Xiqiang Liu

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.

MicroRNA-138 suppresses epithelial–mesenchymal transition in squamous cell carcinoma cell lines

Down-regulation of miR-138 (microRNA-138) has been frequently observed in various cancers, including HNSCC (head and neck squamous cell carcinoma). Our previous studies suggest that down-regulation of miR-138 is associated with mesenchymal-like cell morphology and enhanced cell migration and invasion. In the present study, we demonstrated that these miR-138-induced changes were accompanied by marked reduction in E-cad (E-cadherin) expression and enhanced Vim (vimentin) expression, characteristics of EMT (epithelial-mesenchymal transition). On the basis of a combined experimental and bioinformatics analysis, we identified a number of miR-138 target genes that are associated with EMT, including VIM, ZEB2 (zinc finger E-box-binding homeobox 2) and EZH2 (enhancer of zeste homologue 2). Direct targeting of miR-138 to specific sequences located in the mRNAs of the VIM, ZEB2 and EZH2 genes was confirmed using luciferase reporter gene assays. Our functional analyses (knock-in and knock-down) demonstrated that miR-138 regulates the EMT via three distinct pathways: (i) direct targeting of VIM mRNA and controlling the expression of VIM at a post-transcriptional level, (ii) targeting the transcriptional repressors (ZEB2) which in turn regulating the transcription activity of the E-cad gene, and (iii) targeting the epigenetic regulator EZH2 which in turn modulates its gene silencing effects on the downstream genes including E-cad. These results, together with our previously observed miR-138 effects on cell migration and invasion through targeting RhoC (Rho-related GTP-binding protein C) and ROCK2 (Rho-associated, coiled-coil-containing protein kinase 2) concurrently, suggest that miR-138 is a multi-functional molecular regulator and plays major roles in EMT and in HNSCC progression.

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.

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.

Molecular Characterization of the MicroRNA-138-Fos-like Antigen 1 (FOSL1) Regulatory Module in Squamous Cell Carcinoma

Background: MicroRNA-138 deregulation is a common event in cancer. Results: Our study describes a microRNA regulatory module consisting of tumor suppressor miR-138 and proto-oncogene FOSL1. Conclusion: The deregulation of miR-138-FOSL1 regulatory module may play an important role in cancer initiation and progression. Significance: Our results suggest that microRNAs target both canonical and non-canonical targeting sites located in all areas of mRNA molecules. MicroRNA-138 is one of the most frequently down-regulated microRNAs in cancer. We recently identified 51 candidate targets of microRNA-138 (Jiang, L., Dai, Y., Liu, X., Wang, C., Wang, A., Chen, Z., Heidbreder, C. E., Kolokythas, A., and Zhou, X. (2011) Hum. Genet. 129, 189–197). Among these candidates, Fos-like antigen 1 (FOSL1) is a member of Fos gene family and is a known proto-oncogene. In this study, we first confirmed the microRNA-138-mediated down-regulation of FOSL1 in squamous cell carcinoma cell lines. We then demonstrated the effect of this microRNA-138-FOSL1 regulatory module on downstream genes (homolog of Snail 2 (Snai2) expression and the Snai2-mediated repression of E-cadherin expression), as well as its contributions to tumorigenesis. The microRNA-138-directed recruitment of FOSL1 mRNA to the RNAi-induced silencing complex was confirmed by a ribonucleoprotein-immunoprecipitation assay. Three canonical and three high affinity non-canonical microRNA-138 (miR-138) targeting sites were identified on the FOSL1 mRNA: one in the 5′-UTR, three overlapping sites in the coding sequences, and two overlapping sites in the 3′-UTR. The direct targeting of miR-138 to these sites was confirmed using luciferase reporter gene assays. In summary, we describe an important microRNA regulatory module, which may play an important role in cancer initiation and progression. Our results also provide evidence that microRNAs target both canonical and non-canonical targeting sites located in all areas of the mRNA molecule (e.g. 5′-UTR, coding sequences, and 3′-UTR).

Dysregulation of heat shock protein 27 expression in oral tongue squamous cell carcinoma

BackgroundRecent proteomic studies identified Hsp27 as a highly over-expressed protein in oral squamous cell carcinoma (OSCC). Clinical studies that attempted to evaluate the prognostic values of Hsp27 yielded inconsistent results, which may be due to inclusion of OSCC cases from multiple anatomic sites. In this study, to determine the utility of Hsp27 for prognosis, we focused on oral tongue SCC (OTSCC), one of the most aggressive forms of OSCC.MethodsArchival clinical samples of 15 normal oral tongue mucosa, 31 dysplastic lesions, 80 primary OTSCC, and 32 lymph node metastases were examined for Hsp27 expression by immunohistochemistry (IHC). Statistical analyses were carried out to assess the prognostic value of Hsp27 expression for patients with this disease.ResultsDysregulation of Hsp27 expression was observed in dysplastic lesions, primary OTSCC, and lymph node metastases, and appears to be associated with disease progression. Statistical analysis revealed that the reduced Hsp27 expression in primary tumor tissue was associated with poor differentiation. Furthermore, the higher expression of Hsp27 was correlated with better overall survival.ConclusionOur study confirmed that the dysregulation of Hsp27 expression is a frequent event during the progression of OTSCC. The expression of Hsp27 appears to be an independent prognostic marker for patients with this disease.