Qianting He

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.

AFM nanoindentation detection of the elastic modulus of tongue squamous carcinoma cells with different metastatic potentials

UNLABELLED Although significant advances have been made in understanding the molecular mechanisms that influence tongue squamous cell carcinoma (TSCC) metastasis, less is known about the association between the cellular elastic modulus and TSCC metastasis. Atomic force microscopy (AFM) nanoindentation via the rate-jump method was used to detect the elastic modulus of TSCC cells from patients and cell lines with different metastatic potentials. TSCC cells with higher metastatic potential showed decreases in the elastic modulus compared to TSCC cells with lower metastatic potential. Moreover, the decrease in elastic modulus was accompanied with epithelial-mesenchymal transition (EMT), cytoskeleton (F-actin and β-tubulin) changes, small nucleus size and large nucleus/cytoplasm (N/C) ratio. The present findings demonstrate a close relationship between the cellular elastic modulus and the metastasis of TSCC. The elastic modulus detected by AFM nanoindentation via the rate-jump method can potentially be used to grade the metastatic potential of TSCC. FROM THE CLINICAL EDITOR This team of investigators report the use of an atomic force microscopy-based method to determine the elastic modulus of tongue squamous cell carcinoma cells, and demonstrate that such cells with higher metastatic potential show decreased elastic modulus compared to cells with lower metastatic potential.

Manganese superoxide dismutase induces migration and invasion of tongue squamous cell carcinoma via H2O2-dependent Snail signaling

Our previous studies had revealed that the dysregulation of manganese superoxide dismutase (SOD2) expression was a frequent event in tongue squamous cell carcinoma (TSCC) and may be associated with enhanced metastatic potential. To further evaluate the mechanism of SOD2-mediated metastasis in TSCC, TSCC cell lines with different metastatic potentials (i.e., the highly metastatic UM1 line and the UM2 line, which displays fewer metastases) were used. Compared to UM2 cells, UM1 cells exhibited significantly higher SOD2 activity and intracellular H(2)O(2); higher protein levels of Snail, MMP1, and pERK1/2; lower protein levels of E-cadherin; and no difference in catalase activity. Upon knockdown of SOD2 by RNA interference, UM1 cells displayed significantly reduced migration and invasion abilities; reduced activities of SOD2; lower intracellular H(2)O(2); decreased protein levels of Snail, MMP1, and pERK1/2; and increased protein levels of E-cadherin. The migration and invasion abilities of UM2 and SOD2 shRNA-transfected UM1 cells were enhanced by H(2)O(2) treatment and accompanied by increased protein levels of Snail, MMP1, and pERK1/2 and decreased protein levels of E-cadherin. Moreover the migration and invasion abilities of UM1 cells were decreased after catalase treatment. Thus, we conclude that the SOD2-dependent production of H(2)O(2) contributes to both the migration and the invasion of TSCC via the Snail signaling pathway, through increased Snail, MMP1, and pERK1/2 protein levels and the repression of the E-cadherin protein.

Bmi1 Drives Stem-Like Properties and is Associated with Migration, Invasion, and Poor Prognosis in Tongue Squamous Cell Carcinoma

Bmi1 (B-cell-specific Moloney murine leukemia virus insertion site 1) had been found to involve in self -renewal of stem cells and tumorigenesis in various malignancies. The purpose of this study is to evaluate the role of Bmi1 in the development of tongue squamous cell carcinoma (TSCC) and its functional effect on the migration and invasion of TSCC. Initially, immunohistochemistry revealed that Bmi1 overexpression was a common event in premalignant dysplasia, primary TSCC, and lymph node metastases and was associated with a poor prognosis. A significant correlation between Bmi1 and SOD2 (manganese superoxide dismutase) expression was observed. Side population (SP) cells were used as cancer stem-like cells and further assessed by sphere and colony formation assays, and the expression of stem cell markers. TSCC cells with higher migration and invasion ability (UM1 cell lines) showed a higher proportion of SP cells and Bmi1 expression than TSCC cells with lower migration and invasion ability (UM2 cell lines). Knockdown of Bmi1 in UM1 or SP cells inhibited migration and invasion and decreased the sphere and colony formation, and the expression of stem cell markers and SOD2. Direct binding of C-myc to the Bmi1 promoter was demonstrated by chromatin immunoprecipitation and luciferase assays. Moreover, C-myc knockdown in SP cells inhibited their migration and invasion and decreased the expression of Bmi1 and SOD2. Our results indicate that the deregulation of Bmi1 expression is a frequent event during the progression of TSCC and may have a prognostic value for patients with this disease. The Bmi1-mediated migration and invasion of TSCC is related to cancer stem-like cells and involves the C-myc-Bmi1-SOD2 pathway.

Deregulation of Bmi-1 is associated with enhanced migration, invasion and poor prognosis in salivary adenoid cystic carcinoma.

BACKGROUND Bmi-1 had been found to involve in self renewal of stem cells and tumorigenesis in various malignancies. In this study, we investigated the role of Bmi-1 in the development of salivary adenoid cystic carcinoma (SACC). METHODS At first, we confirmed that the deregulation of Bmi-1 was a frequent event in SACC; up-regulation of Bmi-1 was correlated with clinical stages, vital status and distant metastasis and associated with reduced overall survival and disease free survival. SACC-LM cells, higher migration and invasion abilities, elevated the expression of Bmi-1 protein, epithelial-mesenchymal transition (EMT) related proteins (Snail, Slug and Vimentin) and cancer stem cells (CSCs) related proteins (ABCG2, Notch, ALDH-1, Oct-4, Nanog and Epcam) compared to the SACC-83 cells (lower migration and invasion abilities). The migration and invasion abilities were inhibited in SACC-LM cells upon Bmi-1 knockdown. Meanwhile, Bmi-1 knockdown resulted in simultaneous loss of stem cell markers and EMT markers in SACC-LM cells. CONCLUSION Our studies confirm that Bmi-1 deregulation plays an important role in the development of SACC and contributes to the migration and the invasion abilities of SACC, which is involved in EMT and CSCs. GENERAL SIGNIFICANCE To our knowledge, this is the first study revealing that Bmi-1 deregulation is associated with enhanced migration, invasion and poor prognosis in salivary adenoid cystic carcinoma.

Deregulation of the miR-222-ABCG2 regulatory module in tongue squamous cell carcinoma contributes to chemoresistance and enhanced migratory/invasive potential.

Chemoresistance is often associated with other clinical characteristics such as enhanced migratory/invasive potential. However, the correlation and underlying molecular mechanisms remain unclear. The aim of this study was to elucidate the function of the miR-222-ABCG2 pathway in the correlation between cisplatin (DDP) resistance and enhanced cell migration/invasion in tongue squamous cell carcinoma (TSCC). Using TSCC cell lines and primary cultures from TSCC cases, we first confirmed the correlation among DDP resistance (measured by IC50 values and ABCG2/ERCC1 expression), migratory/invasive potential (assessed by migration/invasion assays) and miR-222 expression. In TSCC cells, siRNA-mediated ABCG2 knockdown led to enhanced DDP responsiveness and reduced migratory/invasive potential, whereas ABCG2 overexpression induced DDP resistance and enhanced cell migration/invasion. Luciferase assays revealed that ABCG2 is a direct target of miR-222. In addition to reducing cell migration/invasion, functional analyses in TSCC cells indicated that miR-222 can reduce expression of the ABCG2 gene and enhance DDP responsiveness. However, co-transfection with ABCG2 cDNA restored both DDP resistance and migration/invasion. Moreover, miR-222 mimics and ABCG2 siRNA inhibited tumor growth and lung metastasis in vivo. Thus, our results verified that DDP resistance is correlated with enhanced migratory/invasive potential in TSCC. ABCG2 is a direct target of miR-222,and deregulation of the miR-222-ABCG2 regulatory module in TSCC contributes to both DDP resistance and enhanced migratory/invasive potential.

SOD2 is a C-myc target gene that promotes the migration and invasion of tongue squamous cell carcinoma involving cancer stem-like cells.

Our previous studies revealed that manganese superoxide dismutase (SOD2) contributes to the migration and invasion of tongue squamous cell carcinoma (TSCC). The purpose of the current study was to further clarify the mechanisms of SOD2 in the migration and invasion of TSCC. Side population (SP) cells were used as cancer stem-like cells and further assessed by sphere and colony formation assays, and the expression of stem cell markers (Bmi1, Nanog and ABCG2). We found that UM1 cells (TSCC cells with increased SOD2 expression, migration and invasion abilities) possessed a higher proportion of SP cells, sphere and colony formation, and expressed a higher level of stem cell markers compared to UM2 cells (reduced SOD2 expression, migration and invasion abilities). SOD2 expression as well as migration and invasion abilities were enhanced in SP cells compared to non-SP cells. Knockdown of SOD2 in UM1 cells or SP cells inhibited the migration and invasion abilities, reduced sphere and colony formation, and the expression of stem cell markers. Direct binding of the C-myc protein to the SOD2 promoter was demonstrated by chromatin immunoprecipitation and luciferase assays. Knockdown of C-myc in UM1 cells inhibited SOD2 expression as well as migration and invasion abilities. Our results indicate that cancer stem-like cells play an important role in the migration and invasion of TSCC. SOD2 is a direct target gene of C-myc and C-myc-SOD2-mediated migration and invasion of TSCC involve cancer stem-like cells.

Folic acid rescue of ATRA-induced cleft palate by restoring the TGF-β signal and inhibiting apoptosis

BACKGROUND Cleft palate is a frequent congenital malformation with a heterogeneous etiology, for which folic acid (FA) supplementation has a protective effect. To gain more insight into the molecular pathways affected by FA, TGF-β signaling and apoptosis in mouse embryonic palatal mesenchymal (MEPM) cells of all-trans retinoic acid (ATRA)-induced cleft palate in organ culture were tested. METHODS C57BL/6J mice embryonic palates were explanted on embryonic day 14 and cultured in DMEM/F12 medium with or without ATRA or FA for 72 h. The palatal fusion was examined by light microscopy. Immunohistochemistry was used to detect TGFβ3/TGF receptor II and caspase 9 in MEPM cells. TUNEL was used to detect apoptosis. RESULTS Similar to development in vivo, palatal development and fusion were normal in control medium. ATRA inhibited palatal development and induced cleft palate, which can be rescued by FA. A higher apoptosis rate and caspase-9 in MEPM cells were detected in the ATRA group than in the control or the ATRA+FA group. Compared with the control or the ATRA+FA group, ATRA had little effect on TGF-β3 in MEPM cells but significantly inhibited TGF-β receptor II. CONCLUSIONS Folic acid can rescue the cultured palates to continue developing and fusing that were inhibited and resulted in cleft palate by ATRA. Apoptosis and TGFβ signaling in MEPM cells were involved in folic acid rescued ATRA-induced cleft palate.

Oleic acid induces apoptosis and autophagy in the treatment of Tongue Squamous cell carcinomas

Oleic acid (OA), a main ingredient of Brucea javanica oil (BJO), is widely known to have anticancer effects in many tumors. In this study, we investigated the anticancer effect of OA and its mechanism in tongue squamous cell carcinoma (TSCC). We found that OA effectively inhibited TSCC cell proliferation in a dose- and time-dependent manner. OA treatment in TSCC significantly induced cell cycle G0/G1 arrest, increased the proportion of apoptotic cells, decreased the expression of CyclinD1 and Bcl-2, and increased the expression of p53 and cleaved caspase-3. OA also obviously induced the formation of autolysosomes and decreased the expression of p62 and the ratio of LC3 I/LC3 II. The expression of p-Akt, p-mTOR, p-S6K, p-4E-BP1 and p-ERK1/2 was significantly decreased in TSCC cells after treatment with OA. Moreover, tumor growth was significantly inhibited after OA treatment in a xenograft mouse model. The above results indicate that OA has a potent anticancer effect in TSCC by inducing apoptosis and autophagy via blocking the Akt/mTOR pathway. Thus, OA is a potential TSCC drug that is worthy of further research and development.

Hexokinase 2 enhances the metastatic potential of tongue squamous cell carcinoma via the SOD2-H 2 O 2 pathway

The glycolytic enzyme hexokinase (HK2), which is aberrantly expressed in various types of tumours, is associated with metastasis. However, its role in the progression and metastasis of tongue squamous cell carcinoma (TSCC) remains unclear. The results of our study showed that HK2 expression is often deregulated in TSCC patients. Increased HK2 expression was associated with tumour stage, clinical stage, lymph node metastasis, but not pathological grade, and reduced overall survival. Microarray and western blotting analyses revealed increases in HK2 expression in TSCC cells with higher metastatic potential. The following effects were observed with HK2 knockdown: inhibition of cell migration and invasion; reduced SOD2 activity and intracellular H2O2 levels; suppression of pERK1/2, Slug and Vimentin expression; and inhibition of tumour growth and lung metastasis in vivo. Conversely, HK2 overexpression promoted cell migration and invasion, increased SOD2 activity and intracellular H2O2, and enhanced expression of pERK1/2, Slug and Vimentin. Thus, our results demonstrate that deregulation of HK2 expression has an important function in the progression of TSCC and may serve as a biomarker of its metastatic potential in TSCC patients. HK2 enhances the metastatic potential of TSCC by stimulating the SOD2-H2O2 pathway.