Biological Functions Of PLK1Regulated By MiR-100and Screening Of Urine Based MicroRNA Biomarker In Bladder Cancer

Bladder cancer is one of the most common human malignancies, and its pathogenesis is complex, involving a large number of genes’expression, aberrant functions and changes in multiple signaling pathways. At present, the molecular genetics of development and progression of bladder cancer is still unclear. In recent years, the pathological relevance and significance of microRNAs (miRNAs) in bladder cancer have attracted more and more attention. In the present study, we comprehensively studied the expression, biological function and molecular mechanisms of miR-100and its downstream target gene, PLK1, in the pathogenesis of bladder cancer. In addition, we also screened urine based miRNA biomarkers to develop a new clinical non-invasive diagnostic method in bladder cancer.Firstly, we used miRNA microarray in bladder cancer tissues and adjacent normal mucosa, and found miRNAs’down-regulation was likely an important mechanism of bladder cancer development. After further screening and validation, we revealed miR-100was generally down-regulated and its expression level, was correlated to tumor stage, tumor grade and lymph node metastasis in bladder cancer. The gain of function study showed enhanced expression of miR-100could not only inhibit cell proliferation, migration, invasion, arrest cell cycle in G2/M phase and promote apoptosis in vitro but also inhibit tumor growth in vivo. These discoveries illustrated new molecular mechanisms of development, invasion and metastasis of bladder cancer, and provided a new therapeutic target for clinical treatment in bladder cancer.Secondly, we predicted target genes of miR-100by bioinformatics, then identified PLK1was a target gene of miR-100in bladder cancer through biological experimental methods. By quantitative RT-PCR, Western Blotting, immunohistochemical techniques and luciferase reporter gene method, we foundmiR-100was partially complement to PLK1mRNA3’ UTR sequence and inhibited thetranslation of PLK1mRNA, which regulated PLK1in the post-transcriptionallevel. The expression level of miR-100was negatively correlated to PLK1protein expression in bladder cancer. In addition, we used one of PLK1inhibitors (BI2536) and found PLK1was directly involved in miR-100inducedcell proliferation, migration and invasion in bladder cancer. This couldprovide a practical theoretical basis for BI2536in the clinical treatmentof bladder cancer. Finally, we established a simple, easy and reproducible urine basedextracellular miRNA extraction process. By urine miRNA microarray, we filteredout a series of high expression miRNA in the urine of patients with bladdercancer. We also found U6can be used as a reference gene in urine miRNA qRT-PCR.After validation of20urine miRNA markers, we found miR-509-5p/miR-124ratioshowed high accuracy, and therefore could be a non-invasive urine biomarkerfor the diagnosis of bladder cancer. In summary, we found that miR-100was generally down-regulated and itsexpression level was correlated to tumor stage, tumor grade and lymph nodemetastasis in bladder cancer. The gain of function study showed enhancedexpression of miR-100could not only inhibit cell proliferation, migration,invasion, arrest cell cycle in G2/M phase and promote apoptosis in vitro butalso inhibit tumor growth in vivo. PLK1was directly involved in miR-100inducedcell proliferation, migration and invasion in bladder cancer. miR-509-5p/miR-124ratio could be a non-invasive urine biomarker for the diagnosis ofbladder cancer. These findings provided a new perspective for the moleculargenetics of bladder cancer pathogenesis and also provided new targets andmethods for the clinical diagnosis and treatment of bladder cancer.