The Study On The Mechanism Of MELK Promoting The Growth Of Bladder Cancer Through ATM/CHK2/p53 Pathway

Background Bladder cancer(BCa)is a common urinary tumor that is biologically and clinically heterogeneous.Defects in the p53 pathway contribute to diagnosis and therapy difficulties as well as adverse clinical outcome in BCa.It has been shown that maternal embryonic leucine zipper kinase(MELK)is overexpressed in multiple human tumors and high level of MELK was correlated with clinically aggressive disease and poor survival,indicating that this kinase is a potential therapeutic target.Although the mechanisms by which MELK mediates aggressive tumor growth are not completely understood,MELK inhibition has been confirmed to lead to the consecutive phosphorylation of ataxia telangiectasia-mutated(ATM)and cell cycle checkpoint kinase 2(CHK2)and to the upregulation of p53 and p21.OTSSP167 is a targeted inhibitor of MELK.It has been observed that OTSSP167 had a strong antitumor effect in a variety of malignant tumors.Objective To investigate the clinical significance,biological function and mechanism of MELK in bladder cancer,and to evaluate the antitumor potential of OTSSP167 in bladder cancer.Methods Bioinformatics was used to analyze the expression of MELK in bladder cancer and predicted its molecular mechanism.The influence of MELK on the prognosis of bladder cancer was analyzed by TCGA,GSE13507 and other databases.The expression of MELK in bladder cancer was detected by RT-PCR,western blot and immunohistochemistry.The effects of MELK and OTSSP167 on the proliferation and migration of bladder cancer were detected in vitro by MTT,plate cloning,Transwell migration and flow cytometry.The effects of MELK and OTSSP167 on the proliferation of bladder cancer in vivo were detected by nude mice bearing tumor model.RT-PCR,western blot,immunofluorescence and restoration experiments were used to explore the mechanism of MELK regulating the growth of bladder cancer and the inhibitory effect of OTSSP167 on MELK.Results MELK was highly expressed in bladder cancer and was associated with tumor progression and poor prognosis.In vitro experiments showed that MELK knockdown inhibited the proliferation and migration of bladder cancer cells,whereas overexpression promoted the proliferation and migration of bladder cancer cells.Cell experiments and tumor bearing experiments in nude mice showed that MELK knockdown could induce G1/S cell cycle arrest in BCa via activating the ATM/CHK2/p53 pathway,thus inhibiting its proliferation.In vitro and in vivo experiments confirmed the antitumor potential of OTSSP167 in bladder cancer,and further confirmed that MELK could regulate the biological behavior of bladder cancer through ATM/CHK2/p53 pathway.The recovery experiment showed that OTSSP167 could restore the biological behavior change of bladder cancer caused by overexpression of MELK.Conclusions Our study results showed that MELK overexpression predicted a poor prognosis in BCa patients.Both in vitro and in vivo studies indicated that MELK silencing or OTSSP167 treatment exhibited anti-tumor effects through abrogating cell proliferation and migration.MELK silencing could induce G1/S cell cycle arrest in BCa via activating the ATM/CHK2/p53 pathway.OTSSP167 had potential antitumor effect in bladder cancer.