The Protective Role And Mechanisms Of CircRNAs In BPA Induced Spermatocyte Toxicity

Part 1 The expression profile of non-coding RNA in mouse spermatocytes apoptosis induced by Bisphenol A[Purpose] To analyze the changes in the expression profile of non-coding RNA in mouse spermatocyte apoptosis induced by Bisphenol A(BPA).We also aimed to analyze the important pathways and related molecules involved,in order to find out target molecules for further research.[Methods] Different concentrations of BPA were exposed to mouse spermatocyte cell line,GC-2.And the control group was 0.1% DMSO solvent treated cells.We used Cell Counting Kit-8(CCK 8)to examine the cell viability and to explore the IC50(half maximal inhibitory concentration)of BPA on GC-2 cells.The concentration of 120 ?M BPA(approximately the concentration of IC50)was applied to treat the cells and detect the DNA replication rates and apoptosis rates.And the RNAs extracted from GC-2 cells exposed with 120 ?M BPA and cells from control group were distributed to three aliquots and subjected to circRNA,miRNA and m RNA deep sequencing.The sequence depth is 10 G,10 M,6G respectively for circRNA,miRNA and m RNA.Differential expressed circRNA host genes and m RNAs were analyzed using R Bioconductor package for GO and KEGG analysis and visualized by ggplot package.The biological function and signal pathway analysis of differentially expressed miRNAs were based on its target genes,and analyzed by DIANA miRPath v.3.0 website.The miRNAs binding to circRNAs were predicted using circ Mir 1.0 software based on miRanda 2010 and RNAhybrid-2.1.2.miRTar Base v.8 database was applied to export the experimentally validated miRNAs.Cytoscape software was used to construct the circRNA-miRNA-m RNA interaction network based on the predicted and actual sequencing results.The four-quadrant scatter plot of circRNAs and host gene m RNAs was drawn by R language to analyze their correlation.Cp G methylation Chip data set(GSE: 79075)was analyzed in association with our circRNA and m RNA sequencing data after downloading BPA treated GC-2 cells from the GEO database,and the correlation between promoter methylation levels and changes in circRNA and m RNA were analyzed.[Results] After 48 hours of BPA treatment,the viability of GC-2 cells decreased in a dose-dependent manner.The IC50 is approximately between 120 ?M to 160 ?M.DNA replication assay and apoptosis analysis by flow cytometry showed 120 ?M BPA significantly reduced the DNA replication capacity and enhanced the apoptosis rates of GC-2 cells.High-throughput sequencing showed that the overall RNA transcription level of GC-2 increased after BPA treatment at 120 M compared with the control group.After BPA treatment,the type,number and expression level of circRNA,miRNA and m RNA were increased as a whole.Up-regulated molecules were more than down-regulated molecules.GO and KEGG analysis showed that circRNA host genes and miRNA target genes with more than two times fold changes were enriched in cell proliferation,apoptosis,DNA damage,DNA repair and other pathways.However,differentially expressed m RNAs screened with the same criteria were not enriched in the same functional pathway as circRNA host genes,suggesting that circRNA may be more sensitive to response than its parent genes under the same treatment conditions.The co-expression analysis of circRNA and its parent gene m RNA showed that the expression fold changes of circRNA were generally greater than those of their host gene m RNAs.And the correlation between the two kinds of molecules was not significant,suggesting that the co-transcription of these two was less likely.Analysis of the methylation Chip and our sequencing data showed that the changes of promoter methylation levels were not correlated with circRNA changes,suggesting that the differences of circRNAs were less likely to be affected by the changes of promoter methylation levels.Spermatogenesis related circRNAs such as circ Dmnt1,circ Kitl,circ Scmh1,circ Top2 a,and circ Clasp2 were significantly altered.At the same time,miRNAs related to spermatogenic function,such as miR-15 b,miR-18 a,and miR-34 family as well as miRNAs related to estrogen pathways,were significantly changed.A large number of functional m RNAs were also significantly changed after BPA treatment,such as Sycp3,Meiob,Kit and other meiosis-related genes were significantly down-regulated after BPA treatment,and the meiotic initiation regulators Sohlh1 and Sohlh2 were also significantly changed.Imprinted genes H19,Igf1 and Igf2 were more than two times down-regulated after BPA treatment.DNA methylation related genes such as Dnmt1,Dnmt3 a and Dnmt3 b did not change at the experimental dose of BPA120 ?M.[Conclusions] BPA treatment induces apoptosis of GC-2 cells and activates the RNA transcription level of GC-2 cells,causing a large number of circRNAs and miRNAs related to cell proliferation and apoptosis,and miRNA to involve in the apoptosis process of the cells.The results suggested that the apoptosis of spermatogenic cells caused by BPA is regulated by non-coding RNA at the epigenetic level.Part 2 circRNA validation and functional screening analysis[Purpose] To validate the significantly differentially expressed circRNAs.And to construct overexpressed plasmids to screen potentially functional circRNAs for further functional studies.[Methods] The screening criteria were first set at fold changes greater two times plus P value less than 0.05.The selected circRNA was specifically amplified using primers designed at "spliced junctions".And the accuracy of the amplified products was verified by Sanger sequencing.qPCR quantitative detection was applied to verify the accuracy of sequencing data.At the same time,primers for linear m RNA of the corresponding genes were designed to detect the changes of the corresponding m RNA of circRNA after the treatment of 120 ?M and 160 ?M BPA,and to compare the expression tendency of these two kinds of molecules.Validated exonic circRNAs were selected to constructoverexpression vectors in batches.The construction strategy was to use commercial circRNA overexpression vector p LCDH-ci R by enzymatic ligation or homologous recombination.The cell viability of after transfection was examined,and functional circRNAs were selected for further study.[Results] In the circRNA sequencing data,25 circRNAs were with a difference of more than two fold changes,and a P value of less than 0.05.For 12 of them we designed specific primers,a single bright band was amplified.qPCR quantification found that 11 of them were in line with the sequencing trend.The m RNAs of these circRNAs corresponding were mostly consistent with the expression trend of circRNA under BPA treatment,but the expression fold changes of circRNAs were greater,especially in the high-dose group(160?M),the response degree of circRNA was greater than that of the corresponding m RNA.Seven of these circRNAs were cloned in the overexpression vector with overexpression efficiency all surpassed two folds.Among them,circ Dcbld2,circMapk1 and circ Tbcld20 have high over-expression efficiency,which can be up to hundreds of times.After the transfection with these 7 vectors,CCK-8 assay was used to detect cell viability.And it was found that circ Dcbld2,circMapk1,circ Mpp6 and circ Tbcld20 can promote cell proliferation,while circ Usp3 can inhibit cell proliferation.circ Cnot6 l and circ Ppm1 b have no significant effect on cell proliferation.The dose of the three circRNA vectors,circ Dcbld2,circMapk1 and circ Tbcld20 was reduced to 1/3 of the original dose,and were transfected together.It was found that the effect of mutual transfection was greater than that of any single circRNA transfection.The downstream miRNAs of the three circRNA was predicted and studied with miRNA sequencing data.Four common target miRNAs including miR-322-5p,miR-497a-5p,miR-6340 and miR-6918-5p as well as a series of common target genes were delineated.circ Dcbld2,circMapk1 and circ Tbcld20 may realize their mutual effects through these target miRNAs and target genes.[Conclusions] The circRNA sequencing results proved to be highly reliable.circRNAsare more sensitive and more active to react than the corresponding target gene m RNAs under BPA treatment.p LCDH-ci R vector can successfully and be conveniently used to over-express a variety of circRNAs,among which circRNAs with short sequences may achieve higher expression multiples.Some elevated circRNAs may play protective roles in BPA-induced GC-2 cell apoptosis.This protective effect of nc RNAs under toxic exposure may be a mechanism by which cells self-regulate and adapt to the environment.At the same time,the synergistic protective effects of circRNAs can be achieved,providing a new perspective for multi-target interference of circRNA in reproductive toxicity.Part 3 The function and downstream mechanism of circMapk1[Purpose] To explore the properties and functions of circMapk1 and the downstream molecular mechanism of apoptosis alleviation.[Methods] The circular properties of circMapk1 were further confirmed by RNase R digestion."Back to back" primers were used to amplify circMapk1 in c DNA and g DNA to confirm the existence of circMapk1.circMapk1 and Mapk1 m RNAs were amplified from c DNA extracted from mice testes at different development stages to determine the expression of circMapk1 during testicular development.GC-2 cells were transfected with circMapk1 overexpression vector,and the overexpression efficiency of circMapk1 was detected by qPCR,and the effect of circMapk1 overexpression on Mapk1 m RNA was also detected.CCK-8 was used to detect the cell viability of GC-2 cells at 24 h,36 h and 48 h after the overexpression of circMapk1,and the synergistic effects of circMapk1 and BPA exposure.DNA replication rate after circMapk1 transfection was detected by Edu incorporation.Annexin V-APC/7-AAD kit was used to detect apoptosis rates in BD flow cytometry.Bioinformatics methods were used to predict the miRNAs and downstream target genes of circMapk1.Double luciferase assay was used to verify the binding of circMapk1 and miRNA.And the co-expression of circMapk1 and downstream miRNA wasdetected by qPCR in the testes of mice at all development stages.miRTar Base was used to predict the target gene of miR-214-3p,and qPCR was used to verify the changes of the target gene after overexpression of miR-214-3p.The intersection of the target gene with obvious down-regulation and the up-regulation of the target genes after overexpression of circMapk1 were screened to obtain the target gene that may work together.p VAX1 vector was used to construct the over-expression vector of these target genes to study their functions and find out the target genes promoting cell proliferation.Furthermore,luciferase reporter assay was used to verify that it was the target gene of miR-214-3p.Western Blot was used to detect the changes of target gene protein after overexpression of miR-214-3p and circMapk1.[Results] After RNase R digestion,m RNAs of linear RNAs such as Actb and Mapk1 were significantly reduced,while circMapk1 was not digested,rather,circMapk1 was significantly enriched due to linear RNAs digestion,which confirmed that circMapk1 was indeed in circular form.circMapk1 can be amplified in c DNA but not in g DNA,which proves that circMapk1 is not a non-specific fragment of the genome.In postnatal mouse testes,the expression of circMapk1 decreased,while the m RNA expression of Mapk1 was relatively stable,suggesting that the expression of circMapk1 is not a transcriptional by-product of Mapk1,but has its own regulation mechanism.CCK-8 assay,Edu assay and flow cytometry assay confirmed that overexpression of circMapk1 can promote the proliferation of GC-2 cells and inhibit the apoptosis caused by BPA.Bioinformatics predicted that circMapk1 could bind to miRNAs such as miR-29a-5p,miR-152-5p and miR-214-3p.The binding of circMapk1 and miR-214-3p was confirmed by luciferase reporter assay.qPCR detected a high correlation between circMapk1 and miR-214-3p expression in mouse testes in different days after birth,further suggesting that circMapk1 may interact with miR-214-3p.Target genes of miR-214-3p were predicted by miRTar Base,and 12 genes related to cell proliferation and apoptosis were screened by examining theirfunctions.qPCR detection revealed that 5 of them were significantly down-regulated.It was found that Akt1,Ctgf and Pkn3 may be the target genes after the intersection of the over-expressed and up-regulated target genes of circMapk1.Functional studies on these three genes revealed that Akt1 overexpression significantly promoted the proliferation of GC-2 cells.The luciferase reporter assay confirmed the interaction between miR-214-3p and Akt1.Western Blot analysis revealed that AKT1 protein can be down-regulated by miR-214-3p and up-regulated by circMapk1,suggesting that circMapk1 may promote cell proliferation by up-regulating AKT1 through sponging miR-214-3p.[Conclusions] circMapk1 can protect against spermatogenic cell apoptosis caused by BPA,and can play a role in up-regulating proliferation-promoting protein AKT1 by sponging miR-214-3p.Part 4 The upstream regulation mechanism of BPA exposure on circRNA expression[Purpose] To study the upstream regulatory mechanism of changes in circRNA expression after BPA exposure.[Methods] The upstream regulation mechanism of BPA exposure on circRNA expression was studied from the perspective of transcriptional regulators and RNA-binding proteins.In terms of transcriptional regulators,2000 bp sequences of the upstream transcriptional initiation site of circRNAs were extracted from UCSC website,and JASPAR transcriptional factor prediction website was used to look up for transcriptional regulators that may bind to circRNA promoter region.qPCR was used to examine whether these transcriptional regulators were elevated after BPA treatment.p VAX1 vector was used to over-express the transcriptional regulatory factor to detect whether circRNA would increase under BPA treatment.In the aspect of RNA-binding protein,we first checked the website of UCSC to find out whether the flanking sequences at both ends of circRNA significantlychanged under BPA exposure are reverse complementary,so as to find the basis for the role of RNA-binding protein.Then,RNA-binding proteins related to circRNA production were searched by literature consultation,and their changes in BPA exposure were detected by qPCR and Western Blot.p VAX1 vector was used for overexpression and si RNA for protein knockdown to determine the impact of RNA binding proteins on circRNA expression.[Results] Through online prediction by JASPAR website,dozens of these circRNA transcriptional regulatory factors that may be jointly regulated the dysregulated circRNAs were obtained.Estrogen receptor 2(ESR2)in estrogen pathway was selected as a possible transcription factor.The m RNA of Esr2 detected by qPCR increased significantly after BPA treatment.Overexpression efficiency of Esr2 by p VAX1 vector can be up to hundreds of times,but no significant changes were detected in circRNAs,suggesting that ESR2 may not be a factor that regulates the increase of these circRNAs under BPA treatment.Among the circRNAs significantly altered by BPA exposure,half of them were found to have repeated complementary matching sites in the flanking intron regions,including circMapk1,which was studied in detail in the previous section.This provides a basis for RNA-binding proteins to regulate the generation of these circRNAs.Literature search revealed that ADAR1 and QKI may be involved in the regulation of circRNAs.si RNA were used to transfect the cells to knock down QKI and ADAR1.The knockdown effect can reach to less than 1/2 by qPCR detection.circRNAs were not significantly affected by ADAR1 knockdown,but circRNAs were significantly increased after low QKI knockdown.On the contrary,the QKI overexpression vector transfected cells showed that the circRNAs were significantly reduced.QKI may regulate the expression of circRNAs.At the same time,Western Blot showed that QKI protein decreased significantly after BPA exposure,and the decrease of QKI protein was more obvious with the increase of BPA concentration,which was consistent with the rising trend of multiple circRNA expressions after BPA exposure.[Conclusions] Compared with the transcription regulatory factors,circRNAs in GC-2 cells may be more likely to be regulated by RNA binding proteins.Under the stimulus of BPA,the expression of QKI protein elevated,increased the expression of some protective circRNA,sponged the apoptosis-promoting of miRNAs,and in turn raised the expression of apoptosis alleviating proteins.This may be the self-adjusting mechanism of cells under BPA exposure.