The Role And Mechanism Research Of PiRNA In Mice Spermatogenesis Injury Induced By Nickel Nanoparticles

In recent years,the incidence of infertility among couples of child-bearing age increased worldwide,and the male reproductive health has become a major public health problem.People began to pay attention to exogenous chemicals-induced reproductive endocrine diseases and adverse pregnancy outcomes.Because of their unique physical,chemical,and biological properties,nickel nanoparticles(Ni NPs)have been used and continue to enter the environment of people ’s living.However,the widespread application of Ni NPs also greatly increased its exposure probability,and brought a potential danger to the environment and human body that have been widely studied.PIWI-interacting RNAs(piRNA),a unique type of small(24-32nucleotides)non-coding RNA,is an important epigenetic molecule.More and more studies have shown that piRNA plays a role in mammalian germ cells and participates in normal spermatogenesis.Therefore,the purpose of this study was to elucidate the role and possible molecular mechanisms of piRNA in mouse spermatogenesis injury induced by Ni NPs.It will provide a theoretical basis for further exploring the regulatory mechanism of Ni NPs-induced cellular damage in the mouse spermatogenesis process,which may offer potential biomarkers for the prevention and treatment of spermatogenic process damage,and is of great significance for the protection of male reproductive health.Study on Ni NPs induced mice spermatogenesis injury and high throughput sequencing in mice.1.Ni NPs induced spermatogenesis injury in miceAdult ICR male mice weighting 18-22 g(n=60)were randomly divided into 5groups,which were control group(normal saline),low-dose group(5 mg/kg Ni NPs),medium-dose group(15 mg/kg Ni NPs),high-dose group(45 mg/kg Ni NPs),and micron nickel group(45 mg/kg micron nickel).After intragastrically administrated with different solution for 4 weeks,all mice were sacrificed to collect blood sample as well as testicular and epididymal tissues.The reproductive toxicity of nano-nickel on male mice was evaluated by a series of parameters,including weight and organ coefficient,testicular marker enzymes,sperm motility,histopathology,and ultrastructural changes under transmission electron microscopy(TEM).Compared with the control group,the testicular and epididymal organ coefficients of mice in each group increased significantly after calculating the body weight of mice and the weight of reproductive organs.The obtained of enzymes showed that testicular marker enzymes in the medium,and high dose Ni NPs groups increased in a dose-dependent manner.The sperm motility parameters in the Ni NPs treated groups showed a downward trend with an increasing dose of Ni NPs exposure,and the number of sperm forward motility in the micro-nickel group was significantly less than that in the control group(P<0.05),suggesting that Ni NPs and micro-nickel may decrease the sperm motility in mice after exposure.Pathological morphogical results of testicular and epididymal tissues showed that the spermatogenesis was seriously impaired as compared with the control group,as demonstrated by the appearance of cell apoptosis and damage in the spermatogenic cells at all levels in the seminiferous tubules in the Ni NPs-treated group,accompanied by a decreased number of spermatogenic cells.Moreover,abnormal morphology and structure of the sperm cells in the seminiferous tubules of the Ni NPs-treated group observed,such as uneven electron density,and mitochondrial damage,as compared with the control group.All of these results demonstrate that Ni NPs exposure can cause mouse sperm damage and affect mice spermatogenesis.2.Screening differential piRNA in Ni NPs induced mice spermatogenesis injury via high-throughput sequencing technologyMice testis tissues in control group and high-dose group were collected,and then total RNA was extracted.Thereafter,the differentially expressed piRNA was selected,and the target differential gene expression and biological function analysis of piRNA were performed via high-throughput sequencing technology.Finally,the key differentially expressed piRNA were selected to explore their possible regulation signal pathways in mice spermatogenesis injury induced by Ni NPs.High-throughput sequencing results of testicular tissue showed that a total of 509 different piRNA were found during Ni NPs-induced spermatogenesis damage in mice,in which 6 piRNA with the high and significantly differential expression folds were selected validated by RT-q PCR.It was verified that piR-mmu-32362259 is stable and highly expressed in mouse testis tissues and GC-1 cell,which is consistent with the results of high-throughput sequencing technology.Moreover,it was predicted that piR-mmu-32362259 regulated a total of 1009 m RNAs.And through Pathway analysis,it was found that piR-mmu-32362259 target gene was significantly enriched in PI3K-AKT signaling pathway.Therefore,we speculate that piR-mmu-32362259 may influence the occurrence and development of mice spermatogenesis injury by regulating the PI3K-AKT signaling pathway.Research on the role and mechanism of PI3K-AKT pathway in Ni NPs induced GC-1 cell damage.GC-1 cells were taken as the research object,and incubated with 0(control group),25,50,75,100 and 200 μg/ml Ni NPs.After exposure 24 hours,the effects of Ni NPs on GC-1 cell activity,apoptosis,and cycle were examined by CCK8 assay and flow cytometry,respectively.The changes of cell morphology and ultrastructure after exposure to nanometer nickel were observed under light microscope and transmission electron microscope,respectively.Furthermore,Western Blot and RT-q PCR were used to detect the exposure effect of Ni NPs on the key proteins and downstream molecules of PI3K-AKT signaling pathway in order to explore the role and mechanism of underlying the effect of Ni NPs on GC-1 cell damage.The results of CCK8 showed that the survival rate of GC-1 cells decreased with the increasing doses of Ni NPs exposure.The results of flow cytometry showed that,compared with the control group,the cell cycle of GC-1 cells in the 25,50,100 and 200 μ g/ml Ni NPs groups was blocked in the G1 phase,which inhibited cell proliferation and promoted apoptosis,and in a dose-dependent manner.The abnormal morphology of the cells was observed,and the number of cells has decreased obviously under light microscope,and the changes of cell microscopy were observed under TEM.Western Blot analysis results showed that the expression levels of PI3 K,AKT,m TOR,p-PI3 K,p-AKT and p-m TOR,as well as downstream molecules CDK4 and Bcl-2 proteins significantly decreased,whereas those of Bax,Caspase-3 and p21 proteins increased significantly.RT-q PCR detection results showed that the transcription levels of p21 and Caspase-3 increased significantly,but those of CDK4,Bcl-2,PI3 K and m TOR decreased,suggesting Ni NPs affects the cell activity,cycle,and apoptosis of mouse spermatogonia by regulating the key proteins in PI3K-AKT signaling pathway and downstream important molecules,ultimately leading leads to cell damage.Reserrch on the role and regulatory mechanism of piRNA in Ni NPs induced GC-1 cell damage.To determine the best MOI for GC-1 cells,lentivirus with low expression of piR-mmu-32362259 was transfected to GC-1 cells.Thereafter,the RT-q PCR technique was used to detect the transcription of piR-mmu-32362259 in GC-1 cells after exposure to Ni NPs.Secondly,CCK8 assay and flow cytometry were used to detect the effects of piR-mmu-32362259 on GC-1 activity,cycle,and apoptosi in the GC-1 cells after esposure to Ni NPs.Finally,Western Blot technology was,respectively,used to detect the effect of piR-mmu-32362259 on key proteins and downstream functional proteins in PI3K-AKT signaling pathway and further to explore the possible regulatory mechanism underlying the effect of piR-mmu-32362259 on Ni NPs-induced GC-1 cell damage.The transfection efficiency was the highest at the time when the MOI of GC-1cell lentivirus transfection was 20.The results of RT-q PCR analysis verified that,compared with the blank group(the normal GC-1 cells),the expression of piR-mmu-32362259 increasing by 3.69 times(P<0.05)in the 100 μg/ml Ni NPs group,3.82 times(P<0.05)in the piR-mmu-32362259-NC group(piR-mmu-32362259-negnative control group),and 1.47 times in the piR-mmu-32362259-KD group(piR-mmu-32362259-knock down group)(P>0.05).The results of CCK8 assay showed that piR-mmu-32362259 gene knockdown has a significant inhibitory effect on Ni NPs-induced reduction of GC-1 cell activity.The results of flow cytometry analysis showed that the apoptotic rate of the 100 g/ml Ni NPs group and the piR-mmu-32362259-NC group was obviously higher than that of the blank group(P<0.05),while the apoptotic rate of the piR-mmu-32362259-KD group was not significantly different from that of the blank group(P>0.05).But the apoptotic rate of piR-mmu-32362259-KD group was significantly lower than that of piR-mmu-32362259-NC group(P<0.05).Additionally,compared with the piR-mmu-32362259-NC group,there was no significant change in the G1 phase of the cell cycle,the S phase increased,and the G2 phase decreased in the piR-mmu-32362259-KD group,suggesting that cell cycle may be arrested at S phase.There were significant differences in the expression levels of PI3 K,AKT,p-PI3 K,p-AKT,p-m TOR,CDK4,Bcl-2,Bax,p21 and Caspase-3 in piR-mmu-32362259-KD group as compared with piR-mmu-32362259-NC group.Collectively,piR-mmu-32362259 may influence the process of Ni NPs-induced GC-1 cell damage by regulating the PI3K-AKT signaling pathway.The purpose of this study was to elucidate the role and possible molecular mechanisms of piRNA in mouse spermatogenesis injury induced by Ni NPs.