among the 125 miRNAs,75 were up-regulated while 50 were down-regulated.In the experiment of mRNA microassay,there are 283 genes altered,including 121 genes up-regulated and 162 genes down-regulated.5.The results of miRNA and mRNA chip are proved to be reliable by RT-PCR.6.The results of GO analysis indicated that target genes of altered miRNAs and mRNAs are involved in cell adhesion,apoptosis,immunity and other related biological processes.KEGG analysis found that target genes of significantly changed miRNAs and mRNAs are mainly involved in metabolism,hormone production,cancer,apoptosis,cell cycle and other related pathways.7.Through the network of miRNA-mRNA,it can be directly seen that miR-29b-3p,miR-29a-3p,miR-29c-3p,miR-1906,miR-182-5p,Gab2,Fos,Igfl,Mania are key miRNAs and genes,which indicates that these miRNAs and genes may play important roles in cell toxicity induced by MC-LR.Conclusion1.A total of 125 miRNAs and 283 genes were altered in mGCs followed by exposure to MC-LR,Which indicated that there was a direct or indirect relationship between miRNA and female mammals’ reproductive toxicity induced by MC-LR.2.Disorder of miRNAs and mRNAs will affect the expression of related genes in the mGCs,so as to interfere with the normal function of mGCs.Because mGCs has important biological functions such as secretion of estrogen,while hormone disorders will lead to infertility,so miRNA plays a crucial role in the healthy development of female reproductive system.Part 2 The role and mechanism of miR-3473g in the synthesis of progesterone in mGCs induced by MC-LRObjectiveTo investigate the mechanism of miR·3473g in MC-LR induced synthesis of progesterone of mGCs in vitro.Methods1.Effects of MC-LR on the secretion of progesterone,the expression of miR-3473g,StAR,CYP1lal in mGCs.(1).The cells were divided into 6 groups,respectively,with 0 μM,0.04 μM,0.2μM,1 μM,5μM,20 MC-LR and 100 nM testosterone and 50 ng/ml follicle stimulating hormone(FSH)co-culture for 48h.After treated,the concentration of progesterone in the cell culture medium secreted by mGCs were detected through ELISA.(2).Using Taqman probe,adopting qRT-PCR to verify the expression of miR-3473g in microarray.The expression of StAR and CYPllal was detected by qRT-PCR in mGCs after different concentration of MC-LR exposure.Western blot was used to detect the protein expression of StAR,CYP11a1.2.The mechanism of miR-3473g in MC-LR-induced elevated progesterone levels in mGCs.(1).Constructing StAR and miR-3473g dual luciferase reporter gene assay system.Using liposome transfection of exogenous miR-3473g mimics or miRNA negative control and recombinant plasmid(wild-type and mutant recombinant plasmid)or empty plasmid into 293T cells,and verify the relationship between miR-3473g and StAR.(2).mGCs were transfected with miR-3473g-mimic,miR-3473g inhibitor and control mimic,and treated with 5 MC-LR at the same time for 48 h.The mRNA levels of miR-3473g,StAR were detected by qRT-PCR.The protein levels of StAR and CYPllal were detected by western blot and immunofluorescence.(3)mGCs were transfected with miR-3473g-mimic,miR-3473g inhibitor and control mimic,and treated with 5 μM MC-LR,100 nM testosterone and 50 ng/ml FSH at the same time for 48 h by liposomal transfection.Concentration of progesterone and estradiol in cell culture medium was detected by ELISA to confirm the role of miR-3473g in MC-LR induced injury of biological function in mGCs.Results1.The reporter assays demonstrated that StAR was the target gene of miR-3473g.2.qRT-PCR was used to verified the expression of miR-3473g.The expression of StAR and CYPllal at mRNA and protein level were increased in mGCs after MC-LR exposure.3.The results of ELISA experiment showed that with increasing concentration of MC-LR,the concentration of progesterone which secreted by mGCs increased.4.When the expression of miR-3473g up-regulated in mGCs,the expression of StAR decreased and the expression of CYPllal were also altered.5.When transfected with miR-3473g mimics,miR-3473g inhibitor or miR-3473g negative control into mGCs by lipofectamine 2000 for 24h5 mGCs treated with 5μM MC-LR,100 nM testosterone and 50 ng/ml FSH for 48h.Results showed that comparing with the mi-NC+MC-LR and in-3473g+MC-LR group,concentration of progesterone significantly decreased in mi-3473g+MC-LR group.The concentration of progesterone in in-3473g+MC-LR group significantly increased compared with in-NC+MC-LR group.6.When transfected with miR-3473g mimics,miR-3473g inhibitor or miR-3473g negative control into mGCs by lipofectamine 2000 for 24h,mGCs treated with 5 μM MC-LR,100 nM testosterone and 50 ng/ml FSH for 48h.We found that the ratio of progesterone to estradiol in MC-LR group was significantly higher than NC group.Comparing to mi-NC+MC-LR and in-3473g+MC-LR group,the ratio of progesterone to estradiol was decreased in mi-3473g+MC-LR group.These results indicated that up-regulated the expression of miR-3473g could reduced the ratio of progesterone to estradiol,inhibiting the premature luteinization of mGCs happened.Conclusion1.In vitro experiments,we confirmed that MC-LR resulted in cytotoxicity to the mGCs.MC-LR could down-regulated the expression of miR-3473g in mGCs,leading to the expression of StAR increased and then inducing the expression of progesterone related genes changed,and eventually increased the level of progesterone in mGCs.2.Up-regulated the expression of miR-3473g could inhibit the increase of progesterone secreted induced by MC-LR in mGCs.3.The expression of miR-3473g could affect the ratio of progesterone to estradiol.Up-regulated the expression of miR-3473g could reduce the ratio of progesterone to estradiol,and inhibit the premature luteinization of mGCs occurred.Features and innovations in this study1.The effects of MC-LR on the expression of miRNA and mRNA in GCs were studied by microarray technique for the first time and we analyzed aberrant expression of miRNAs and mRNAs and the biological process and signaling pathways which these miRNAs and mRNAs involved in by bioinformatics in mGCs after MC-LR exposure.We found key miRNAs and mRNAs by constructing the miRNA-mRNA network.2.We first found that miR-3473g played a crucial role in MC-LR induced toxicity of mGCs,and confirmed StAR was the target gene of miR-3473g.The molecular mechanism of miR-3473g in MC-LR induced unbalanced level of progesterone in mGCs was proposed. |
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