| Objective: Prostate cancer(PCa)ranks second malignant tumors among male in worldwide,and its mortality is the fifth.Although the prevalence of prostate cancer in China is only sixth among men,it is a rare and persistently rising tumor.The main reason for this phenomenon due to with the development of China's economy,the dietary structure of residents has changed,the average life expectancy has been extended,and physical examination has become more and more popular.The incidence of “rich disease” in this traditional concept has naturally increased.Prostate cancer has gradually become a major disease in urology.Traditional prostate cancer detection is screened of prostate specific antigen(PSA),and histopathological diagnosis is performed using prostate biopsy and prostate cancer score.The level of PSA and the pathological score are related to the prognosis of the tumor,however with the heterogeneity of patients and the development of the drug,this prognosis indicator is not as good as before.PCa is a hormone-dependent tumor,and most tumor cells depend on the androgen receptor pathway for survival and proliferation.Therefore,the current treatment for PCa mainly inhibits the function of androgen receptor by blocking the synthesis of androgen.Although the effect of androgen blockade treatment is significant,as the tumor evolves,the PSA of many patients recidivation and enters the stage of lethal-resistant castration-resistant prostate cancer(CRPC)ultimately.Reactive oxygen species(ROS)are widely distributed in the body.It can be used as an intracellular signal molecule,involved in the transduction of various signal pathways.It can damage biological macromolecules such as proteins,lipids and DNA with its own accumulation.ROS exceeds cause DNA damage leading to a decrease in the cell's self-repair ability,which may bring about malignant mutations and promote cells migration to malignant tumor.There is a dynamic balance between the mechanism of intracellular ROS production and clearance.Once the balance is broken,the cells will die.In most cases,ROS initiates the apoptotic process by attacking the mitochondria.In PCa,ROS levels in the tumor microenvironment are higher than normal prostates due to the age of the patient and the unhealthy habits.In the process of androgen blockade,chemotherapy and radiotherapy,apoptosis also produces more ROS,which can be transmitted to the cells that have not been destroyed by the tumor microenvironment,causing a chain reaction.How ROS in these environments affect tumor progression remains to be further studied.Mitogen-activated protein kinase(MAPK)is widely present in eukaryotic cells and can be activated by various factors.It is a conserved and complex tertiary kinase.When activated,they can cause phosphorylation of secondary kinases.Activated second kinase further phosphorylates primary kinases.Kinases regulate the function of transcription factors,thereby affecting cellular function.Among them,the primary kinase JNK can cause apoptosis,and the primary kinase ERK1/2 can promote cell survival.Prostate associated gene 4(PAGE4)is a stress-responsive protein that is active during embryonic development and naturally occurs in mature testis but not in normal prostate.PAGE4 is highly expressed in benign prostatic hyperplasia,prostatic precancerous lesions,and early prostate cancer,while low expressed in CRPC and metastatic prostate cancer.That means,the distribution of PAGE4 is closely related to the degree of malignancy of PCa.PAGE4 is also an intrinsically disordered protein with no fixed three-dimensional morphology.Phosphorylation kinases alter the morphology of intrinsically disordered proteins by phosphorylating their different amino acid sites,thereby affecting protein function.Some studies have shown that PAGE4 can reduce the production of ROS in PCa.Therefore,in-depth study of the role of PAGE4 in PCa and analysis of its distribution can provide a reference for judging the prognosis of prostate cancer,and may open a new horizon for the treatment of PCa.Methods: In this study,hydrogen peroxide was used to simulate ROS stimulation,and the expression of PAGE4 was observed in prostate cancer cells.The model of prostate cancer cell line overexpressing PAGE4 was established to investigate the response and function of PAGE4 in prostate cancer cells after ROS treatment.Single cell electrophoresis was used to observe the effect of PAGE4 after ROS-induced DNA damage;flow cytometry was used to detect the effect of PAGE4 on ROS-induced apoptosis.RNA sequencing technology was used to observe the changes in gene transcription levels of prostate cancer cells stimulated by ROS,and the signal pathway regulated by PAGE4 was analyzed by bioinformatics technology.Signaling pathways regulated by PAGE4 were detected at the cellular level by Western blotting and verified by immunohistochemistry in patient tissues.The effect of PAGE4 on mouse prostate cancer xenografts was observed in in vivo model,and the malignant genes related to prostate cancer in transplanted tumors were analyzed.The prognosis of PAGE4 and prostate cancer was analyzed in a tumor genomic mapping database.Results: 1.ROS stimulation can cause elevated levels of PAGE4 in prostate cancer cells,and the expression level of PAGE4 is positively correlated with the dose and treatment time of H2O2.This increase in the amount of PAGE4 expression can be inhibited by the reducing agent.2.In the prostate cancer cell model with conditional overexpression of PAGE4,PAGE4 can inhibit the production of endogenous ROS in cells.Exogenous ROS can still increase PAGE4,suggesting that the increased expression of PAGE4 by ROS stimulation by post-transcriptional regulated.Different phosphorylation levels of PAGE4 have different inhibitory effects on endogenouse ROS.3.Overexpressed PAGE4 can significantly reduce the DNA damage of prostate cancer cells induced by ROS.The length and DNA content of comet tail in single cell electrophoresis are less than those control group.Overexpressed PAGE4 can also significantly reduce the prostate cancer cells apoptosis caused by ROS.4.RNA sequencing suggests that ROS treatment,the DNA damage repair pathway was active and the MAPK pathway activated in prostate cancer cells with high PAGE4 expression.5.Detection of key molecules of MAPK pathway,JNK and its upstream and downstream molecules were inhibited by PAGE4 overexpression,while ERK1/2 was activated by PAGE4.Activation of ERK1/2 by PAGE4 was also observed in patient tissues.6.PAGE4 inhibited the apoptosis of prostate cancer xenografts in mice,showing a growth advantage.In the transplanted tumors overexpressing PAGE4,the expression of malignant related genes in prostate cancer was lower than control group,suggesting that PAGE4 promotes cancer growth but inhibits the effect of cancer evolution.7.Prostate cancer patients with high expression of PAGE4 have a longer disease-free survival.Conclusion: 1.In prostate cancer cells,ROS can cause PAGE4 increase,which can reduce the production of endogenous ROS and promote the balance of ROS in cells.2.PAGE4 reduces the production of endogenous ROS mainly through post-transcriptional level regulation,PAGE4 specific kinase CLK2 and HIPK1 by changing the phosphorylation of PAGE4 and altering its spatial structure.3.Overexpressed PAGE4 can significantly reduce ROS-induced DNA damage and apoptosis,indicating that ROS treatment,PAGE4 increase is a self-protective mechanism.4.Overexpression of PAGE4 can enhance the expression of DNA damage repair related genes,mainly activating the MAPK pathway.5.Cell and tissue levels can be observed activation of the MAPK pathway by PAGE4 in prostate cancer cells.6.PAGE4 can promote the growth of subcutaneous xenograft prostate cancer cells,but inhibit the evolution of prostate cancer cells to malignant tumors.7.In patients with high PAGE4 tend to have a better prognosis.This may be because PAGE4 decreases cancer cell mutations by reducing DNA damage,thereby reducing its malignant evolution,patients acquire longer disease-free survival. |
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