lncRNA RPSAP52 induced the development of tongue squamous cell carcinomas via miR-423-5p/MYBL2

Growing lncRNAs have been noted to involve in the initiation and development of several tumours including tongue squamous cell carcinomas (TSCCs). However, the biological role and mechanism of lncRNA RPSAP52 were not well-explained. We indicated that RPSAP52 was higher in TSCC samples compared with that in control samples. The higher expression of RPSAP52 was positively correlated with higher T stage and TNM stage. Ectopic expression of RPSAP52 induced TSCC cell growth and cycle and induced cytokine secretion including IFN-γ, IL-1β and IL-6, IL-8, IL-10 and TGF-β. We found that the overexpression of RPSAP52 suppressed miR-423-5p expression in SCC-4 cell. miR-423-5p was lower in TSCC samples compared with that in control samples, and miR-423-5p level was negatively correlated with higher T stage and TNM stage. Pearson's correlation indicated that miR-423-5p was negatively associated with that of RPSAP52 in TSCC tissues. Furthermore, MYBL2 was one direct gene of miR-423-5p and elevated expression of miR-423-5p suppressed MYBL2 expression and ectopic expression of RPSAP52 increased MYBL2 expression in SCC-4 cell. Finally, we illustrated that RPSAP52 overexpression promoted TSCC cell growth and cycle and induced cytokine secretion including IFN-γ, IL-1β and IL-6, IL-8, IL-10 and TGF-β via modulating MYBL2. These data provided new insight into RPSAP52, which may be one potential treatment target for TSCC.     

miR-19a and miR-424 target TGFBR3 to promote epithelial-to-mesenchymal transition and migration of tongue squamous cell carcinoma cells

Previous studies indicate that TGFBR3 (transforming growth factor type III receptor, also known as betaglycan), a novel suppressor of progression in certain cancers, is down-regulated in tongue squamous cell carcinoma (TSCC). However, the role of this factor as an upstream regulator in TSCC cells remains to be elucidated. The present study was designed to elucidate whether TGFBR3 gene expression is regulated by two microRNA molecules, miR-19a and miR-424. The study also aimed to determine if these microRNAs promote migration of CAL-27 human oral squamous cells. Immunohistochemistry (IHC) and western blot analyses demonstrated that TGFBR3 protein levels were dramatically down-regulated in clinical TSCC specimens. Conversely, bioinformatics analyses and qRT-PCR results confirmed that both miR-19a and miR-424 were markedly up-regulated in clinical TSCC specimens. In this study, we observed that transfection of a TGFBR3-containing plasmid dramatically inhibited epithelial-to-mesenchymal transition (EMT) and migration in CAL-27 cells. Co-immunoprecipitation analyses also revealed that TGFBR3 forms a complex with the β-arrestin 2 scaffolding protein and IκBα. Furthermore, overexpression of TGFBR3 decreased p-p65 expression and increased IκBα expression; these effects were subsequently abolished following knockdown of β-arrestin 2. Moreover, over-expression of miR-19a and miR-424 promoted migration and EMT in CAL-27 cells. We also observed that the promotion of EMT by miR-19a and miR-424 was mediated by the inhibition of TGFBR3. Our study provides evidence that miR-19a and miR-424 play important roles in the development of TSCC. These results expand our understanding of TGFBR3 gene expression and regulatory mechanisms pertaining to miRNAs.     

IFI16 restoration in hepatocellular carcinoma induces tumour inhibition via activation of p53 signals and inflammasome

Objective

Interferon‐inducible 16 (IFI16) is known to involve in p53‐dependent tumour suppression and also the formation of inflammasome, which function, however, remains controversy during carcinogenesis as a pattern recognition receptor for tumour death‐derived free DNA. In this study, we investigated the anti‐tumour role of IFI16 in hepatocellular carcinoma (HCC).

Materials and methods

Hepatocellular carcinoma tissues (n = 20) and corresponding non‐neoplastic tissues (n = 20) were collected to determine the expression of IFI16. After the transfection of pcDNA3.1‐IFI16 into Huh7 and SMMC7721 cells in vitro, the influence of IFI16 overexpression on cell vitality, colony formation, apoptosis and migration were analysed. The role effect of IFI16 in vivo was further investigated.

Results

The expression of IFI16 was significantly decreased in tumour tissues and cell lines. Overexpression of IFI16 induced decrease of cell vitality, colony formation and increased apoptosis with impaired ability of migration. Mechanistically, IFI16 could activate p53 at Ser15 to up‐regulate the p21^WAF1/CIP1 level to inhibit tumour growth and migration, which was restored by the p53 inhibitor Pifithrin‐α (20 μmol/L). Moreover, IFI16‐induced tumour cell death promoted the recruitment of inflammasome complex to enhance tumour inhibition, but the caspase‐1 inhibitor Ac‐YVAD‐CMK (50 μmol/L) could suppress this process in HCC. The results in vivo indicated that restored expression of IFI16 in tumour cells effectively promote tumour regression, which could be partly abrogated by the inhibition of activation of p53 signals or induced inflammasome.

Conclusion

IFI16 is a tumour suppressor in HCC via activation of p53 signals and inflammasome.

     

IFI16 is an essential mediator of growth inhibition, but not differentiation, induced by the leukemia inhibitory factor/JAK/STAT pathway in medullary thyroid carcinoma cells

Activation of Ras or Raf in the human medullary thyroid carcinoma (MTC) cell line, TT, induces growth arrest and differentiation via two parallel, yet independent, pathways. One of these pathways is intracellular and the other is a cell-extrinsic, autocrine/paracrine pathway mediated by the leukemia inhibitory factor (LIF)/JAK/STAT pathway. Here, we show that IFI16 is a necessary and sufficient downstream effector for LIF effects in MTC cells, specifically required for the LIF/JAK/STAT pathway-induced growth inhibition in these cells. IFI16 was induced by Raf or LIF. Dominant-negative STAT3 could block the induction, indicating that Raf can induce IFI16 only via the cell-extrinsic pathway. Knock-down of IFI16 using siRNA abrogated LIF-induced changes in cellular levels of E2F1, cyclin D1, and p21WAF/CIP1, and cell cycle arrest. In addition, adenovirus-mediated overexpression of IFI16 was sufficient to induce growth arrest. In contrast to its essential role for LIF-mediated growth arrest, IFI16 was not required for differentiation induced by LIF. Knock-down of IFI16 could not block changes in differentiation markers of the MTC cells, including calcitonin, RET, and cell morphology. Our study identifies IFI16 as an essential growth-specific effector of the cell-extrinsic growth inhibitory pathway of Ras/Raf signaling in MTC cells.     

Overexpression of transforming growth factor type?III receptor restores TGF-β1 sensitivity in human tongue squamous cell carcinoma cells

The transforming growth factor type III receptor (TβRIII), also known as β-glycan, is a multi-functional sensor that regulates growth, migration and apoptosis in most cancer cells. We hereby investigated the expression of TβRIII in clinical specimens of tongue squamous cell carcinoma (TSCC) and the underlying mechanism that TβRIII inhibits the growth of CAL-27 human oral squamous cells. The TSCC tissues showed a significant decrease in TβRIII protein expression as detected by immunohistochemistry (IHC) and western blot analysis. Transfection of TβRIII-containing plasmid DNA dramatically promoted TGF-β1 (10 ng/ml)-induced decrease in cell viability, apoptosis and cell arrest at the G₀-/G₁-phase. Moreover, transient overexpression of TβRIII enhanced the TGF-β1-induced cyclin-dependent kinase inhibitor 2b (CDKN2b) and p38 protein activity, but did not affect the activities of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase 1/2 (JNK1/2) in CAL-27 cells. These results suggest overexpression of TβRIII receptor restored TGF-β1 sensitivity in CAL-27 cells, which may provide some new insights on exploiting this molecule therapeutically.     

miR-181a–Twist1 pathway in the chemoresistance of tongue squamous cell carcinoma

Although many researches have been undertaken to disclose the mechanisms of chemoresistance, the mechanisms remain unclear. The aim of this study is to elucidate the role of miR-181a–Twist1 pathway in the chemoresistance of tongue squamous cell carcinoma (TSCC). We found that cisplatin-induced chemoresistance in TSCC cell lines underwent EMT (epithelial–mesenchymal transition) and was accompanied by enhancing metastatic potential (migration and invasion in vitro), miR-181a downregulation and Twist1 upregulation. Functional analyses indicated that miR-181a reversed chemoresistance, inhibited EMT and metastatic potential in TSCC cells. Twist1 was confirmed as a direct miR-181a target gene by luciferase reporter gene assays. Twist1 knockdown by siRNA led to a reversal of the chemoresistance, inhibited EMT and metastatic potential in TSCC cells. Our study demonstrates that miR-181a–Twist1 pathway may play an important role in the development of cisplatin-chemoresistance, with EMT and an increase the metastatic potential of TSCC cells.     

LncRNA GACAT1 induces tongue squamous cell carcinoma migration and proliferation via miR-149

Recent studies have observed that lncRNAs (long non-coding RNAs) are involved in the progression of various tumours including tongue squamous cell carcinoma (TSCC). Recently, a new lnRNA, GACAT1, has been firstly identified in gastric cancer. However, its potential role in TSCC remains unknown. In this reference, we observed that GACAT1 was overexpressed in TSCC samples and cell lines. Of 25 TSCC specimens, GACAT1 expression was overexpressed in 18 patients (18/25, 72%) compared to non-tumour specimens. Ectopic expression of GACAT1 induced cell growth and migration and promoted epithelial to mesenchymal transition in TSCC. In addition, ectopic expression of GACAT1 decreased miR-149 expression in SCC1 cell. We observed that miR-149 expression was down-regulated in TSCC cell lines. Moreover, we observed that GACAT1 expression was negatively correlated with miR-149 expression. GACAT1 overexpression induced TSCC cell growth and migration via regulating miR-149 expression. These data provided that GACAT1 played an oncogenic role in the progression of TSCC partly through modulating miR-149 expression.     

LUCAT1 as an oncogene in tongue squamous cell carcinoma by targeting miR-375 expression

Emerging studies suggested that lncRNAs play a crucial molecular role in cancer development and progression. LncRNA LUCAT1 has been proved as oncogenic molecular in lung cancer, glioma, osteosarcoma, renal carcinoma and oesophageal squamous cell carcinoma. However, its roles and function mechanisms in tongue squamous cell carcinoma (TSCC) are still unknown. We showed that the expression of LUCAT1 was up-regulated in the TSCC cells and tissues and the higher LUCAT1 expression was associated with the poor overall survival (OS). Knockdown expression of LUCAT1 suppressed TSCC cell proliferation, cycle and migration. In addition, we demonstrated that miR-375 overexpression inhibited the luciferase activity of LUCAT1 wild-type and knockdown LUCAT1 promoted the miR-375 expression in TSCC cell. Furthermore, we indicated that miR-375 expression was down-regulated in the TSCC cell lines and tissues and the lower expression of miR-375 was associated with poor OS. The expression of miR-375 was inversely correlated with LUCAT1 expression in the TSCC tissues. Knockdown LUCAT1 promoted TSCC cell proliferation, cell cycle and migration partly through regulating miR-375 expression. In summary, this study suggested the tumorigenic effect of lncRNA LUCAT1 in TSCC cells by targeting miR-375 expression.     

Long noncoding RNA LINC00961 inhibited cell proliferation and invasion through regulating the Wnt/β-catenin signaling pathway in tongue squamous cell carcinoma

Tongue squamous cell carcinoma (TSCC) is the most frequent style of oral squamous cell carcinoma. However, the molecular mechanisms and function of LINC00961 in the TSCC progression remain unknown. In this study, we proved that LINC00961 expression was downregulated in TSCC cells (Tca8113, SCC1, SCC-4, and SCC-15) compared with normal tissue. In addition, we showed that LINC00961 expression was downregulated in TSCC samples compared with matched normal tissues. Moreover, ectopic expression of LINC00961 decreased TSCC cell growth and invasion and suppressed epithelial-mesenchymal transition in TSCC cell. Furthermore, we indicated that overexpression of LINC00961 decreased β-catenin expression. Knockdown of LINC00961 promoted cell proliferation and invasion partly via promoting the Wnt/β-catenin signaling pathway. These results suggested that LINC00961 was downregulated in TSCC tissues and acted as a tumor suppressor gene in the development of TSCC.     

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.     

The interferon-inducible IFI16 gene inhibits tube morphogenesis and proliferation of primary, but not HPV16 E6/E7-immortalized human endothelial cells

Immunohistochemical analysis has demonstrated that the human IFI16 gene, in addition to the hematopoietic tissues, is highly expressed in endothelial cells and squamous stratified epithelia. In this study, we have developed a reliable HSV-derived replication-defective vector (TO-IFI16) to efficiently transduce IFI16 into primary human umbilical vein endothelial cells (HUVEC), which are usually poorly transfectable. HUVEC infection with TO-IFI16 virus suppressed endothelial migration, invasion and formation of capillary-like structures in vitro. In parallel, sustained IFI16 expression inhibited HUVEC cell cycle progression, accompanied by significant induction of p53, p21, and hypophosphorylated pRb. Further support for the involvement of these pathways in IFI16 activity came from the finding that infection with TO-IFI16 virus does not impair the in vitro angiogenic activity and cell cycle progression of HUVEC immortalized by HPV16 E6/E7 oncogenes, which are known to inactivate both p53 and pRb systems. This use of a reliable viral system for gene delivery into primary human endothelial cells assigns a potent angiostatic activity to an IFN-inducible gene, namely IFI16, and thus throws further light on antiangiogenic therapy employing IFNs.     

Anti-tumor effect of carrimycin on oral squamous cell carcinoma cells in vitro and in vivo

Purpose: Carrimycin is a newly synthesized macrolide antibiotic with good antibacterial effect. Exploratory experiments found its function in regulating cell physiology, proliferation and immunity, suggesting its potential anti-tumor capacity. The aim of this study is to investigate the anti-tumor effect of carrimycin against human oral squamous cell carcinoma cells in vitro and in vivo.Methods: Human oral squamous cell carcinoma cells (HN30/HN6/Cal27/HB96 cell lines) were treated with gradient concentration of carrimycin. Cell proliferation, colony formation and migration ability were analyzed. Cell cycle and apoptosis were assessed by flow cytometry. The effect of carrimycin on OSCC in vivo was investigated in tumor xenograft models. Immunohistochemistry, western blot assay and TUNEL assays of tissue samples from xenografts were performed. The key proteins in PI3K/AKT/mTOR pathway and MAPK pathway were examined by western blot.Results: As the concentration of carrimycin increased, the proliferation, colony formation and migration ability of OSCC cells were inhibited. After treating with carrimycin, cell cycle was arrested in G0/G1 phase and cell apoptosis was promoted. The tumor growth of xenografts was significantly suppressed. Furthermore, the expression of p-PI3K, p-AKT, p-mTOR, p-S6K, p-4EBP1, p-ERK and p-p38 were down-regulated in vitro and in vivo.Conclusions: Carrimycin can inhibit the biological activities of OSCC cells in vitro and in vivo, and regulate the PI3K/AKT/mTOR and MAPK pathways.     

siRNA enhances DNA-mediated interferon lambda-1 response through crosstalk between RIG-I and IFI16 signalling pathway

In addition to silencing specific genes, small interfering RNA (siRNA) transfection is also associated with the non-specific induction of inflammatory cytokines and type I interferon. Those so-called “off-target” effects have considerable implications for the interpretation of in vitro studies and clinical application of siRNA. The present study attempted to develop a better understanding of the mechanism involved in these off target effects. Synthesized siRNA significantly enhances DNA-mediated interferon lambda-1 response (IFN-λ1/IL-29), a newly characterized antiviral interferon in non-immune or primary immune cells. This enhancement was most pronounced by double-stranded siRNA with at least a 2-nucleotide overhang at one 3′ terminus in a dose-dependent manner, while the presence of DNA was indispensable. A pull-down assay using biotinylated siRNA- or DNA-conjugated beads indicated that retinoic acid-inducible gene I (RIG-I) and interferon gamma-inducible protein 16 (IFI16) were involved in the sensing of siRNA and DNA, respectively. Co-immunoprecipitation analysis further revealed that RIG-I and IFI16 formed a complex via siRNA, and the dissociation of IFI16 from this complex in the presence of DNA activated the downstream STING-TBK1-IRF3 (stimulator of interferon genes – tank-binding kinase 1 – interferon regulatory factor 3) pathway, shedding light on a new physiological signalling pathway to activate innate immunity. Collectively, these findings may provide rational information for siRNA-induced innate immunity, with important implications for developing siRNA-based reagents to control human diseases.     

IFI6 Inhibits Apoptosis via Mitochondrial-Dependent Pathway in Dengue Virus 2 Infected Vascular Endothelial Cells

Dengue hemorrhagic fever (DHF)/Dengue shock syndrome (DSS) is a fatal infectious disease that demands an effective treatment. Interferon (IFN)-stimulated genes (ISGs) induced by dengue virus (DENV) exert antiviral effects. Among ISGs, IFN-α inducible gene 6 (IFI6) was increased in DENV infected human umbilical vascular endothelial cells (HUVECs) by microarray analysis in our previous study. However, its function is incompletely understood. In this study, we confirmed that IFI6 was markedly induced in DENV infection of both primary HUVECs and EA.hy926 cell lines. Recombinant EA.hy926 cell lines in which IFI6 was either over-expressed (IFI6+/+) or knocked-down (IFI6-/-) were generated. The activation of caspase-3 and intrinsic apoptosis-related protein caspase-9 were down-regulated in IFI6+/+ but up-regulated in IFI6-/- cells at 24–48 hrs post-infection. After incubation with DENV for 48 hrs, the mitochondrial membrane potential (Δψ(m)) was more stable in IFI6+/+ cells but reduced in IFI6-/- cells, as assayed by fluorescence staining with JC-1. We observed that Bcl-2 expression was increased in IFI6+/+ and decreased in IFI6-/- cells. By contrast, Bax expression was decreased in IFI6+/+ and increased in IFI6-/- cells. It is presumed that the anti-apoptotic function of IFI6 is expressed by regulating the rheostatic balance between bcl-2/bax expression and inhibition of Δψ(m) depolarization during DENV infection of vascular endothelial cells(VECs). In addition, the pro-apoptotic protein X-linked Inhibitor of Apoptosis (XIAP)-Associated Factor 1(XAF1) expression had been reported to be up-regulated and led to the induction of apoptosis in DENV2-infected VECs,but the relationship between XAF1 and IFI6 dengue virus-induced apoptosis in VECs warrants further study.