The MiRNA Changes During The Effect Of Cadmium On Alternative Splicing Of Kitl Pre-mRNA In Mice Ovarian Granulosa Cells

Objective:We aim to investigate the influence of cadmium in different concerntration and the exposure time on Kitl pre-mRNA alternative solicing in ovarian granulosa cells in mice,and analyze the expression of the miRNA,which were involved in regulating the expression of Kitl gene, for providing evidences for miRNA epigenetic studies of cadimum in ovarian granulosa cells.Methods:1、The detection of Kitl mature mRNA different subtypes. The 26-day-old female ICR mices’ ovarian granulosa cells were extracted under the sterile condition and cultured in medium in vitro. Then these cells were randomized to exposure to Cd(CdCl2) according to block-split-plot experimental design methods when the cells were adherent stably and their shapes were stable. The primary processing is exposure doses(0 μM、10 μM、20 μM、40 μM) and the split-plot is different exposure time( 0 h、2h、4 h、6 h、8 h). Each experiment repeated three times. Then Kitl mRNA different subtypes species were detected by PCR and agarose gel electrophoresis;expression of different subtypes in each group were observed by qRT-PCR. Expression of each isoform of Kitl gene mRNA was confirmed by RT-PCR.2、Screening of miRNA expression profile related to Kitl gene expression regulation and bioinformatics analysis.(1)Predicted the miRNA targets of genes of alternative splicing important factors hnRNP A1、hnRNP D、hnRNP L、hnRNP K/J、SR based on databases of miRanda、mi RDB、miRWalk and Targetscan, which are available on the web site at http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk/mi RNApredictedtarget.html.(2)Researched miRNA related to Kitl gene or similar to Kitl gene’s function on the basis of relative literatures, then compared with Kitl predicted results to further screening purpose of miRNA.(3)Detected the miRNA expression profile of mices’ ovarian granulosa cells which had been exposured to 0 and 10 μM Cd 4 hours by utilizingmiRCURYMLNAArray, and the differentially expressed miRNA(Fold charge over 2)was analysised By clustering.(4)Took downstream target genes of the intersectional miRNA of these predictional, literatures’ and the microarray’s data to performed bioinformatics analysis by using DAVID online tools, which including gene ontology(GO) analysis and KEGG pathway analysis.At the same time, due to limitations of the study time, we picked out several miRNA,which the SUM value of prediction were high, and the literature,the chip both had good clues, for the next PCR validation.3、The expression of mmu-miR-27a-3p, mmu-mi R-34b-5p, mmu-miR-297a-3p,mmu-miR-129-5p, mmu-miR-107-3p were detected in the effect of cadmium on Kitl pre-mRNA alternative splicing in mice ovarian granulosa cells.Results:1、The different subtypes of Kitl mature mRNA.(1)Consistent with the control group, the infected group were only amplify two segments:the Kitl1(970 bp) and Kitl2(886 bp).And the Kitl1, Kitl2 mRNA expression level in groups of different exposure doses, have no statistically significant difference(P > 0.05), but the change of the Kitl1/ Kitl2 mRNA ratio has statistically significant(P < 0.05);(2)The Kitl1、Kitl2 mRNA expression level and the changes of Kitl1 / Kitl2 mRNA ratio in different exposure time groups have statistically significant(P < 0.05), and they all has an interaction between dose and time(P < 0.01).(3) Compared with the control group, the magnitude and trends change of Kitl1, Kitl2 expression levels and the Kitl1 / Kitl2 ratio with time in all treated groups are significantly different.2、Screening of miRNA expression profile related to Kitl gene expression regulation and bioinformatics analysis.(1)according to target mi RNA gene prediction,Kitl gene has 470 miRNA, and among them, 29 miRNA can be found in all four databases(SUM = 4);we obtaine 12 miRNA after hnRNP A1, hnRNP, hnRNP K/D,hnRNP L J, SR target mi RNA gene prediction(SUM≥2, Intersected), and besides mmu-miR-370, the rest are all the Kitl target miRNA(SUM = 3).(2)According to the literature data, there are many same miRNAs with the Kitl target miRNA gene prediction results.(3)Following treatment with 10 μM Cd, a total of 335 mi RNA were deregulated(FC≥2), of which 191 were upregulated and 144 were downregulated, and58 had a greater than 10-fold change.( 4)The number of intersectional miRNA of predictional, literatures’ and the microarray’s data was 38(SUM≥3). Through furtherscreen out several highly homology of miRNA, this experiment finally get 29 miRNA associated with Kitl gene expression regulation. Among these 29 miRNA,mmu-miR-27a-3p 、 mmu-miR-34b-5p 、 mmu-miR-129-5p 、mmu-miR-297a-3p/mmu-miR-297b-3p/mmu-miR-297c-3p 、 mmu-miR-107-3p have better tips.(5)GO analysis results show that the function of target genes of 29 miRNA mainly enriched in the biological process of cell metabolism regulation, mRNA transcription regulation, after interleukin-6 mediated signal transduction, cell cycle, cell proliferation, differentiation and migration; and these target genes of 29 miRNA participate in the cytotoxicity process of cadmium by molecular functions such as combining regulation of nucleic acids, protein and ion, actin rely on ATP activity and transcription inhibitory activity, and the product the expression of target genes involved in the construction of cell components, such as biofilm formation, macromolecular complexes, intracellular organelles, membrane-bound organelles and nucleic acid; The results of pathway enrichment analysis showed that these miRNA target genes mainly occurred in the Ras signaling pathways, Rap1 signal Pathway, Fox O signaling pathways,the Hippo signaling Pathway and MAPK signal Pathway, regulation and control of actin cytoskeleton, stem cell signaling pathways regulating polymorphism, local adhesion and carcinogenic Pathway, these metabolic pathways closely involved in the cytotoxic mechanism of cadmium, including cell proliferation, differentiation and apoptosis regulation, stress, inflammatory reaction and so on.3、Compared with the control group, the real expression of these five miRNA in the infected group(4 h, 10 μM) had no statistically significant difference,but their change tendencys were the same.Conclusions:Under this experimental condition:1、Cadmium exposure can affect Kitl pre-mRNA alternative splicing of ovarian granulosa cells in mice,thus affecting the expression and regulation of gene Kitl,interfering normal function of granulosa cells.2、Cadmium might characteristically change mi RNA expression profiling in mice ovarian granulosa cells; Among these differentially expressed miRNA, 29 miRNA might closely associated with Kitl gene expression, and their downstream target genes may be closely involved in cytotoxic effect of cadmium, such as cell proliferation differentiation and apoptosis regulation, stress, inflammatory reaction and so on.3、Under this experimental condition(4 h, 10 μM), mmu-miR-27-a-3p,mmu-miR-34b-5p, mmu-mi R-297-a-3p, mmu-miR-129-5p, mmu-miR-107-3p all don’t participate in the change of the process of alternative splicing of Kitl pre-mRNA by cadmium in mice’s ovarian granulosa cells.