ROS、piRNA-DQ759395 Regulate The Role Of P53 In Cadmium-induced Spermatogenic Cell Cycle Abnormalities

Objective:To study the effect and possible mechanism of cadmium on the cycle of spermatogenic cells（GC-2 spd cell）by regulating P53 through ROS and piRNA-DQ759395.Methods:1.Effects of cadmium exposure on piRNA expression in testis tissue and prediction of target genesSix 6-week-old SD male rats were divided into control group and experimental group by random number table,and were fed adaptively for 7 days.The experimental group was given 3 mg/（kg·day）cadmium chloride（Cd Cl2）solution by gavage.After exposure to the drug,the control group was given the same dose of distilled water,and testicular tissue was collected after continuous exposure for 28 days;total RNA was extracted from the right testis tissue by Trizol method;Arraystar Rat piRNA Array RN4 gene chip was used for hybridization sequencing to detect differentially expressed piRNA Then,RT-q PCR was used to verify the expression of piRNA;finally,GO enrichment analysis and KEGG pathway analysis were used to find the target genes of differentially expressed piRNA to determine the effect of cadmium exposure on testicular piRNA expression and predict target genes.2.The role of piRNA-DQ759395/P53 in cadmium-induced GC-2 spd cell cycle abnormalitiesCCK-8 assay was used to determine the median lethal dose of Cd Cl2 to GC-2 spd cells,and the exposure concentration of subsequent experiments was determined;DCFH-DA probe was used to detect the content of ROS induced by different doses of cadmium in cells;flow cytometry was used to observe cadmium Abnormal cell cycle caused by induced cells;Western Blot technology was used to detect the expression levels of related proteins（P53,p-P53,P21,cyclin D1 and cyclin E1）.At the same time,in order to clarify the roles of ROS,piRNA,and P53 in cadmium-induced cell cycle abnormalities,cells were pretreated with reactive oxygen species inhibitor（NAC）and P53 transcriptional activity inhibitor（PFT-a）for 2 h,and then treated with cadmium.24h,use flow cytometry and Western Blot technology to detect related proteins;use transient transfection technology to transfect antagomir-759395 into GC-2 spd to silence piRNA-DQ759395,use flow cytometry and Western Blot technology to detect related proteins.Results:1.Effects of cadmium exposure on piRNA expression in testis tissue and prediction of target genes（1）Gene chip results showed that 272 piRNAs were up-regulated in rat testis tissue exposed to Cd Cl₂（3 mg/kg·day）（Fold Change>2,P<0.05）;（2）The 4 up-regulated piRNAs with large differential expression were selected for verification.RT-q PCR results showed that the detection results of 4 up-regulated piRNAs（DQ614103,DQ759395,DQ765261,DQ607852）were consistent with the piRNA chip results,and the difference was statistically significant（P<0.01）.Based on the results of gene chip and RT-q PCR,piRNA-DQ759395 was selected for further analysis.（3）Through GO enrichment analysis,it was found that piRNA-DQ759395 target genes are mainly involved in 67 biological processes such as apoptosis,regulation of circadian rhythm,mitosis,lipid metabolism,negative regulation of glucocorticoid-mediated signaling pathways,and cell cycle（P<0.05）;the analysis results of target gene binding characteristics showed that piRNA-DQ759395 has a binding site with P53.2.The role of piRNA-DQ759395/P53 in cadmium-induced GC-2 spd cell cycle abnormalities（1）The median lethal dose of Cd Cl₂to GC-2 spd cells was 13.867μmol/L,so the highest dose of Cd Cl₂in the subsequent experiments was 10μmol/L,and divided into control group（serum-free medium without Cd Cl₂）and 3 experimental groups（The serum-free medium contains 2.5,5,and 10μmol/L Cd Cl₂,respectively）;（2）After exposure to Cd Cl₂for 24 hours,compared with the control group,the intracellular ROS in the 5 and 10μmol/L Cd Cl₂exposure groups increased,and the difference was statistically significant（P<0.05 or P<0.01）;q PCR results showed that,Compared with the control group,the expression level of piRNA-759395 in the10μmol/L Cd Cl₂group was up-regulated,and the difference was statistically significant（P<0.05）.（3）The results of flow cytometry showed that compared with the control group,the number of cells in the G1 phase in the 10μmol/L Cd Cl₂group was significantly increased,and the difference was statistically significant（P<0.01）.Compared with the 10μmol/L Cd Cl₂experimental group,the expression levels of P53,p-P53,and P21in the experimental group were all increased,and the differences in the 5 and 10μmol/L Cd Cl₂groups were statistically significant（P<0.05 or P<0.01）.（4）After the antioxidant NAC pretreatment,the results of flow cytometry showed that compared with the 10μmol/L Cd Cl₂exposure dose group,the number of cells in the G1 phase of the cell cycle decreased,and the difference was statistically significant（P<0.05）;Detection of related proteins showed that the protein expressions of P53,p-P53 and P21 were down-regulated（P<0.05）,and the protein expressions of cyclin D1 and cyclin E1 were up-regulated（P<0.05）.（5）After GC-2 spd cells were transiently transfected with piRNA DQ759395inhibitor,q PCR results showed that compared with the control group,the expression level of piRNA-759395 was down-regulated after transfection with antagomir-759395,and the difference was statistically significant（P<0.001）;the results of flow cytometry showed that compared with the control group,the number of cells in the G1phase of the cell cycle decreased,and the difference was statistically significant（P<0.05）.The protein expression levels of P53,p-P53,and P21 were down-regulated,and the difference was statistically significant（P<0.05 or P<0.01）,the protein expression levels of cyclin D1 and cyclin E1 were up-regulated（P<0.01）.（6）The results of flow cytometry showed that compared with the 10μmol/L Cd Cl2 exposure dose group,the number of cells in the G1 phase of the cell cycle decreased after pretreatment with P53 transcriptional activity inhibitor（PFT-a）,and the difference was statistically significant.Significant（P<0.05）;after PFT-a pretreatment of GC-2 spd,compared with the 10μmol/L Cd Cl2 exposure dose group,the protein expression levels of P53,p-P53,and P21 were down-regulated（P<0.05）.The expression level of cyclin E1 protein was up-regulated（P<0.05）.Conclusions:1.In vivo exposure to cadmium can induce abnormal expression of some piRNAs in rat testis,and piRNA-DQ759395 may affect the cell cycle through its target gene P53.2.Cadmium induces abnormal cell cycle of GC-2 spd,and its mechanism may be that,on the one hand,cadmium induces the increase of ROS in GC-2 spd cells;Activates P21,which ultimately has an effect on the cell cycle.