MiR-200c And MiR-141 In Human Bladder Cancer

Research background:MicroRNAs (miRNAs) are a class of small non-coding RNAs and consist of 20-25 ribonucleotides. The mature miRNAs with full or partial complementarity to the target mRNAs direct the cleavage of target mRNAs or act as repressors of translation, involved in modulating the development, differentiation, proliferation and apoptosis of cells. Urinary bladder cancer is among the most common tumors in urinary system. According to the statistics of National Cancer Institute, 68810 new cases were diagnosed and 14100 patients died in 2008, costing $2.9 billion annually in treating this disease. Unfortunately, patients with advanced disease face a 5 year survival rate of approximately 20–40% despite wide range of treatment modalities including surgery, chemotherapy and radiotherapy. Recent advances in molecular pathology have revealed that bladder cancer is a disease of multiple gene aberrations, including amplification and overexpression of oncogenes and mutation or deletion of tumor suppressor genes, which result in the deregulation of signaling transduction pathways, leading the tumor cells to escape from the normal growth control mechanism and present the malignant phenotype of invasiveness and increased angiogenesis. Some microRNAs were demonstrated to be dysregulated in diverse cancer subtypes including hepatocellular carcinoma, lung cancer, breast cancer, colorectal cancer and other cancers. However, the expression and function of microRNAs in bladder cancer are still scarce.Research objection:To find the dysregulated microRNAs in bladder cancer by microRNA array and verify some of these differentially expressed microRNAs in a enlarged sample population by realtime RT-PCR. Furthermore, to explore the functional aspects of some microRNAs in bladder cancer cell lines in vitro.Research Methods:1. microRNA array screening . We collected 3 pairs of bladder cancer tissues and its para-tumor control. Small RNA was extracted using mirVANA microRNA extraction kit. RNA was labeled by Cy3 and hybridized with Agilent microRNA array. After incubation and washing, the array was scanned by Agilent scanner, raw data was obtained using Feature Extraction software and further analyzed. The results were displayed as the ratio of the microRNA signal in tumor to that in para-tumor tissues. Then cluster analysis was done by cluster 3.0 software using selected data.2. Verifying of the expression of miR-200c and miR-141 in bladder cancer: We verified part of the dysregulated microRNAs in bladder cancer by realtime RT-PCR, which included 20 tumor tissues , matching normal bladder mucosa, bladder cancer T24 cell, J82 cell, EJ cell and 5637 cell. Total RNA was extracted using TRIzol according to protocol, then reversely transcribed using gene specific stem-loop RT primer. The cDNA was then quantified by a taqman probe based relative quantitative study using RNU6B as the internal control. The relative gene expression was calculated and compaired using student t test.3. Expression of Sox2 ,miR-200c in bladder cancer: Sox2 was predicted to be one of the targets of miR-200 family using bioinformatics. Immunohistochemisty of Sox2 was examined in bladder cancer tissue in which the expression of miR-200c has been verified. Westernblot of Sox2 was examined in T24 cell line with relative lower expression of miR-200c.Research Results:1. MicroRNA array screening found that certain microRNAs were more than 3 folds upregulated in bladder cancer, such as miR-141, miR-200c, miR-96, miR-182, miR-183,et al. Certain microRNAs, such as miR-125b, miR143, miR-145, miR-99a and miR-10 were more than 3 folds downregulated in bladder cancer。2. miR-200c and miR-141 quantification showed higher expression of miR-200c and miR-141 in bladder cancer samples than normal bladder mucosa beside the bladder cancer tissue. The expression of miR-200c and miR-141 is higher in superficial baldder cancer than invasive bladder cancer; it is lower in bladder cancer sample with higher grade (G2+G3) than lower grade (G1). Likewise, 5637 cell, which is relative well-differentiated and less malignant, show higher expression of miR-141 and miR-200c than T24, J82 and EJ cell, which are relative poor-differentiated and more malignant.3. Bladder cancer tissue with relative lower expression of miR-200c showed strong expression of Sox2. Few positivity can be seen in bladder cancer tissue with relative higher expression of miR-200c. Sox2 can also be detected in T24 cell with lower expression of miR-200c.Research Conclusions:1. Multiple microRNAs are dysregulated in urinary bladder cancer, including miR-200c, miR-141, miR-143, miR-145, et al.2. The expression of miR-200c and miR-141 suggests that they may participate in the progression of bladder cancer.3. The expression of Sox2 in bladder cancer tissue and T24 cellsuggests it,s participation in the genesis and progression of bladder cancer. Sox2 may be the one of the target of miR-200c and and one therapeutic target in the future.