The Inhibitory Role Of MiRNA-100in Bladder Cancer Carcinogenesis

【Background】Bladder cancer(BC) is the most common tumor of the urinary system in China, which is a greatthreat to the survival of the patients with the disease. It stands at ninth for the incidence of allmalignant tumors throughout the world, while at sixth among men and after tenth among women. Itis estimated that bladder cancer will account for69,250new cases of cancer and14,990cancer-related deaths in the United States during2011. Among men, it is the fourth most commoncancer and is the eighth leading cause of death from cancer. The ratio of men to women thatdevelop bladder cancer is approximately3:1. The disease presents in two different forms: non–muscle-invasive tumors (stage Ta and T1) and muscle-invasive cancers (stage T2-4). Non–muscle-invasive tumors form a heterogeneous group, spanning from completely benign,noninvasive papillary tumors that rarely progress, to papillary lamina propria–invasive high-gradetumors, which progress to muscle-invasive cancer in up to60%at long-term follow-up. Tumorgrade is also an important prognostic factor for BC patients. Although low grade tumor ismorphologically well differentiated, high grade cancers are poorly differentiated and moreaggressive. To date, the mechanisms of BC carcinogenesis have not been fully investigated.Therefore, better understanding of the pathogenesis is essential for the development of noveleffective therapies for BC.MicroRNAs (miRNAs) are a class of small non-coding RNA molecules of20–22nucleotidesthat regulate gene expression through translational repression and mRNA degradation. So far,1527human miRNAs are registered at miRBase release18.0(http://www.mirbase.org/).They Althoughtheir biological functions remain largely unknown, recent studies show that miRNAs play a criticalrole as important regulators of all hallmarks of cancer, and that about half of the human miRNAsare located in cancer-associated genomic regions and can function as tumor suppressor genes oroncogenes depending on their targets. To date, several human miRNAs have been shown to bedysregulated in BC, such as miR-125b, miR-133a, miR-143, miR-218, and miR-221, whichcontribute to the development and progression of BC.A preliminary study showed that downregulation of miR-100was common in low grade BCtissues compared with normal tissues by realtime rtPCR. However, its biological functions were notdeeply investigated. Moreover, the role of miR-100in cancer cells seemed controversial as itserved as an oncogene in acute myelogenous leukemia but as a tumor suppressor in cervical cancer.Until now, no functional evidence of miR-100in BC has been documented. 【Object】To study the effects of miR-100on the development and malignant progression ofbladder cancer and to investigate the potential mechanisms it participates. Provide basictheory for miR-100as a diagnostic/prognostic biologic marker and therapeutic factor forBC.【Methods】1. Human tissues and cell lines. Tissue specimens from77BC patients,9BC cell lines were usedand a primary BC cell line921T was established.2. microRNA array analysis. Ten paired bladder cancer tissues (5non-muslce-invasive and lowgrade;5invasive and high grade) were adopted and miRNA profiling was performed using20GeneChipTMmiRNA Array.3. RNA extraction and quantitative realtime RT-PCR. To further confirm the microarray results,several targets with higher fold-change in the data (miR-100,-145,-182, and-210) wereselected and examined in an expanded BC cohort.4. Plasmid construction, Lentivirus Production and Transduction. The miR-100/control lentiviruswas purchased.Oligonucleotide transfection pre-miR-100precursor and its negative control,cholesterol-conjugated miR-100mimics for in vivo RNA delivery and its negative controls,and miR-100inhibitor and mTOR siRNA were synthesized.5. Cell growth, wound-healing, and matrigel invasion assays. A cell proliferation assay wasperformed with the Cell Counting Kit-8. Cell migration activity was evaluated bywound-healing assay. A cell invasion assay was carried out using modified Boyden Chambers.6. Cell cycle and Annexin Ⅴ assays using FCM7. Tumor formation in nude mice. Human BC-bearing female nude mice with subcutaneouspassage of EJ and intravesical passage of5637were used for evaluating the antitumor effect ofmiR-100in vivo.8. Tumor metastases in nude mice. New metastatic tumor focus as well as the orthotopic BCtissue of the mice would be harvested and used for evaluating the antimetastatic effects ofmiR-100and the metastatic effects of its target gene(s) in vivo.9. Bioinformatics. To investigate the predicted target genes, we used the TargetScan program.Fisher's exact test was used to analyze the target genes according to the Gene Ontology (GO)which is the key functional classification of NCBI. Similarly, Pathway analysis was used tofind out the significant pathway of the differential genes according to KEGG. Later, we use the KEGG database to build the network of genes. The mTOR pathway(mTOR-miR-Network) wasbuilt finally.10. Luciferase assay, Western blot and Immunohistochemical assay.11. Statistical analysis.【Results】1. Expression of miR-100is frequently decreased in human BC tissues. Of the miRNAs analyzed,20exhibited significantly differential expression in non-muscle-invasive and low grade BCtissues while22in muscle-invasive high grade samples, respectively.10miRNAs were foundas an intersection set of the two set of significantly dysregulated miRNAs. Several targets withhigher fold-change in the data (miR-100,-145,-182, and-210) were examined in an expandedBC cohort consisting of67pairs of tumor tissues and corresponding adjacent normal tissues.MiR-100and-145expression was stably down-regulated while miR-182up-regulated.2. MiR-100suppresses BC cell proliferation in vitro and in vivo. Overexpression of miR-100cansignificantly decrease BC cell proliferation whereas RNAi-mediated silencing of miR-100increased cell growth ratio. To further confirm the above findings, three in vivo models wereused, which indicated that miR-100exerts a growth-inhibiting function in human BC.3. mTOR is a direct target of miR-100. All of the ten miRNAs within the intersection set wererendered for systemic bioinformatic analysis. Among these, the potential target gene mTOR(FRAP1) was rendered for further study. The relative luciferase activity of the reportercontaining wild-type mTOR3'-UTR was significantly suppressed when miR-100wascotransfected. Later, we examined9BC cell lines by qRT-PCR. Obviously, as Pearson'scorrelation test indicated, there were inverse correlations between miR-100and the mTORmRNA level. Western blot and qRT-PCR were performed to examine the effect ofoverexpression or knockdown of miR-100on the mRNA and protein levels of mTOR in threebladder cancer cell lines, showing miR-100may act as a tumor suppressor throughdown-regulating the expression of mTOR in bladder cancer cells.4. Cell cycle and apoptosis. EJ cells in miR100-treated group and the si-mTOR treated group wasarrested in the G0/G1phase. No evidence of apoptosis in any of the3groups tested by AnnexinⅤ assays. Moreover, overexpression of miR-100by pre-treated with Lenti-miR-100couldmake5637cells more sensitive to the exposure of doxorubicin setting and enhance thecell-apoptotic effect of doxorubicin.5. Overexpression of miR-100leads to reduce in tumor cell migration and invasion. 6. Cancerous metastatic model further suggested the function of miR-100and mTOR. miR-100was more than5-fold down-regulated in all the three metastatic BC cells (P<0.01) while theexpression of mTOR was up-regulated correspondingly,, compared with that in921T-2cells.【Conclusions】1. MiR-100was frequently down-regulated in BC tissues, suggesting its potential tumorsuppressor role in BC regardless of any tumor stage or tumor grade.2. Modified the expression level of miR-100in BC cells could correspondingly affected theirmalignant phenotype, including suppressing BC cells into S phase to decrease cell proliferation,reducing EJ/5637cell migration and invasion, reducing the tumorigenesis capacity in vivo.SiRNA-mediated mTOR gene silencing reduced the malignant phenotype of BC cells,suggesting that miR-100may become a potentially therapeutic factor for BC treatment.3. As a hot research point, the mTOR signaling pathway has also been increasingly foundimplicated in BC tumorigenesis. mTOR was a direct target of miR-100in BC cells. Growthinhibitory effect of miR-100was mediated by repressing mTOR expression, and was attributedto the regulation of cell cycle progression, particularly at the G1-to-S transition.4. In addition, data from the cancerous metastatic model further suggested that miR-100andmTOR regulated cell motility and were associated with tumor metastasis. Our findings onmiR-100are encouraging and suggest that this miRNA could be a prognostic factor ofmetastases and potential target for the treatment of BC in future.Taken together, this study identified miR-100as a growth suppressive miRNA in human BC,at least, partly through repression of mTOR. Our data provide further evidence of a pivotal roleof miRNAs in BC tumorigenesis. As miR-100is downregulated in BC, reintroduction of thismature miRNA into the tumor tissue could provide a therapeutic strategy by reducing theexpression of target genes. Although miRNA-based therapeutics is still in their infancy, ourfindings on miR-100are encouraging and suggest that this miRNA could be a potential targetfor the treatment of BC in future.