microRNA-21 modulates cell proliferation and sensitivity to doxorubicin in bladder cancer cells

Transitional cell carcinomas (TCCs) of the urinary bladder are common malignancies with a high recurrence rate. Since microRNA-21 (miR-21) may contribute to tumorigenesis and chemoresistance in many cancer types, we aimed to investigate its efficacy in TCCs. The expression of miR-21 and its target PTEN was determined by real-time qRT-PCR and western blotting, respectively in tumor tissues as well as adjacent non-tumor mucosa. The effect of miR-21 on cell proliferation and chemosensitivity to doxorubicin were measured using the MTT method. Cell apoptosis induced by doxorubicin was investigated using flow cytometry in the T24 cell line. BCL-2, AKT and pAKT were detected by western blotting for analysis of potential mechanisms. miR-21 was significantly up-regulated in tumor tissues while PTEN was expressed in lower levels compared to non-tumor tissues. A negative correlation between expression of miR-21 and PTEN was established in vivo. Cell proliferation and chemoresistance to doxorubicin were promoted by overexpression of miR-21 in T24 cells. BCL-2 up-regulation could be achieved by miR-21 overexpression, which prevented T24 cells from apoptosis induced by doxorubicin. Furthermore, the miR-21 induced BCL-2 up-regulation could be cancelled by the PI3K inhibitor LY294002. These data verified the oncogenic role of miR-21 in TCCs and may usher in new therapeutic strategies in treating this disease.     

miR-203在结直肠癌中的表达及其功能分析

目的 探讨microRNA-203（miR 203）在结直肠癌中的表达情况及其临床意义,并在结直肠癌细胞中验证其功能。方法 采用实时荧光定量PCR（qRT-PCR）检测52例结直肠癌及对应癌旁组织中miR-203的水平,分析miR-203表达与临床病理参数及术前癌胚抗原的关系;检测miR 203在3种结直肠癌细胞株SW480、Lovo和HT 29及人正常结肠黏膜上皮细胞NCM460中的表达情况,选取miR-203水平最低的细胞分别转染miR-203模拟物（mimics）和miR-203 control,采用MTT法、流式细胞仪PI／Annexin V双染法和Transwell法检测转染miR 203对细胞增殖、凋亡和侵袭能力的影响。结果 结直肠癌组织中miR-203的相对表达量为0.372±0.019,低于癌旁组织的miR-203水平,且其表达与TNM分期、淋巴结转移、肿瘤大小及术前癌胚抗原水平均有关（P＜0.05）;与NCM460细胞相比（其miR-203表达水平设为1.00）,结直肠癌细胞SW480、Lovo和HT-29的miR 203相对表达量依次为0.26±0.07、0.44±0.05和0.32±0.04,选取SW480细胞进行后续实验。MTT检测显示,转染miR-203 mimics 48、72和96 h的SW480细胞吸光值均低于转染miR-203 control组,差异均有统计学意义（P＜0.05）;且转染miR-203 mimics 48和96 h的SW480细胞凋亡率高于转染miR-203 control组,但穿膜细胞数低于转染miR-203 control组（P＜0.05）。结论 miR-203在结直肠癌组织和细胞中低表达,上调miR 203水平可抑制结直肠癌细胞的增殖、侵袭并诱导凋亡。     

The expression and function of microRNA-203 in lung cancer

We aimed to determine the expression of microRNA-203 (miR-203) in human lung cancer cell lines and to evaluate the effects of miR-203 by targeting survivin, on the lung cancer cell line 95-D to provide potential new strategies for treating lung cancer. The expression of miR-203 was detected using quantitative real-time PCR (qRT-PCR) in the in vitro cultured lung cancer cells A549, HCC827, NCI-H1299, and 95-D as well as in normal human bronchial epithelial cells. Following a 72-h transfection with the miR-203 precursor in 95-D lung cancer cells, the change in miR-203 expression was detected using qRT-PCR and the resulting effect on survivin protein expression was ascertained by Western blot analysis. The influence of miR-203 on the viability of 95-D lung cancer cells was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The effect of miR-203 on 95-D cell proliferation was analyzed using flow cytometry. The consequences of miR-203 expression on 95-D cell apoptosis were analyzed by Annexin V/propidium iodide double staining coupled with flow cytometry. The role of miR-203 in the invasive potential of 95-D cells was studied using a transwell chamber assay. A luciferase reporter gene system was used to verify that survivin is a target gene for miR-203. By qRT-PCR, the expression of miR-203 was lower in lung cancer cells than in normal bronchial epithelial cells (p?<?0.01), and the expression of miR-203 in 95-D lung cancer cells was significantly higher after a 72-h transfection with the miR-203 precursor (p?<?0.01). After a 72-h transfection with the miR-203 precursor, survivin protein levels in 95-D cells were significantly decreased (p?<?0.01). Cell viability, as assessed with an MTT assay, decreased following an increase in miR-203 expression (p?<?0.05). The flow cytometry results indicated that after miR-203 expression increased, the cell proliferation index decreased (p?<?0.05) and the number of apoptotic cells increased (p?<?0.01). Increased miR-203 expression led to a significant decrease in the number of cells that migrated through a transwell chamber membrane (p?<?0.01). The luciferase reporter gene system demonstrated that the relative luciferase activity significantly decreased after transfection with the miR-203 precursor (p?<?0.05). The expression of miR-203 is downregulated in lung cancer cells. miR-203 negatively regulates survivin protein expression and inhibits the proliferation and invasion of lung cancer cells. Therapeutic strategies that enhance miR-203 expression or silence survivin could potentially benefit lung cancer patients.     

Novel molecular targets regulated by tumor suppressors microRNA-1 and microRNA-133a in bladder cancer

Our expression signatures of human cancer including bladder cancer (BC) revealed that the expression of microRNA-1 (miR-1) and microRNA-133a (miR-133a) is significantly reduced in cancer cells. In the human genome, miR-1 and miR-133a are located on the same chromosomal region (miR-1-2 and miR-133a-1 on 18q11.2, and miR-1-1 and miR-133a-2 on 20q13.33) called cluster. In this study, we identified the novel molecular targets commonly regulated by miR-1 and miR-133a in BC. Genome-wide molecular target search and luciferase reporter assays showed that prothymosin-α (PTMA) and purine nucleoside phosphorylase (PNP) are directly regulated by miR-1 and miR-133a. Silencing of these two genes significantly inhibited cell proliferation and invasion, and increased apoptosis in BC cells. Immunohistochemistry showed that PTMA expression levels were significantly higher in BC compared to normal bladder epitheliums. PTMA and PNP were identified as new target genes regulated by the miR-1 and miR-133a cluster in BC. These genes may function as oncogenes contributing to cell proliferation and invasion in BC. Tumor suppressive miR-1 and miR-133a-mediated novel molecular targets may provide new insights into the potential mechanisms of BC oncogenesis.     

microRNA-1301-mediated inhibition of tumorigenesis

The relatively recent discovery of microRNAs has added a completely new dimension to the study of the regulation of tumor cells, but how they control cell behavior remains largely elusive. HepG2 cells were assigned to the miR-1301 group and the control group. RT-PCR, Western blotting, wound healing, the Transwell chamber migration and MTT assays, and apoptosis detection assays were used to analyze cell behavior of HepG2 cells after miR-1301 mimic transfection. Our study showed that miR-1301 was downregulated in HepG2 cells, and that miR-1301 inhibited migration and invasion of HepG2 cells and promoted cellular apoptosis after transfection with miR-1301 mimics. In addition, p53 mRNA and p53 protein expression was upregulated, and Bcl-2 and Bcl-xL mRNA and protein expression was downregulated in the miR-1301 group. These results indicate that miR-1301 may be an inhibitor of tumorigenesis in HepG2 cells.     

Cyclooxygenase-2 modulates the insulin-like growth factor axis in non-small-cell lung cancer

Constitutive overexpression of cyclooxygenase-2 (COX-2) occurs frequently in several different malignancies, including lung, colon, breast, and prostate cancer. Clinical studies have established elevated serum insulin-like growth factor (IGF-I) content and IGF-I:IGF-binding protein 3 (IGFBP-3) ratio as risk factors for these same malignancies. Therefore, we sought to determine the link between COX-2 expression and the IGF axis in COX-2 gene-modified human non-small-cell lung cancer (NSCLC) cells. Overexpression of COX-2 in NSCLC cells enhanced the antiapoptotic and mitogenic effects of IGF-I and IGF-II, facilitated the autophosphorylation of the type 1 IGF receptor, increased class IA phosphatidylinositol 3'-kinase activity, and decreased expression of IGFBP-3. Thus, these findings show that COX-2 augments the stimulatory arm of the IGF axis.     

Circulating microRNA-203 predicts metastases,early recurrence,and poor prognosis in human gastric cancer

Background

Metastasis is a major cause of death in patients with gastric cancer (GC). MicroRNAs (miRNAs) relating to the epithelial–mesenchymal transition (EMT) control GC progression and metastasis. The aim of this study was to evaluate serum EMT-associated miRNAs for metastatic and prognostic noninvasive biomarkers in GC.

Methods

In the first step of this study (preliminary experiments), we selected candidate miRNAs associated with metastasis by analyzing the expression of the miR-200 family (miR-200a, miR-200b, miR-200c, miR-141, and miR-429) and miR-203 in serum samples from stage I (n = 12) and stage IV (n = 12) GC patients. The second phase involved the independent validation of candidate miRNAs in serum specimens from 130 patients with GC and 22 controls.

Results

Based on the preliminary experiments, miR-203 was selected as the candidate serum miRNA that was most closely associated with metastasis. Validation analysis revealed that serum miR-203 levels were significantly lower in stage IV than stage I–III GC patients. Serum miR-203 expression was significantly lower in GC patients with a higher T stage, vessel invasion, and lymph node, peritoneal, and distant metastases. Low expression of serum miR-203 was significantly associated with poor disease-free and overall survival. Multivariate analysis revealed that low serum miR-203 expression was an independent predictive marker for lymph node, peritoneal, and distant metastases and a poor prognosis in patients with GC.

Conclusions

Serum miR-203 has the potential to serve as a noninvasive biomarker for prognosis and to predict metastasis in patients with GC.

     

Role of Neuropilin-2-mediated signaling axis in cancer progression and therapy resistance

Neuropilins (NRPs) are transmembrane proteins involved in vascular and nervous system development by regulating angiogenesis and axon guidance cues. Several published reports have established their role in tumorigenesis. NRPs are detectable in tumor cells of several cancer types and participate in cancer progression. NRP2 is also expressed in endothelial and immune cells in the tumor microenvironment and promotes functions such as lymphangiogenesis and immune suppression important for cancer progression. In this review, we have taken a comprehensive approach to discussing various aspects of NRP2-signaling in cancer, including its regulation, functional significance in cancer progression, and how we could utilize our current knowledge to advance the studies and target NRP2 to develop effective cancer therapies.

     

The importance of integrin-linked kinase in the regulation of bladder cancer invasion

It is important to understand the molecular mechanisms of bladder cancer progression not only to prevent cancer progression but also to detect new therapeutic targets against advanced bladder cancer. The integrin-linked kinase (ILK) is a major signaling integrator in mammalian cells and plays an important role in epithelial-mesenchymal transition (EMT) of human cancers, but its mechanisms are not completely understood. In this study, we investigated the importance and mechanisms of ILK in bladder cancer progression. When the expression of ILK in bladder cancer cell lines and N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced murine bladder cancer was evaluated, ILK has a tendency to be overexpressed in invasive cell lines and invasive BBN-induced murine bladder cancer. Overexpression of ILK in 253J bladder cancer cells suppressed E-cadherin expression, resulting in the promotion of cell invasion. Conversely, ILK knockdown by siRNA suppresses cell invasion in invasive bladder cancer cells through the regulation of E-cadherin or matrix metalloprotease 9 (MMP-9). To regulate E-cadherin expression, our results showed that the glycogen synthase kinase 3β (GSK3β)-Zeb1 pathway may play an important role downstream of ILK. Finally, the results of a human bladder tissue microarray (TMA) showed that ILK expression correlates with the invasiveness of human bladder cancer. Our study suggests that ILK is overexpressed in invasive bladder cancer and plays an important role in the EMT of bladder cancer via the control of E-cadherin and MMP-9 expression. ILK may be a new molecular target to suppress tumor progression in advanced and high-risk bladder cancer patients.     

Dual regulation by microRNA-200b-3p and microRNA-200b-5p in the inhibition of epithelial-to-mesenchymal transition in triple-negative breast cancer

Epithelial to mesenchymal transition (EMT) involves loss of an epithelial phenotype and activation of a mesenchymal one. Enhanced expression of genes associated with a mesenchymal transition includes ZEB1/2, TWIST, and FOXC1. miRNAs are known regulators of gene expression and altered miRNA expression is known to enhance EMT in breast cancer. Here we demonstrate that the tumor suppressive miRNA family, miR-200, is not expressed in triple negative breast cancer (TNBC) cell lines and that miR-200b-3p over-expression represses EMT, which is evident through decreased migration and increased CDH1 expression. Despite the loss of migratory capacity following re-expression of miR-200b-3p, no subsequent loss of the conventional miR-200 family targets and EMT markers ZEB1/2 was observed. Next generation RNA-sequencing analysis showed that enhanced expression of pri-miR-200b lead to ectopic expression of both miR-200b-3p and miR-200b-5p with multiple isomiRs expressed for each of these miRNAs. Furthermore, miR-200b-5p was expressed in the receptor positive, epithelial breast cancer cell lines but not in the TNBC (mesenchymal) cell lines. In addition, a compensatory mechanism for miR-200b-3p/200b-5p targeting, where both miRNAs target the RHOGDI pathway leading to non-canonical repression of EMT, was demonstrated. Collectively, these data are the first to demonstrate dual targeting by miR-200b-3p and miR-200b-5p and a previously undescribed role for microRNA processing and strand expression in EMT and TNBC, the most aggressive breast cancer subtype.     

miR-203a通过靶向抑制CDK6调控膀胱癌细胞增殖及放射敏感性

目的:探讨miR-203a在膀胱癌(BC)细胞系(RT-112、T24、5637、UM-UC-3细胞)中的表达及对细胞增殖及放射敏感性的影响。方法:将miR-203a mimics、miR-203a inhibitor、CDK6 siRNA、CDK6表达质粒及相应阴性对照(NC)转染入BC细胞中。实时荧光定量PCR检测...     

The microRNA-218~Survivin axis regulates migration,invasion, and lymph node metastasis in cervical cancer

Cervical cancer is the third most common cancer in women worldwide. In the present study, global microRNA profiling for 79 cervical cancer patient samples led to the identification of miR-218 down-regulation in cervical cancer tissues compared to normal cervical tissues. Lower miR-218 expression was associated significantly with worse overall survival (OS), disease-free survival (DFS), and pelvic/aortic lymph node recurrence. In vitro, miR-218 over-expression decreased clonogenicity, migration, and invasion. Survivin (BIRC5) was subsequently identified as an important cervical cancer target of miR-218 using in silico prediction, mRNA profiling, and quantitative real-time PCR (qRT-PCR). Concordant with miR-218 over-expression, survivin knockdown by siRNA decreased clonogenicity, migration, and invasion. YM155, a small molecule survivin inhibitor, significantly suppressed tumor growth and lymph node metastasis in vivo. Our findings demonstrate that the miR-218~survivin axis inhibits cervical cancer progression by regulating clonogenicity, migration, and invasion, and suggest that the inhibition of survivin could be a potential therapeutic strategy to improve outcome in this disease.     

Intratumoral CRH modulates immuno-escape of ovarian cancer cells through FasL regulation

Although corticotropin-releasing hormone (CRH) and Fas ligand (FasL) have been documented in ovarian carcinoma, a clear association with tumour progression and immuno-escape has not been established. FasL plays an important role in promoting tumour cells' ability to counterattack immune cells. Here, we examined immunohistochemically the expression of CRH, CRHR1, CRHR2 and FasL in 47 human ovarian cancer cases. The ovarian cancer cell lines OvCa3 and A2780 were further used to test the hypothesis that CRH might contribute to the immune privilege of ovarian tumours, by modulating FasL expression on the cancer cells. We found that CRH, CRHR1, CRHR2 and FasL were expressed in 68.1, 70.2, 63.8 and 63.8% of the cases respectively. Positivity for CRH or FasL expression was associated with higher tumour stage. Finally, CRH increased the expression of FasL in OvCa3 and A2780 cells through CRHR1 thereby potentiated their ability to induce apoptosis of activated peripheral blood lymphocytes. Corticotropin-releasing hormone produced by human ovarian cancer might favour survival and progression of the tumour by promoting its immune privilege. These findings support the hypothesis that CRHR1 antagonists could potentially be used against ovarian cancer.     

姜黄素抑制乳腺癌细胞增殖与下调Twist蛋白相关

目的:评价姜黄素的抗乳腺癌作用并初步探讨可能的作用机制.方法:免疫组化法检测82对乳腺癌和癌旁病理组织标本中Twist蛋白表达及其差异;CCK8法观察姜黄素对乳腺癌细胞增殖的影响;并利用细胞免疫荧光检测姜黄素对乳腺癌细胞Twist蛋白表达的影响.结果:乳腺癌组织中Twist蛋白表达量平均积分为(214.6±67.8)%,癌旁组织中Twist蛋白表达量平均积分为(104.5±71.2)%,统计分析显示乳腺癌组织Twist蛋白表达量比癌旁组织显著增高(P=0.007).50 μmol/L姜黄素处理MCF-7和MDA-MB-231两株乳腺癌细胞后,两株乳腺癌细胞的增殖受到显著抑制(P<0.05).与空白对照组和溶剂对照组相比,姜黄素组的两株乳腺癌细胞中Twist蛋白表达水平均明显减少(P<0.05).结论:姜黄素抑制乳腺癌细胞增殖的作用与下调Twist蛋白表达有关.