The Role Of PiRNA/Bcl-xL And AMPK/ULK In Autophagy Of Spermatogenic Cells Induced By Cadmium

Objective:To study cadmium-induced autophagy in spermatocytes and its possible mechanism.Methods:1.The effect of cadmium exposure on the expression of piRNA in testis tissue and the prediction of target genesSix 6-week-old SD rats were divided into the control group and the experimental group using a random number table.After 7 days of adaptive feeding,the experimental group was given a 3mg/kg/day CdCl₂solution by intragastric administration,and the control group was given intragastric administration with the same dose of distilled water.After 28 days of continuous exposure,testicular tissues were collected.Trizol method was used to extract total RNAs from testicular tissue;and use the Arraystar Rat piRNA Array RN4 gene chip for hybridization sequencing to detect differentially expressed piRNA;then use RT-q PCR to verify the expression of piRNA;finally,use GO enrichment analysis and KEGG Pathway analysis to look for target genes of differentially expressed piRNA.In order to explore the effect of cadmium exposure on the expression of piRNA in testis tissue and the prediction of target genes.2.The role of piRNA-DQ765261/Bcl-x L and AMPK/ULK in cadmium-induced autophagy in GC-2spd cellsFirst,the CCK-8 experiment was used to determine the half-lethal dose of CdCl₂to mouse primary spermatocytes cell（GC-2spd）,and determines the exposure concentration for subsequent experiments;then Chemiluminescence was used to detect ROS produced by GC-2spd cells induced by different doses of cadmium;and transmission electron microscope was used to observe the autophagosomes in cadmium-induced cells;finally Western Blot was used to detect the expression levels of autophagy-related proteins（Beclin1 and LC3Ⅱ/I）and autophagy signaling pathway-related proteins（Bcl-x L,p-AMPKαand p-ULK1）.Through these experiments,the specific molecular mechanism of cadmium-induced autophagy in spermatocytes was explored from the morphological and molecular levels.Results:1.The effect of cadmium exposure on the expression of piRNA in testis tissue and the prediction of target genes（1）Gene chip results show that after the testis tissues of rats exposed to CdCl₂（3mg/kg·day）,the expressions of 272 piRNA show up-regulated,following with the expressions of 402 piRNA show down-regulated（Fold Change>2,P<0.05）;（2）Four up-regulated piRNAs and 4 down-regulated piRNAs with larger differential expression are selected to verify,by RT-q PCR,the results show that the expression results of 3 up-regulated piRNAs（DQ765261,DQ737820 and DQ614103）are consistent with the gene chip results,and these difference are statistically significant（P<0.05 or P<0.01）;Considering gene chip and RT-q PCR results,it is found that the piRNA with the largest expression difference is piRNA-DQ765261.（3）Through GO enrichment analysis,it is found that the target gene of piRNA-DQ765261 is mainly involved in 46 biological processes,such as transcription regulation using DNA as template,positive regulation of neuronal differentiation,cell response to glucose starvation,negative regulation of apoptosis process,cell proliferation,and response to hypoxia（P<0.05）;the analysis of target gene’s binding characteristics showed that piRNA-DQ765261 contains binding sites with Bcl-x L.2.The role of piRNA-DQ765261/Bcl-x L and AMPK/ULK in cadmium-induced autophagy in GC-2spd cells（1）The median lethal dose of CdCl₂to GC-2spd cells is 13.36μmol/L.Thus the subsequent experiments are divided into control group（the serum-free medium does not contain CdCl₂）and 3 experimental groups（the serum-free medium contains 2.5,5,and 10μmol/L CdCl₂,respectively）;（2）After 24 hours of exposure to CdCl₂,the number of autophagosomes and autophagolysosomes increases in the experimental groups（2.5,5,and 10μmol/L CdCl₂）compared with the control group;（3）Compared with the control group,the intracellular ROS increases in the 2.5and 10μmol/L CdCl₂groups,which are statistically significant（P<0.05 or P<0.01）;（4）Detections of autophagy-related proteins show that compared with the control group,the expressions level of Beclin1 in the experimental groups increase in a dose-dependent manner,while only the 10μmol/L CdCl₂group has a statistically significant difference（P<0.05）;simultaneously the expressions of LC3Ⅱ/I increase in a dose-dependent manner,and the differences of the 5 and 10μmol CdCl₂groups are statistically significant（P<0.05 or P<0.01）.（5）Results of autophagy signaling pathway related proteins show that compared with the control group,the expressions of p-AMPKαprotein in the experimental group increase in a dose-dependent manner,and the difference of the 5 and 10μmol/L CdCl₂groups are statistically significant（P<0.05 or P<0.01）;the expressions level of p-ULK1 in the experimental group increase in a dose-dependent manner,and the difference of the 10μmol/L CdCl₂group is statistically significant（P<0.05）;the expressions of Bcl-x L protein in the experimental groups decrease in a dose-dependent manner,and the differences of the 5 and 10μmol/L CdCl₂group are statistically significant（P<0.05 or P<0.01）.Conclusions:1.Cadmium exposure in vivo can induce the up-regulation of piRNA（DQ765261,DQ737820 and DQ614103）expression in rat testis.piRNA-DQ765261 may participate in the autophagy process of spermatogenic cells through its target gene Bcl-x L.2.Cadmium exposure in vitro can induce autophagy in GC-2spd cells.Cadmium can induce the increase of intracellular ROS.On the one hand,ROS may regulate the down-regulation of Bcl-x L protein expression through piRNA-DQ765261,on the other hand,ROS may activate the AMPK/ULK signaling pathway;jointly promote the expression of Beclin1,thereby inducing autophagy...