Regulation of vascular endothelial growth factor receptor-2 expression in pancreatic cancer cells by Sp proteins

Vascular endothelial growth factor receptor-2 (VEGFR2/KDR) is an important mediator of angiogenesis, and VEGFR2 mRNA is expressed in several pancreatic cancer cell lines. Deletion analysis of the VEGFR2 promoter in Panc-1, AsPC-1, and MiaPaCa-2 pancreatic cancer cells shows that the proximal region of the promoter is primarily responsible for VEGFR2 expression, and two GC-rich sites at -58 and -44 are critical elements in all three cell lines. Panc-1, AsPC-1, and MiaPaCa-2 cells also express Sp1, Sp3, and Sp4 proteins which bind to the GC-rich region of the VEGFR2 promoter in electrophoretic mobility shift and chromatin immunoprecipitation assays. RNA interference with small inhibitory RNAs for Sp1, Sp3, and Sp4 decreases VEGFR2 mRNA and reporter gene activity in transfection assays, confirming that VEGFR2 expression in pancreatic cancer cells is regulated by Sp proteins. These results suggest that VEGFR2 cannot only be targeted by receptor tyrosine kinase inhibitors but also by drugs that downregulate Sp proteins or block Sp-dependent transactivation.     

MicroRNA-149 Inhibits Proliferation and Cell Cycle Progression through the Targeting of ZBTB2 in Human Gastric Cancer

An increasing body of evidence indicates that miR-149 can both suppress and promote tumor growth depending on the tumor type. However, the role of miR-149 in the progression of gastric cancer (GC) remains unknown. Here we report that miR-149 is a tumor suppressor in human gastric cancer. miR-149 expression is decreased in GC cell lines and clinical specimens in comparison to normal gastric epithelial cell and tissues, respectively. The expression levels of miR-149 also correlate with the differentiation degree of GC cells and tissues. Moreover, ectopic expression of miR-149 in gastric cancer cells inhibits proliferation and cell cycle progression by down-regulating ZBTB2, a potent repressor of the ARF-HDM2-p53-p21 pathway, with a potential binding site for miR-149 in its mRNA''s 3′UTR. It is also found that ZBTB2 expression increases in GC cells and tissues compared to normal gastric epithelial cell and tissues, respectively. Silencing of ZBTB2 leads to suppression of cell growth and cell cycle arrest in G0/G1 phase, indicating that ZBTB2 may act as an oncogene in GC. Furthermore, transfection of miR-149 mimics into gastric cancer cells induces down-regulation of ZBTB2 and HDM2, and up-regulation of ARF, p53, and p21 compared to the controls. In summary, our data suggest that miR-149 functions as a tumor suppressor in human gastric cancer by, at least partially through, targeting ZBTB2.     

MiR-27 as a Prognostic Marker for Breast Cancer Progression and Patient Survival

Background

MicroRNA-27a (miR-27a) is thought to be an onco-microRNA that promotes tumor growth and metastasis by downregulating ZBTB10. The potential predictive value of miR-27a was studied in breast cancer patients.

Methods

The expression of miR-27a and ZBTB10 was examined in 102 breast cancer cases using in situ hybridization (ISH) and immunohistochemistry techniques and were evaluated semi-quantitatively by examining the staining index. The Correlation of miR-27a and ZBTB10 expression was analyed by Spearman Rank Correlation. The association of miR-27a and ZBTB10 expression with clinicopathological characteristics was analyzed using the χ² test, and their effects on patient survival were analyzed by a log-rank test and the Kaplan-Meier method. Univariate and multivariate Cox regression analyses were used to evaluate the prognostic values of miR-27a and ZBTB10.

Results

miR-27a was markedly up-regulated in invasive breast cancers that expressed low levels of ZBTB10 (P<0.001). A reverse correlation between miR-27a and ZBTB10 was also observed in breast cancer tissue samples (r_s = −0.478, P<0.001). Furthermore, the expression of miR-27a and ZBTB10 was significantly correlated with clinicopathological parameters, including tumor size, lymph node metastasis and distant metastasis (P<0.05), but not with receptor status. Patients with high miR-27a or low ZBTB10 expression tended to have significantly shorter disease-free survival times (57 months and 53 months, respectively, P <0.001) and overall survival times (58 months and 55 months, respectively, P <0.001). Univariate and multivariate analysis showed that both miR-27a and ZBTB10 were independent prognostic factors of disease-free survival in breast cancer patients (P <0.001), while only miR-27a was an independent predictor of overall survival (P <0.001).

Conclusions

High miR-27a expression is associated with poor overall survival in patients with breast cancer, which suggests that miR-27a could be a valuable marker of breast cancer progression.     

MicroRNA-22 regulates hypoxia signaling in colon cancer cells

MicroRNAs (MiRNAs) are short, non-coding RNA that regulate a variety of cellular functions by suppressing target protein expression. We hypothesized that a set of microRNA regulate tumor responses to hypoxia by inhibiting components of the hypoxia signaling pathway. We found that miR-22 expression in human colon cancer is lower than in normal colon tissue. We also found that miR-22 controls hypoxia inducible factor 1α (HIF-1α) expression in the HCT116 colon cancer cell line. Over-expression of miR-22 inhibits HIF-1α expression, repressing vascular endothelial growth factor (VEGF) production during hypoxia. Conversely, knockdown of endogenous miR-22 enhances hypoxia induced expression of HIF-1α and VEGF. The conditioned media from cells over-expressing miR-22 contain less VEGF protein than control cells, and also induce less endothelial cell growth and invasion, suggesting miR-22 in adjacent cells influences endothelial cell function. Taken together, our data suggest that miR-22 might have an anti-angiogenic effect in colon cancer.     

Aspirin Inhibits Colon Cancer Cell and Tumor Growth and Downregulates Specificity Protein (Sp) Transcription Factors

Acetylsalicylic acid (aspirin) is highly effective for treating colon cancer patients postdiagnosis; however, the mechanisms of action of aspirin in colon cancer are not well defined. Aspirin and its major metabolite sodium salicylate induced apoptosis and decreased colon cancer cell growth and the sodium salt of aspirin also inhibited tumor growth in an athymic nude mouse xenograft model. Colon cancer cell growth inhibition was accompanied by downregulation of Sp1, Sp3 and Sp4 proteins and decreased expression of Sp-regulated gene products including bcl-2, survivin, VEGF, VEGFR1, cyclin D1, c-MET and p65 (NFκB). Moreover, we also showed by RNA interference that β-catenin, an important target of aspirin in some studies, is an Sp-regulated gene. Aspirin induced nuclear caspase-dependent cleavage of Sp1, Sp3 and Sp4 proteins and this response was related to sequestration of zinc ions since addition of zinc sulfate blocked aspirin-mediated apoptosis and repression of Sp proteins. The results demonstrate an important underlying mechanism of action of aspirin as an anticancer agent and, based on the rapid metabolism of aspirin to salicylate in humans and the high salicylate/aspirin ratios in serum, it is likely that the anticancer activity of aspirin is also due to the salicylate metabolite.     

MicroRNA-24 Suppression of N-Deacetylase/N-Sulfotransferase-1 (NDST1) Reduces Endothelial Cell Responsiveness to Vascular Endothelial Growth Factor A (VEGFA)

Heparan sulfate (HS) proteoglycans, present at the plasma membrane of vascular endothelial cells, bind to the angiogenic growth factor VEGFA to modulate its signaling through VEGFR2. The interactions between VEGFA and proteoglycan co-receptors require sulfated domains in the HS chains. To date, it is essentially unknown how the formation of sulfated protein-binding domains in HS can be regulated by microRNAs. In the present study, we show that microRNA-24 (miR-24) targets NDST1 to reduce HS sulfation and thereby the binding affinity of HS for VEGFA. Elevated levels of miR-24 also resulted in reduced levels of VEGFR2 and blunted VEGFA signaling. Similarly, suppression of NDST1 using siRNA led to a reduction in VEGFR2 expression. Consequently, not only VEGFA binding, but also VEGFR2 protein expression is dependent on NDST1 function. Furthermore, overexpression of miR-24, or siRNA-mediated reduction of NDST1, reduced endothelial cell chemotaxis in response to VEGFA. These findings establish NDST1 as a target of miR-24 and demonstrate how such NDST1 suppression in endothelial cells results in reduced responsiveness to VEGFA.     

MiR-4282 inhibits tumor progression through down-regulation of ZBTB2 by targeting LIN28B in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is the most aggressive type of head and neck cancer with an unsatisfactory 5-year survival rate. MicroRNAs are a group of small noncoding RNAs reported to serve important roles in carcinogenesis, inhibiting certain gene expression via targeting the 3′-untranslated region of messenger RNAs (mRNAs). MiR-4282 has been newly discovered to be a tumor suppressor in colorectal cancer, but it has never been studied in OSCC. The present study aimed to uncover the role of miR-4282 in OSCC. We first confirmed that miR-4282 was downregulated in OSCC and validated its prognostic significance. Through gain-of-function assays, miR-4282 was discovered to inhibit proliferation, migration, and epithelial-to-mesenchymal transition, and induce apoptosis. By mechanistic research, we predicted via bioinformatics tools and confirmed by luciferase reporter and pulldown assays that miR-4282 targeted LIN28B, an RNA-binding protein, which has been reported to regulate RNA stability in cancers. Furthermore, we confirmed the interaction between LIN28B and zinc finger and BTB domain containing 2 (ZBTB2), and validated that miR-4282 regulated mRNA stability of ZBTB2 by inhibiting LIN28B. Rescue assays proved that miR-4282 inhibited tumor progression through LIN28B/ZBTB2 axis. In vivo assays proved that miR-4282 inhibited tumor growth in OSCC. In conclusion, the present study revealed that miR-4282 inhibited tumor progression through downregulation of ZBTB2 by targeting LIN28B in OSCC cells, indicating miR-4282 as a novel biomarker for OSC.     

Retracted: MicroRNA-204-3p represses colon cancer cells proliferation,migration, and invasion by targeting HMGA2

Colon cancer is a detrimental neoplasm of the digestive tract. MicroRNAs (miRNAs) as central regulators have been discovered in colon cancer. Nonetheless, the impact of miR-204-3p on colon cancer remains indistinct. The research attempted to uncover the impacts of miR-204-3p on colon cancer cells growth, migration, and invasion. miR-204-3p expression level in colon cancer tissues and diverse colon cancer cell lines were testified by the quantitative real-time polymerase chain reaction. Exploration of the impacts of miR-204-3p on cell growth, migration, invasion, and their associated factors through assessment of CCK-8, flow cytometry, Transwell, and western blot, respectively. High mobility group AT-hook 2 (HMGA2) expression was then detected in Caco-2 cells after miR-204-3p mimic and inhibitor transfection, additionally dual-luciferase activity was implemented to further uncover the correlation between HMGA2 and miR-204-3p. The impact of HMGA2 on Caco-2 cell growth, migration, and invasion was finally assessed. We found that repression of miR-204-3p was discovered in colon cancer tissues and HCT116, SW480, Caco-2, HT29 and SW620 cell lines. MiR-204-3p overexpression mitigated Coca-2 cell viability, facilitated apoptosis, simultaneously adjusted CyclinD1 and cleaved caspase-3 expression. Cell migration, invasion, and the associated factors were all suppressed by miR-204-3p overexpression. Reduction of HMGA2 was presented in Caco-2 cells with miR-204-3p mimic transfection, and HMGA2 was predicated to be a target gene of miR-204-3p. Besides, HMGA2 silence showed the inhibitory effect on Caco-2 cells growth, migration, and invasion. In conclusion, miR-204-3p repressed colon cancer cell growth, migration, and invasion through targeting HMGA2.     

MicroRNA-147 Induces a Mesenchymal-To-Epithelial Transition (MET) and Reverses EGFR Inhibitor Resistance

Background

The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer progression and may promote resistance to therapy. An analysis of patients (n = 71) profiled with both gene expression and a global microRNA assessment (∼415 miRs) identified miR-147 as highly anti-correlated with an EMT gene expression signature score and postulated to reverse EMT (MET).

Methods and Findings

miR-147 was transfected into colon cancer cells (HCT116, SW480) as well as lung cancer cells (A-549). The cells were assessed for morphological changes, and evaluated for effects on invasion, motility, and the expression of key EMT markers. Resistance to chemotherapy was evaluated by treating cells with gefitinib, an EGFR inhibitor. The downstream genes regulated by miR-147 were assayed using the Affymetrix GeneChip U133 Plus2.0 platform. miR-147 was identified to: 1. cause MET primarily by increasing the expression of CDH1 and decreasing that of ZEB1; 2. inhibit the invasion and motility of cells; 3. cause G1 arrest by up-regulating p27 and down-regulating cyclin D1. miR-147 also dramatically reversed the native drug resistance of the colon cancer cell line HCT116 to gefitinib. miR-147 significantly repressed Akt phosphorylation, and knockdown of Akt with siRNA induced MET. The morphologic effects of miR-147 on cells appear to be attenuated by TGF-B1, promoting a plastic and reversible transition between MET and EMT.

Conclusion

miR-147 induced cancer cells to undergo MET and induced cell cycle arrest, suggesting a potential tumor suppressor role. miR-147 strikingly increased the sensitivity to EGFR inhibitor, gefitinib in cell with native resistance. We conclude that miR-147 might have therapeutic potential given its ability to inhibit proliferation, induce MET, as well as reverse drug sensitivity.     

miR-200 Inhibits lung adenocarcinoma cell invasion and metastasis by targeting Flt1/VEGFR1

The microRNA-200 (miR-200) family is part of a gene expression signature that predicts poor prognosis in lung cancer patients. In a mouse model of K-ras/p53-mutant lung adenocarcinoma, miR-200 levels are suppressed in metastasis-prone tumor cells, and forced miR-200 expression inhibits tumor growth and metastasis, but the miR-200 target genes that drive lung tumorigenesis have not been fully elucidated. Here, we scanned the genome for putative miR-200 binding sites and found them in the 3'-untranslated region (3'-UTR) of 35 genes that are amplified in human cancer. Mining of a database of resected human lung adenocarcinomas revealed that the levels of one of these genes, Flt1/VEGFR1, correlate inversely with duration of survival. Forced miR-200 expression suppressed Flt1 levels in metastasis-prone lung adenocarcinoma cells derived from K-ras/p53-mutant mice, and negatively regulated the Flt1 3'-UTR in reporter assays. Cancer-associated fibroblasts (CAFs) isolated from murine lung adenocarcinomas secreted abundant VEGF and enhanced tumor cell invasion in coculture studies. CAF-induced tumor cell invasion was abrogated by VEGF neutralization or Flt1 knockdown in tumor cells. Flt1 knockdown decreased the growth and metastasis of tumor cells in syngeneic mice. We conclude that miR-200 suppresses lung tumorigenesis by targeting Flt1.     

Transforming growth factor‐β1 modulates metalloproteinase‐2 and ‐9, nitric oxide,RhoA and α‐smooth muscle actin expression in colon adenocarcinoma cells

Colon carcinoma invasiveness is a process involving cell–cell and cell–matrix alterations, local proteolysis of the ECM (extracellular matrix) or changes in cytokine and growth factor levels. In order to evaluate the role of TGF‐β1 (transforming growth factor‐β1) and small G protein RhoA in tumour progression, the influence of TGF‐β1 treatment or RhoA‐associated kinase inhibitor on the production of NO (nitric oxide) and MMP‐2 and MMP‐9 (metalloproteinases‐2 and ‐9) was analysed in three human colon adenocarcinoma cell lines (HT29, LS180, SW948) representing different stages of tumour development. All the tested cell lines produced low amounts of MMP‐2 and MMP‐9. rhTGF‐β1 and the synthetic Rho kinase inhibitor (Y‐27632) decreased MMP‐2 secretion by colon cancer cells, especially in the most advanced stage of colon cancer. rhTGF‐β1 decreased NO secretion by cells, while Y‐27632 had no effect on it. Immunoblotting with anti‐RhoA antibodies followed by densitometry revealed that RhoA levels were slightly increased after incubation of colon carcinoma cells (SW948) with rhTGF‐β1. rhTGF‐β1 induced α‐smooth muscle actin (α‐SMA) expression, especially in high Duke's grade of colon cancer, while Y‐27632 blocked it. Summing up, in colon carcinoma cells, TGF‐β1 and RhoA protein may regulate tumour invasiveness measured as MMP, NO and α‐SMA expression or assayed using motility data and may be a good target for cancer therapy.     

Vector-based miR-15a/16-1 plasmid inhibits colon cancer growth in vivo

miR-15 (microRNA 15) and miR-16 are frequently deleted or down-regulated in many cancer cell lines and various tumour tissues, suggesting that miR-15a/16-1 plays important roles in tumour progression and might be a method for cancer treatment. We have developed a vector-based plasmid to explore the anti-tumour efficacy of miR-15a/16-1 in colon cancer in vivo. It is proposed that miR-15a and miR-16-1 target cyclin B1 (CCNB1), which associates with several tumorigenic features such as survival and proliferation. The levels of miR-15a and miR-16-1 in colon cancer cells were inversely correlated with CCNB1 expression, and there was consensus between miR-15a/16-1 and CCNB1 mRNA sequences by analysing homology. Vector-based miR-15a/16-1 expression plasmid was constructed and transfected into HCT 116 and SW620 colon cancer cells in vitro. The effects produced on cell viability and angiogenesis were analysed using flow cytometric analysis, colony formation analysis and tube formation analysis. CCNB1 expression down-regulation was checked by Western blotting. Systemic delivery of miR-15a/16-1 plasmids encapsulated in cationic liposome led to a significant inhibition of subcutaneous tumour growth and angiogenesis in tumour tissues, whereas no effects were observed with liposome carrying the non-specific plasmid. In summary, miR-15a/16-1 has been applied in colon cancer treatment in vivo, and resulted in effective colon tumour xenografts growth arrest and angiogenesis decrease. These findings suggest that systemic delivery of vector-based miR-15a/16-1 expression plasmid can be an approach to colon cancer therapy.     

miR-22 represses cancer progression by inducing cellular senescence

Cellular senescence acts as a barrier to cancer progression, and microRNAs (miRNAs) are thought to be potential senescence regulators. However, whether senescence-associated miRNAs (SA-miRNAs) contribute to tumor suppression remains unknown. Here, we report that miR-22, a novel SA-miRNA, has an impact on tumorigenesis. miR-22 is up-regulated in human senescent fibroblasts and epithelial cells but down-regulated in various cancer cell lines. miR-22 overexpression induces growth suppression and acquisition of a senescent phenotype in human normal and cancer cells. miR-22 knockdown in presenescent fibroblasts decreased cell size, and cells became more compact. miR-22-induced senescence also decreases cell motility and inhibits cell invasion in vitro. Synthetic miR-22 delivery suppresses tumor growth and metastasis in vivo by inducing cellular senescence in a mouse model of breast carcinoma. We confirmed that CDK6, SIRT1, and Sp1, genes involved in the senescence program, are direct targets of miR-22. Our study provides the first evidence that miR-22 restores the cellular senescence program in cancer cells and acts as a tumor suppressor.