Anxun Wang

MicroRNA-138 suppresses invasion and promotes apoptosis in head and neck squamous cell carcinoma cell lines.

Metastasis is a critical event in the progression of head and neck squamous cell carcinoma (HNSCC). To identify microRNAs associated with HNSCC metastasis, six paired HNSCC cell lines with different metastatic potential were examined. Using microarrays, a panel of differentially expressed microRNAs was identified, including reduction of miR-138 in highly metastatic cells. Ectopic transfection of miR-138 suppressed cell invasion and led to cell cycle arrest and apoptosis. Knockdown of miR-138 enhanced cell invasion and suppressed apoptosis. Thus, our results suggested miR-138 acts as a tumor suppresser and may serve as a therapeutic target for HNSCC patients at risk of metastasis.

Downregulation of the Rho GTPase signaling pathway is involved in the microRNA‐138‐mediated inhibition of cell migration and invasion in tongue squamous cell carcinoma

Tumor metastasis is the dominant cause of death in cancer patients, including patients with oral tongue squamous cell carcinoma (TSCC). Previously, we reported that reduced miR‐138 level is correlated with enhanced metastatic potential in TSCC cells. Here, we demonstrate that miR‐138 suppresses TSCC cell migration and invasion by regulating 2 key genes in the Rho GTPase signaling pathway: RhoC and ROCK2. Direct targeting of miR‐138 to specific sequences located in the 3′‐untranslated regions of both RhoC and ROCK2 mRNAs was confirmed using luciferase reporter gene assays. Ectopic transfection of miR‐138 reduced the expression of both RhoC and ROCK2 in TSCC cells. These reduced expressions, in consequence, led to the reorganization of the stress fibers and the subsequent cell morphology change to a round bleb‐like shape as well as the suppression of cell migration and invasion. In contrast, knockdown of miR‐138 in TSCC cells enhanced the expression of RhoC and ROCK2, which resulted in an altered, elongated cell morphology, enhanced cell stress fiber formation and accelerated cell migration and invasion. Taken together, our results suggest that miR‐138 plays an important role in TSCC cell migration and invasion by concurrently targeting RhoC and ROCK2, and miR‐138 may serve as a novel therapeutic target for TSCC patients at risk of metastatic disease.

Down-regulation of the microRNA-99 family members in head and neck squamous cell carcinoma

OBJECTIVES MicroRNA deregulation is a critical event in head and neck squamous cell carcinoma (HNSCC). Several microRNA profiling studies aimed at deciphering the microRNA signatures of HNSCC have been reported, but there tends to be poor agreement among studies. The objective of this study was to survey the published microRNA profiling studies on HNSCC, and to assess the commonly deregulated microRNAs in an independent sample set. MATERIALS AND METHODS Meta-analysis of 13 published microRNA profiling studies was performed to define microRNA signatures in HNSCC. Selected microRNAs (including members of miR-99 family) were evaluated in an independent set of HNSCC cases. The potential contributions of miR-99 family to the tumorigenesis of HNSCC were assessed by in vitro assays. RESULTS We identified 67 commonly deregulated microRNAs. The up-regulation of miR-21, miR-155, miR-130b, miR-223 and miR-31, and the down-regulation of miR-100, miR-99a and miR-375 were further validated in an independent set of HNSCC cases with quantitative RT-PCR. Among these validated microRNAs, miR-100 and miR-99a belong to the miR-99 family. Our in vitro study demonstrated that restoration of miR-100 to the HNSCC cell lines suppressed cell proliferation and migration, and enhanced apoptosis. Furthermore, ectopic transfection of miR-99 family members down-regulated the expression of insulin-like growth factor 1 receptor (IGF1R) and mechanistic target of rapamycin (mTOR) genes. CONCLUSION In summary, we described a panel of frequently deregulated microRNAs in HNSCC, including members of miR-99 family. The deregulation of miR-99 family contributes to the tumorigenesis of HNSCC, in part by targeting IGF1R and mTOR signaling pathways.

Identification and experimental validation of G protein alpha inhibiting activity polypeptide 2 (GNAI2) as a microRNA-138 target in tongue squamous cell carcinoma

MicroRNA deregulation is a critical event in tumor initiation and progression. The down-regulation of microRNA-138 has been frequently observed in various cancers, including tongue squamous cell carcinoma (TSCC). Our previous studies suggest that deregulation of miR-138 is associated with the enhanced proliferation and invasion in TSCC cells. Here, we seek to identify the targets of miR-138 in TSCC, and explore their functional relevance in tumorigenesis. Our genome-wide expression profiling experiments identified a panel of 194 unique transcripts that were significantly down-regulated in TSCC cells transfected with miR-138. A comprehensive screening using six different sequence-based microRNA target prediction algorithms revealed that 51 out of these 194 down-regulated transcripts are potential direct targets for miR-138. These targets include: chloride channel, nucleotide-sensitive, 1A (CLNS1A), G protein alpha inhibiting activity polypeptide 2 (GNAI2), solute carrier family 20, member 1 (SLC20A1), eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1), and Rho-related GTP-binding protein C (RhoC). GNAI2 is a known proto-oncogene that is involved in the initiation and progression of several different types of tumors. Direct targeting of miR-138 to two candidate binding sequences located in the 3′-untranslated region of GNAI2 mRNA was confirmed using luciferase reporter gene assays. Knockdown of miR-138 in TSCC cells enhanced the expression of GNAI2 at both mRNA and protein levels. In contrast, ectopic transfection of miR-138 reduced the expression of GNAI2, which, in consequence, led to reduced proliferation, cell cycle arrest and apoptosis. In summary, we identified a number of high-confident miR-138 target genes, including proto-oncogene GNAI2, which may play an important role in TSCC initiation and progression.

Pharmacokinetic characteristics and anticancer effects of 5-Fluorouracil loaded nanoparticles

BackgroundIt is expected that prolonged circulation of anticancer drugs will increase their anticancer activity while decreasing their toxic side effects. The purpose of this study was to prepare 5-fluorouracil (5-FU) loaded block copolymers, with poly(γ-benzyl-L-glutamate) (PBLG) as the hydrophobic block and poly(ethylene glycol) (PEG) as the hydrophilic block, and then examine the 5-FU release characteristics, pharmacokinetics, and anticancer effects of this novel compound.Methods5-FU loaded PEG-PBLG (5-FU/PEG-PBLG) nanoparticles were prepared by dialysis and then scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to observe the shape and size of the nanoparticles, and ultraviolet spectrophotometry was used to evaluate the 5-FU in vitro release characteristics. The pharmacokinetic parameters of 5-FU/PEG-PBLG nanoparticles in rabbit plasma were determined by measuring the 5-FUby high-performance liquid chromatography (HPLC). To study in vivo effects, LoVo cells (human colon cancer cell line) or Tca8113 cells (human oral squamous cell carcinoma cell line) were implanted in BALB/c nude mice that were subsequently treated with 5-FU or 5-FU/PEG-PBLG nanospheres.Results5-FU/PEG-PBLG nanoparticles had a core-shell spherical structure with a diameter of 200 nm and a shell thickness of 30 nm. The drug loading capacity was 27.1% and the drug encapsulation was 61.5%. Compared with 5-FU, 5-FU/PEG-PBLG nanoparticles had a longer elimination half-life (t1/2, 33.3 h vs. 5 min), lower peak concentration (C, 4563.5 μg/L vs. 17047.3 μg/L), and greater distribution volume (VD, 0.114 L vs. 0.069 L). Compared with a blank control, LoVo cell xenografts and Tca8113 cell xenografts treated with 5-FU or 5-FU/PEG-PBLG nanoparticles grew slower and had prolonged tumor doubling times. 5-FU/PEG-PBLG nanoparticles showed greater inhibition of tumor growth than 5-FU (p < 0.01). In the PEG-PBLG nanoparticle control group, there was no tumor inhibition (p > 0.05).ConclusionIn our model system, 5-FU/PEG-PBLG nanoparticles changed the pharmacokinetic behavior of 5-FU, thus increasing its anticancer activity. 5-Fluorouracil loaded nanoparticles have potential as a novel anticancer drug that may have useful clinical applications.

MicroRNA Deregulations in Head and Neck Squamous Cell Carcinomas

ABSTRACT Objectives Head and neck/oral cancer, predominantly head and neck squamous cell carcinoma (HNSCC), is the sixth most common cancer in the world. While substantial advances have been made to define the genomic alterations associated with head and neck/oral cancer, most studies are focused on protein coding genes. The aim of this article is to review the current literature on identified genomic aberrations of non-coding genes (e.g., microRNA) in head and neck/oral cancer (HNOC), and their contribution to the initiation and progression of HNOC. Material and Methods A comprehensive review of the available literature relevant to microRNA deregulation in HNSCC/HNOC, was undertaken using PubMed, Medline, Scholar Google and Scopus. Keywords for the search were: microRNA and oral cancer, microRNA and squamous cell carcinoma, microRNA deregulation and oral cancer, microRNA and carcinogenesis in the head and neck/oral cavity. Only full length articles in the English language were included. Results We recently identified a panel of microRNA deregulations that were consistently observed in HNSCC [Chen et al., Oral Oncol. 2012;48(8):686-91], including 7 consistently up-regulated microRNAs (miR-21, miR-7, miR-155, miR-130b, miR-223, miR-34b), and 4 consistently down-regulated microRNAs (miR-100, miR-99a, miR-125b, miR-375). In this review, we will first provide an overview on microRNA and HNSCC. We will then provide a comprehensive review on the roles of microRNA deregulations in HNSCC. The functional significance of the identified HNSCC-associated microRNAs and a number of other relevant microRNAs (e.g., miR-138, miR-98, miR-137, miR-193a and miR-218) will be discussed in detail. Conclusions Based on current literature, microRNA deregulation plays a major role in head and neck/oral cancer.

Tumor budding correlates with poor prognosis and epithelial-mesenchymal transition in tongue squamous cell carcinoma

BACKGROUND Tumor budding is a readily detectable histopathological feature and has been recognized as an adverse prognostic factor in several human cancers. However, the prognostic value of tumor budding in tongue squamous cell carcinoma (TSCC) has not been reported. The purpose of this study was to assess the correlation of tumor budding with the clinicopathologic features, and the known molecular biomarkers (E-cadherin and Vimentin), as well as to evaluate its prognostic significance for TSCC. METHODS Archival clinical samples of 230 patients with TSCC were examined for tumor budding. Immunohistochemistry analyses were performed to examine the expression of E-cadherin and Vimentin. Statistical analyses were carried out to assess the correlation of tumor budding with clinicopathologic parameters and patient survival. The potential association between tumor budding and alterations of E-cadherin and Vimentin expression was also assessed. RESULTS Of the 230 TSCC cases examined, tumor budding was observed in 165 cases (71.7%), with a mean tumor bud count of 7.5 (range from 1 to 48 buds). High-intensity budding (≥5 tumor buds) was observed in 111 cases (48.3%). Statistical analysis revealed that tumor budding was associated with tumor size (P < 0.05), differentiation (P < 0.05), clinical stage (P < 0.05), lymph node metastasis (P < 0.01), and correlated with reduced overall survival. In addition, significant associations were observed among tumor budding and the deregulation of E-cadherin (P < 0.001) and Vimentin (P < 0.001). CONCLUSIONS Tumor budding, which associates with epithelial-mesenchymal transition, is a frequent event and appears to be an independent prognostic factor in TSCC.

MicroRNA-24 targeting RNA-binding protein DND1 in tongue squamous cell carcinoma

Deregulations of microRNA have been frequently observed in tongue squamous cell carcinoma (TSCC), but their roles in tumorigenesis are not entirely clear. Here, we reported the up‐regulation of miR‐24 in TSCC. MiR‐24 up‐regulation reduced the expression of RNA‐binding protein dead end 1 (DND1). Knockdown of miR‐24 led to enhanced expression of DND1. The direct targeting of miR‐24 to the DND1 mRNA was predicted bioinformatically and confirmed by luciferase reporter gene assays. Furthermore, the miR‐24‐mediated change in DND1 expression suppressed the expression of cyclin‐dependent kinase inhibitor 1B (CDKN1B), and also led to enhanced proliferation and reduced apoptosis in TSCC cells.

Polycomb group protein EZH2‐mediated E‐cadherin repression promotes metastasis of oral tongue squamous cell carcinoma

Enhancer of Zeste homolog 2 (EZH2) is a critical component of the polycomb‐repressive complex 2 (PRC2) that regulates many essential biological processes, including embryogenesis and many developmental events. The oncogenic role of EZH2 has recently been implicated in several cancer types. In this study, we first confirmed that the over‐expression of EZH2 is a frequent event in oral tongue squamous cell carcinoma (OTSCC). We further demonstrated that EZH2 over‐expression is correlated with advanced stages of the disease and is associated with lymph node metastasis. Statistical analysis revealed that EZH2 over‐expression was correlated with reduced overall survival. Furthermore, over‐expression of EZH2 was correlated with reduced expression of tumor suppressor gene E‐cadherin. These observations were confirmed in vitro, in which knockdown of EZH2‐induced E‐cadherin expression and reduced cell migration and invasion. In contrast, ectopic transfection of EZH2 led to reduced E‐cadherin expression and enhanced cell migration and invasion. Furthermore, EZH2 may act on cell migration in part by suppressing the E‐cadherin expression. Taken together, these data suggest that EZH2 plays major roles in the progression of OTSCC, and may serve as a biomarker or therapeutic target for patients at risk of metastasis.

MicroRNA-181a suppresses salivary adenoid cystic carcinoma metastasis by targeting MAPK-Snai2 pathway

BACKGROUND To date microRNAs and their contribution to the onset and propagation of salivary adenoid cystic carcinoma (SACC) are limited. The objective of this study was to identify miR-181a and its mechanism in the metastasis of SACC. METHODS At first microarray and quantitative RT-PCR were used to investigate microRNA profiles and miR-181a in paired SACC cell lines with different metastatic potential. Then the effect of miR-181a on metastatic potential of SACC was investigated. MiR-181a target genes and Snai2 promoter activity were investigated using luciferase reporter gene assays. Western blot was used to detect MAPK-Snai2 pathway-related protein level. RESULTS A panel of deregulated microRNAs (including miR-181a) was identified in paired of SACC cell lines. Functional analysis indicated that miR-181a inhibited SACC cell migration, invasion and proliferation in vitro, and it suppressed tumor growth and lung metastasis in vivo. Direct targeting of miR-181a to MAP2K1, MAPK1 and Snai2 was confirmed by luciferase reporter gene assays. MiR-181a mimic inhibited the expression of MAP2K1, MAPK1 and Snai2 in SACC cells. MAP2K1 or MAPK1 siRNA suppressed Snai2 gene promoter activity and reduced Snai2 expression and the metastatic potential of SACC cells. CONCLUSIONS Our results indicate that miR-181a plays an important role in the metastasis of SACC, and may serve as a novel therapeutic target for SACC. MiR-181a regulates the MAPK-Snai2 pathway both through direct cis-regulatory mechanism and through indirect trans-regulatory mechanism. GENERAL SIGNIFICANCE To our knowledge, this is the first study revealing that miR-181a deregulation mediated the metastasis of SACC by regulating MAPK-Snai2 pathway.