Yiwei Lin

Cyclin-dependent kinase 4 is a novel target in micoRNA-195-mediated cell cycle arrest in bladder cancer cells

miRNAs are a class of small‐noncoding RNAs capable of negatively regulating gene expression. Here, we found that miR‐195 is down‐regulated in human bladder cancer tissue versus normal adjacent tissue. To better characterize the role of miR‐195 in bladder cancer, we conducted gain of function analysis by transfecting bladder cancer cell line T24 with chemically synthesized miR‐195 mimic. We identified CDK4, an early G1 cell cycle regulator, as a novel target of miR‐195. Selective over‐expression of miR‐195 could induce G1‐phase arrest in T24 cells, and subsequently inhibit T24 cell growth. These findings indicate that miR‐195 could be a potential tumor suppressor in bladder cancer.

MicroRNA-124-3p inhibits cell migration and invasion in bladder cancer cells by targeting ROCK1

BackgroundIncreasing evidence has suggested that dysregulation of certain microRNAs (miRNAs) may contribute to human disease including carcinogenesis and tumor metastasis in human. miR-124-3p is down-regulated in various cancers, and modulates proliferation and aggressiveness of cancer cells. However, the roles of miR-124-3p in human bladder cancer are elusive. Thus, this study was conducted to investigate the biological functions and its molecular mechanisms of miR-124-3p in human bladder cancer cell lines, discussing whether it has a potential to be a therapeutic biomarker of bladder cancer.MethodsThree human bladder cancer cell lines and samples from ten patients with bladder cancer were analyzed for the expression of miR-124-3p by quantitative RT--PCR. Exogenetic overexpression of miR-124-3p was established by transfecting mimics into T24, UM-UC-3 and J82 cells, after that cell proliferation and cell cycle were assessed by MTT assay, flow cytometry and Colony-forming assay. Cell motility and invasion ability were evaluated by wound healing assay and transwell assay. Tissue microarray, and immunohistochemistry with antibodies against ROCK1, MMP2 and MMP9 was performed using the peroxidase and DAB methods. The target gene of miR-124-3p was determined by luciferase assays, quantitative RT--PCR and western blot. The regulation of epithelial-to-mesenchymal transition by miR-124-3p was analyzed by western blot.ResultsmiR-124-3p is frequently down-regulated in bladder cancer both in three bladder cancer cell lines, T24, UM-UC-3, J82 and clinical samples. Overexpression of miR-124-3p induced G1-phase arrest in T24, UM-UC-3 and J82 cell lines and suppressed cell growth in colony-forming assay. miR-124-3p significantly repressed the capability of migration and invasion of bladder cancer cells. In addition, ROCK1 was identified as a new target of miR-124-3p. ROCK1, MMP2, MMP9 were up-regulated in bladder cancer tissues. Furthermore, we demonstrated miR-124-3p could inhibit bladder cancer cell epithelial mesenchymal transfer, and regulated the expression of c-Met, MMP2, MMP9.ConclusionsmiR-124-3p can repress the migration and invasion of bladder cancer cells via regulating ROCK1. Our data indicate that miR-124-3p could be a tumor suppressor and may have a potential to be a diagnostics or predictive biomarker in bladder cancer.

MicroRNA-449a acts as a tumor suppressor in human bladder cancer through the regulation of pocket proteins

Frequent downregulation of microRNA-449a (miR-449a) was detected in 14 human bladder cancer tissues. The restoration of miR-449a inhibited cell growth and induced G1-phase arrest in T24 and 5637 human bladder cancer cells. CDK6 and CDC25a were downregulated after miR-449a treatment, resulting in the functional accumulation of the pocket proteins Rb and p130. The growth of T24 tumor xenografts was suppressed by exogenous miR-449a, and the nuclear proliferation antigen Ki-67 was downregulated in miR-449a-treated tumors. These results suggest a tumor-suppressive role for miR-449a in human bladder cancer.

MicroRNA-409-3p Inhibits Migration and Invasion of Bladder Cancer Cells via Targeting c-Met

There is increasing evidence suggesting that dysregulation of certain microRNAs (miRNAs) may contribute to tumor progression and metastasis. Previous studies have shown that miR-409-3p is dysregulated in some malignancies, but its role in bladder cancer is still unknown. Here, we find that miR-409-3p is down-regulated in human bladder cancer tissues and cell lines. Enforced expression of miR-409-3p in bladder cancer cells significantly reduced their migration and invasion without affecting cell viability. Bioinformatics analysis identified the pro-metastatic gene c-Met as a potential miR-409-3p target. Further studies indicated that miR-409-3p suppressed the expression of c-Met by binding to its 3′-untranslated region. Silencing of c-Met by small interfering RNAs phenocopied the effects of miR-409-3p overexpression, whereas restoration of c-Met in bladder cancer cells bladder cancer cells overexpressing miR-409-3p, partially reversed the suppressive effects of miR-409-3p. We further showed that MMP2 and MMP9 may be downstream effector proteins of miR-409-3p. These findings indicate that miR-409-3p could be a potential tumor suppressor in bladder cancer.

MicroRNA-490-5p inhibits proliferation of bladder cancer by targeting c-Fos.

MicroRNAs (miRNAs) are non-protein-coding sequences that play a crucial role in tumorigenesis by negatively regulating gene expression. Here, we found that miR-490-5p is down-regulated in human bladder cancer tissue and cell lines compared to normal adjacent tissue and a non-malignant cell line. To better characterize the function of miR-490-5p in bladder cancer, we over-expressed miR-490-5p in bladder cancer cell lines with chemically synthesized mimics. Enforced expression of miR-490-5p in bladder cancer cells significantly inhibited the cell proliferation via G1-phase arrest. Further studies found the decreased c-Fos expression at both mRNA and protein levels and Luciferase reporter assays demonstrated that c-Fos is a direct target of miR-490-5p in bladder cancer. These findings indicate miR-490-5p to be a novel tumor suppressor of bladder cancer cell proliferation through targeting c-Fos.

miR-26a inhibits proliferation and motility in bladder cancer by targeting HMGA1.

It is increasingly clear that microRNAs play a crucial role in tumorigenesis. Recently, emerging evidence suggested that miR‐26a is aberrantly expressed in tumor tissues. In our study, frequent down‐regulation of miR‐26a was observed in 10 human bladder cancer tissues. Forced expression of miR‐26a in the bladder cancer cell line T24 inhibited cell proliferation and impaired cell motility. High mobility group AT‐hook 1 (HMGA1), a gene that modulates cell cycle transition and cell motility, was verified as a novel target of miR‐26a in bladder cancer. These findings indicate an important role for miR‐26a in the molecular etiology of bladder cancer and implicate the potential application of miR‐26a in bladder cancer therapy.

Downregulation of microRNA-182-5p contributes to renal cell carcinoma proliferation via activating the AKT/FOXO3a signaling pathway

BackgroundEmerging evidence has suggested that dysregulation of miR-182-5p may contribute to tumor development and progression in several types of human cancers. However, its role in renal cell carcinoma (RCC) is still unknown.MethodsQuantitative RT-PCR was used to quantify miR-182-5p expression in RCC clinical tissues. Bisulfite sequencing PCR was used for DNA methylation analysis. The CCK-8, colony formation, flow cytometry, and a xenograft model were performed. Immunohistochemistry was conducted using the peroxidase and DAB methods. A miR-182-5p target was determined by luciferase reporter assays, quantitative RT-PCR, and Western blotting.ResultsmiR-182-5p is frequently down-regulated in human RCC tissues. Epigenetic modulation may be involved in the regulation of miR-182-5p expression. Enforced expression of miR-182-5p in RCC cells significantly inhibited the proliferation and tumorigenicity in vitro and in vivo. Additionally, overexpression of miR-182-5p induced G1-phase arrest via inhibition of AKT/FOXO3a signaling. Moreover, FLOT1 was confirmed as a target of miR-182-5p. Silencing FLOT1 by small interfering RNAs phenocopied the effects of miR-182-5p overexpression, whereas restoration of FLOT1 in miR-182-5p -overexpressed RCC cells partly reversed the suppressive effects of miR-182-5p.ConclusionsThese findings highlight an important role for miR-182-5p in the pathogenesis of RCC, and restoration of miR-182-5p could be considered as a potential therapeutic strategy for RCC therapy.

microRNA-330 inhibits cell motility by downregulating Sp1 in prostate cancer cells.

microRNAs (miRNAs), small non-coding RNAs, have emerged as key regulators of a large number of genes. The present study aimed to explore novel biological functions of miR-330 in the human prostate cancer cell lines DU145 and PC3. We confirmed that miR-330 was downregulated and inversely correlated with specificity protein 1 (Sp1) expression. Overexpression of miR-330 by transfection of a chemically synthesized miR-330 mimic induced a reduction in expression levels of the Sp1 protein, accompanied by significant suppression of cellular migration and invasion capability. In addition, the Sp1-knockdown experiments presented similar phenomena. Finally, the luciferase reporter assay validated Sp1 as the direct target of miR-330. These findings indicate that miR-330 acts as an anti-metastatic miRNA in prostate cancer.

Resveratrol confers resistance against taxol via induction of cell cycle arrest in human cancer cell lines.

Resveratrol, which is highly concentrated in the skin of grapes and is abundant in red wine, has been demonstrated to account for several beneficial properties, including antioxidant, anticoagulant, anti-inflammatory and anticancer effects. Taxol is a microtubule-stabilizing drug that has been extensively used as effective chemotherapeutic agents in the treatment of solid tumors. Here, we investigated whether the combination of the two compounds would yield increased antitumor efficacy in human cancer cells. Unexpectedly, resveratrol effectively prevented tumor cell death induced by taxol in 5637 bladder cancer cells. This pronounced antagonistic function of resveratrol against taxol was associated with changes in multiple signal transduction pathways, but not with tubulin polymerization. Importantly, cell cycle analysis showed that resveratrol prevented the cells from entering into mitosis, the phase in which taxol exerts its action. Furthermore, resveratrol blocked the cytotoxic effects of vinblastine but not cisplatin in 5637 cells. Interestingly, resveratrol pre-treatment followed by taxol resulted in synergistic cytotoxicity. Finally, we extended our studies to various human cancer cell lines. Taken together, our results indicate that resveratrol may have the potential to negate the therapeutic efficacy of taxol and suggest that consumption of resveratrol-related products may be contraindicated during cancer therapy with taxol.

Silencing of mutant p53 by siRNA induces cell cycle arrest and apoptosis in human bladder cancer cells

Backgroundp53 is the most frequently mutated tumor-suppressor gene in human cancers. It has been reported that mutations in p53 result not only in the loss of its ability as a tumor suppressor, but also in the gain of novel cancer-related functions that contribute to oncogenesis. The present study evaluated the potential of silencing of mutant p53 by small interfering RNA in the treatment of bladder cancer cells in vitro.MethodsWe used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to assess cell viability and flow cytometry to detect cell cycle alterations and apoptosis. The related molecular mechanisms were assessed by western blotting. We also used the MTT assay and flow cytometry to investigate if silencing of mutant p53 by knockdown with small interfering (si)RNA would change the sensitivity to cisplatin treatment.ResultsUsing 5637 and T24 human bladder cancer cell lines characterized by mutations in p53, we found that silencing of the mutant p53 by RNA interference induced evident inhibition of cell proliferation and viability, which was related to the induction of G2 phase cell cycle arrest and apoptosis. Moreover, our study also showed that the p53-targeting siRNA cooperated with cisplatin in the inhibition of bladder cancer cells.ConclusionsThese findings suggest that RNA interference targeting mutant p53 may be a promising therapeutic strategy for the treatment of bladder cancer.