The Role And Mechanisms Of The PI3K/AKT/mTOR Pathway In Microcystin-LR-induced Malignant Transformation Of Mouse Spermatogonia

ObjectiveMicrocystin-LR(MC-LR)is a cyanotoxin with tumor-promoting activity.Exposure to cyanotoxins from the environment may be an important trigger for seminoma.This study aimed to investigate the effect of MC-LR on the malignant transformation of spermatogonia,and to reveal the regulatory role and mechanisms of PI3K/AKT/m TOR pathway in MC-LR-induced malignant transformation of mouse spermatogonia,providing etiological clues for the development of spermatogonia..Methods1.Exposure concentration and method of MC-LR on GC-1 cellsThe effect of different concentrations of MC-LR on the proliferation ability of mouse spermatogonia cell line(GC-1)was detected by CCK8 to determine the concentration of MC-LR exposure in subsequent experiments.GC-1 cells were continuously cultured with MC-LR and passaged every 3 days,so that the cells were always exposed to 20 n M MC-LR until the 35 th generation.The 15 th,25th and 35 th generation cells were marked as passage 15,25 and 35 for subsequent experiments.2.Effects of MC-LR multigenerational exposure on the proliferation,migration and invasion ability of GC-1 cellsThe accumulation of MC-LR in cells was detected by western blot and immunofluorescence,and the morphological changes of cells were observed.Altered cell proliferation,migration and invasion abilities after MC-LR multigenerational exposure were detected by cell proliferation assay,scratch assay and Transwell assay.3.Nude mouse tumorigenesis assay of MC-LR multigenerational exposure GC-1cellsAfter 35 generations of MC-LR exposure,the GC-1 cells and the same generation control group cells were injected subcutaneously into nude mice.The weight and volume of the tumors were measured following 28 days of feeding.Hematoxylin-eosin(H&E)staining was used for the pathological analysis of tumor tissues,and immunohistochemistry was used to measure the tumor marker Ki67 in tumor tissues.4.Effects of MC-LR multigenerational exposure on PI3K/AKT/m TOR pathway and downstream proteins in GC-1 cellsActivation of PI3K/AKT/m TOR pathway proteins(PIK3CA,PIK3 CB,AKT,m TOR)and downstream proteins related to proliferation,migration and invasion(c-MYC,CDK4,CCND1,MMP14)in GC-1 cells was detected by Western Blot after multigenerational exposure of MC-LR.5.Role and mechanisms of PI3K/AKT/m TOR pathway in MC-LR-induced malignant transformation of GC-1 cellsAfter a 24 h pretreatment with the PI3 K inhibitor Wortm on the 35 th generation GC-1 cells following MC-LR exposure and the same generation control group.The levels of PI3K/AKT/m TOR pathway and downstream proteins were detected by Western Blot.The distribution of cell-cycle was detected by propidium iodide staining and flow cytometry.Results1.MC-LR can enter and accumulate in GC-1 cellsMC-LR could significantly enhance the proliferation ability of GC-1 cells at the concentration of 20 n M(P<0.05),and the subsequent dose was determined to be 20 n M.Western Blot and immunofluorescence results showed that the bands and fluorescence intensity of MC-LR were enhanced after 15,25 and 35 generations of MC-LR exposure of GC-1 cells(P<0.05).2.MC-LR multigenerational exposure induced malignant transformation of GC-1 cellsMicroscopic observation revealed that after 35 generations of MC-LR exposure to GC-1 cells,the GC-1 cells showed stacked growth and morphological changes such as enlarged nuclei and indistinct demarcation between nucleus and cytoplasm.The proliferation,migration and invasion abilities of GC-1 cells were enhanced after35 generations of MC-LR exposure(P<0.05).The results of tumorigenic assay in nude mice showed that MC-LR exposed group had higher volume and weight of subcutaneously transplanted tumors than the control group(P<0.05).H&E staining and immunohistochemistry results showed that tumor cells in MC-LR group were more diffusely distributed and disorderly arranged.Pathological examination showed that the subcutaneous xenografts in MC-LR group were highly malignant seminoma.The expression level of tumor marker Ki67 was higher than the control group(P<0.05).3.Multigenerational exposure of MC-LR activates PI3K/AKT/m TOR pathway and downstream proteins in GC-1 cellsWestern blot showed that,compared with the same generation control group,the PI3K/AKT/m TOR pathway proteins(PIK3CA,PIK3 CB,AKT,m TOR)and their downstream proteins related to proliferation,migration and invasion(c-MYC,CDK4,CCND1,MMP14)were significantly activated in MC-LR-treated GC-1 cells for 35 generations.4.PI3K/AKT/m TOR pathway is involved in MC-LR-induced malignant transformation of GC-1 cellsThe PI3K/AKT/m TOR pathway and its downstream proteins activated by MC-LR in GC-1 cells were significantly inhibited after pretreatment with Wortm(P<0.05).The proliferation,migration and invasion abilities of GC-1 cells enhanced after MC-LR exposure are decreased by Wortm pretreatment(P<0.05).5.PI3K/AKT/m TOR pathway-mediated cell cycle dysregulation involvement in the malignant proliferation of GC-1 cells induced by MC-LRAfter 35 generations of MC-LR treatment of GC-1 cells,the distribution of G0/G1 phase decreased and the distribution of S phase increased(P<0.05).Wortm pretreatment of malignantly transformed GC-1 cells increased the distribution of cells in G0/G1 phase and decreased the distribution of cells in S phase(P<0.05).Conclusion1.MC-LR multigenerational exposure activates PI3K/AKT/m TOR pathway to induce malignant transformation of mouse spermatogonia.2.PI3K/AKT/m TOR pathway-mediated cell cycle dysregulation is involved in MC-LR-induced malignant proliferation of mouse spermatogonia.