Studies On The Expression Of PKM2in Bladder Urothelial Cancer And Its Effects On Glucose Metabolism And Biological Behaviors Of Bladder Cancer Cell Lines

Background:Bladder cancer is one of the most common malignant tumors in urogenital system, of which bladder urothelial carcinoma accounts for90%approximately.The incidence of bladder cancer is in an upward trend in the past decades. Though studied extensively, the molecular mechanism for the growth and spreading in bladder cancer remains unclear.Altered metabolism is fundamental to the growth and survival of cancer cells. The M2splice isoform of pyruvate kinase is a key regulator of Warburg effect in cancer cells.Most cancer cells has increased expression of PKM2, which positions PKM2as an attractive target for cancer therapy. However, until now there has been little information about the expression and function of PKM2in bladder cancer.Objectives:(1) To investigate the expression of PKM2in human bladder cancer tissues and cell lines with regard to related clinicopathologic significance.(2) To construst a lentivirus vector-based RNA interference of PKM2and verify the effect of RNAi vectors.(3) To analyze the alterations of glucose metabolism and biological behaviors upon the knockdown of PKM2in bladder cell lines.(4) To explore the potential role of PKM2in the spreading.of bladder cancerMethods:(1) Thirty six cases of bladder cancer tissues and matched normal adjacent tissues were collected. The expression of PKM2was detected by immunohistochemistry, Western-blot and quantitative real time polymerase chain reaction (Real-time PCR).The relationship between clinicopathologic parameters and PKM2expression was analyzed.(2) The pLV lentivirus vector with PKM2shRNA was constructed. Western-blot and real time-PCR were used to measure the interference effects of RNAi on PKM2expression.(3) The effects of PKM2knockdown on T24cell proliferation, apoptosis, ROS,migration, invasion and spreading were assessed by CCK8assay, transwell assay, flow cytometry and nude mouse xenografts and orthotopic model.(4) Western-blot and Real-time PCR were performed to measure the expression of E-cadherin,vimentin and fibronectin after the down-regulation of PKM2.Results:(1) Bladder cancer tissues and cell lines have increased expression of PKM2compared with matched normal adjacent tissue and normal bladder cell line respectively. The expression of PKM2correlate with stage and grade of bladder cancer.(2)Three PKM2shRNA and one control shRNA with pLV lentivirus vector were constructed. All the PKM2shRNA vector significantly downregulate the expression of PKM2, of which PKM2shRNA1performs best. And the control shRNA show no effects on the expression of PKM2.(3) Knockdown of PKM2decreases the glucose consumption and lactate production and suppress cell proliferation, migration, invasion while increase the ROS level and apoptosis in T24bladder cancer cells in vitro. Knockdown of PKM2leads to smaller xenografts tumor volume in nude mouse models.And in the Orthotopic models, knockdown of PKM2lead to smaller tumor volume as well as lesser metastastis formation.(4) Knockdown of PKM2decreases the expression of vimentin and fibronectin while increases E-cadherin expression.Conclusions:(1) PKM2protein and mRNA have a significantly high expression in bladder tissues and cell lines with positive corelation with tumor grade and stage. PKM2may be involved in the occurence and progression of bladder cancer.(2) Knockdown of PKM2impair the glucose metabolism and suppress cell proliferation, migration, invasion and spreading in vitro and in vivo and lead to the ROS accumulation and apoptosis. PKM2may serve as a potential therapeutic target for the treatment of bladder cancer.(3) Knockdown of PKM2decrease the expression of vimentin and fibronectin while increase the expression of E-cadherin,indicating that PKM2may play a role in the epithelial-mesenchymal transition.PKM2may promote invasion and metastasis via inducing the EMT process in bladder cancer. Figures:26; tables:27; references:85.