Effects Of MicroRNA-34a On Invasive Urothelial Carcinoma Of Bladder By Targeting Notch1

MicroRNAs (miRNAs or miRs) are a class of21~25nucleotide,non-coding and highly conserved RNAs that post-transcriptionallyregulate gene expression through messenger RNA (mRNA) degradationand translational repression. It has been reported that miRNAs participatein various oncological processes such as carcinogenesis, carcinomaprogression and metastasis. Most recently, miR-34a has been found asone of the key miRNAs that function as tumor suppressor in diversecarcinomas. An estimated386,300new cases and150,200deaths frombladder cancer occurred in2008. Bladder cancer is the fifth mostcommen carcinoma in the world, the second most commen urino-genitaltract carcinoma and the second lethal cause in urino-genital tractcarcinomas. A previous study from our laboratory concerning Notchsignaling pathway, which has been the subject of intensive research, found that silencing Notch1expression can inhibit cell proliferation andinduce cell cycle arrest at G1of bladder cancer cells and Notch1genemight act as a tumor gene in invasive bladder cancer. It has been wellestablished diverse Notch mediators, but certainly other mediators ofNotch have yet to be discovered. Therefore, the potential Notchmediators such as miRNAs in invasive urothelial carcinoma of bladder(UCB) triggered our initial interest. Our invastigation contains three partsas follows:Part one: Expression patterns of miR-34a and Notch1in invasiveUCB tissues.A previous study from our laboratory concerning Notch signalingpathway found that silencing Notch1expression can inhibit cellproliferation and induce cell cycle arrest at G0-G1of bladder cancer cells.A study has indicated that miR-34a inhibits tumor growth by targetingNotch1in glioma cells. In order to investigate the relation between miR-34a and Notch1in invasive UCB, we examined the miR-34a andNotch1expression levels in invasive UCB tissues and in patient-matchedadjacent bladder tissues by quantitative real-time RT-PCR (qRT-PCR)and western blot. We found that miR-34a expression is significantlydownregulated in invasive UCB tissues than in patient-matched adjacentbladder tissues, which is negatively correlated with Notch1expression.Interestingly, parallel results from other studies also revealed the similarnegative correlations between miR-34a and Notch1in tumors. Thus weinfer that miR-34a might function as a tumor suppressor in invasive UCBby targeting Notch1.Part two: Prediction and validation of binding site betweenmiR-34a and Notch1.We inferred that miR-34a might function as a tumor suppressor ininvasive UCB by targeting Notch1through negatively correlatedexpression pattern in invasive UCB tissues. To further precise the binding site between miR-34a and Notch1, we used4publicly availablealgorithms to predict and luciferase assay to valid the prediction. Humanurinary transitional carcinoma cell lines, T24and5637were used as invitro cell models. Luciferase assays showed that miR-34a transfectionsignificantly down-regulated the normalized Notch13’UTR luciferaseactivities in T24and5637, which validated the prediction that miR-34abinds to the3’-untranslated region (3’UTR) of Notch1. When themiR-34a expression plasmid was transfected into T24and5637, mRNAlevels of Notch1and protein levels of Notch1were down-regulated. Thuswe further conclude that miR-34a exerts their regulatory effects bybinding to the3’-untranslated region (3’UTR) of Notch1.Part three: Effects of miR-34a on invasive UCB by targetingNotch1In order to illucidate the effects of miR-34a on invasive UCB, we usedT24and5637as in vitro cell models. We found that both forced-expressing miR-34a and silencing Notch1in bladder tumor cellsstrongly inhibited cell proliferation, cell cycle progression, cell migrationand cell invasion through cell proliferation assay, cell cycle assay,transwell migration assay and transwell invasion assay. Thus weconclude that miR-34a functions as a potential tumor suppressor ininvasive UCB by targeting Notch1.Taken together, our study has explored the crucial tumor suppressiverole of miR-34a in invasive urothelial bladder carcinoma. Here weindicate that microRNA-34a expression is down-regulated in bladdertumor tissues and it inhibits cell proliferation, cell cycle progression, cellmigration and cell invasion of bladder cancer cells by antagonizingNotch1, which may indicate its tumor-suppressive function in invasiveUCB. These new findings of our research work may provide novelinsights in the dianosis, clinical treatment and prognosis of invasiveurothelial carcinoma of bladder.