| This post-graduate research project is composed of two parts of study.The first part (Chapter 2) is titled as’Muscle-invasive bladder cancer-associated aberrant expression of microRNAs and the biomedical significances’. The objectives are, to select and validate microRNAs (miRNAs) that were differentialy expressed in the tumor tissues between muscle-invasive bladder cancer (MIBC) and non-muscle-invasive bladder cancer (NMIBC); to examine the free miRNAs in urine supernatant as potential biomarkers for discriminating the two types of bladder cancer; and to explore their functions.Based on data derived from miRNA microarray analysis on 45 cases of bladder cancer, 10 miRNAs (hsa-miR-199a-3p, has-miR-132, hsa-miR-141*, hsa-miR-200c*, hsa-miR-30e, hsa-miR-30b, hsa-miR-98, hsa-miR-331-3p, hsa-miR-151-3p, hsa-miR-151-5p) were revealed differentialy expressed between the MIBC and NMIBC. Among the 10 miRNAs, seven were verified by Real-time PCR with the 45 bladder cancers analyzed by miRNA microarray previously, and then conducted to validate in an independent sample set with 54 bladder cancers. The independent sample set validation showed that 3 miRNAs, hsa-miR-199a-3p, hsa-miR-30e and hsa-miR-30b were also differentialy expressed between the MIBC and NMIBC. These 3 miRNAs were subsequently measured by Real-time PCR in urine supernatant samples of 96 bladder cancer patients, and hsa-miR-30e and hsa-miR-30b presented distinct quantitative levels between the MIBC and NMIBC patients preoperatively. While ROC analysis implicates urinary cell-free hsa-miR-30b and hsa-miR-30e as potential diagnosis biomarkers to differentiate the two types of bladder cancer. On the other hand, target prediction indicated that hsa-miR-199a-3p may have multi-regulating roles on crucial molecular pathways of bladder cancer. Therefore, Luciferase and CCK8 assay were utilized to investigate the effects of hsa-miR-199a-3p on the expression of its putative targets and their correlation with proliferation. In vitro, hsa-miR-199a-3p was implicated repressing the level of SGK2-3’UTR, and promoting proliferation in bladder cancer cell lines T24 and 5637.The second part (Chapter 3) is titled as ’Bladder cancer-associated DNA copy number changes in 5 gene fragments and their significance’. The aim is to explore whether or not the DNA copy number changes in 5 gene fragments are specific abnormalities for bladder cancer in the Chinese. Real-time PCR was conducted to gauge DNA copy number change in the 5 gene fragments among commercial genome DNA of peripheral blood leucocyte (PBL) derived from Caucasian, genome DNA of PBL derived from Chinese health volunteers, the PBL and the corresponding tumor tissue of bladder cancer patients.As a result, comparing with the Caucasia PBL, all the gene fragments revealed copy number changings with decreased CEP63, increased FOSL2, GHR and PAQR6, in the PBL of Chinese health volunteers, except ZFAND3. Comparing with the PBL of Chinese healthy volunteers, in the tumor tissue samples of the bladder cancer patients, the copy number of CEP63 (P<0.001), GHR (P=0.019) and PAQR6 (P=0.001) increased; whereas FOSL2 (P=0.41) and ZFAND32 (P=0.062) revealed no statistical difference. Comparing with the PBL of Chinese healthy volunteers, the copy number of CEP63 (P<0.001) and PAQR6 (P<0.001) increased but FOSL2 (P<0.001) and ZFAND32 (P<0.001) decreased in the PBL of bladder cancer patients. Comparing with the PBL of the bladder cancer patients, gain of copy number was observed with CEP63 (P<0.001), FOSL2 (P<0.001), GHR (P<0.001) and ZFAND32 (P<0.001), while PAQR6 (P=0.325) exhibited no statistical difference, in the corresponding tumor tissues.As implicated, the copy number variation of CEP63, FOSL2, GHR and PAQR6 was found between Chinese (Han) and Caucasian. Copy number gain of CEP63 and GHR may involve in carcinogenesis of bladder cancer; while gain of PAQR6 was possibly associated with the genetic sensibility for this malignancy. |
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