Dental pulp cells cocultured with macrophages aggravate the inflammatory conditions stimulated by LPS.

BACKGROUND: Pulp inflammation is complex interactions between different types of cells and cytokines. To mimic the interactions of different types of cells in inflamed dental pulp tissues, dental pulp cells (DPCs) were cocultured with different ratios of macrophages (THP-1) or LPS treatment. METHODS: DPCs were cocultured with various ratios of THP-1, then photographed cell morphology and determined cell viability by MTT assay at preset times. Total RNA was also extracted to measure the inflammation marker-IL-6 and IL-8 expressions by RT-Q-PCR. The DPCs and THP-1 were treated with 0.01 - 1µg/ml lipopolysaccharide (LPS) and extract RNA at preset times, and detected IL-6 and IL-8 expression. DPCs were cocultured with various ratios of THP-1 with 0.1 µg/mL LPS, and detected IL-6 and IL-8 expression after 24 and 48 h. The data were analyzed by unpaired t-test or Mann-Whitney test. Differences were considered statistically significant when p < 0.05. RESULTS: THP-1 and DPCs coculture models did not suppress the viability of DPCs and THP-1. Cocultured with various ratios of THP-1 could increase IL-6 and IL-8 expressions of DPCs (p = 0.0056 - p < 0.0001). The expressions of IL-6 and IL-8 were stronger in higher ratio groups (p = 0.0062 - p < 0.0001). LPS treatment also induced IL-6 and IL-8 expressions of DPCs and THP-1 (p = 0.0179 - p < 0.0001 and p = 0.0189 - p < 0.0001, separately). Under the presence of 0.1 µg/mL LPS, DPCs cocultured with THP-1 for 24 h also enhanced IL-6 and IL-8 expression (p = 0.0022). After cocultured with a higher ratio of THP-1 for 48 h, IL-6 and IL-8 expressions were even stronger in the presence of LPS (p = 0.0260). CONCLUSIONS: Coculturing dental pulp cells and macrophages under LPS treatment aggravate the inflammatory process. The responses of our models were more severe than traditional inflamed dental models and better represented what happened in the real dental pulp. Utilizing our models to explore the repair and regeneration in endodontics will be future goals.

Biodentine but not MTA induce DSPP expression of dental pulp cells with different severity of LPS-induced inflammation.

OBJECTIVES: To explore the inflammatory and differentiation response in inflamed dental pulp cells (DPCs) induced by lipopolysaccharide (LPS) under different conditions with Biodentine and mineral trioxide aggregate (MTA) treatment. MATERIALS AND METHODS: DPCs were treated with 0.001-1 µg/mL LPS for different periods to induce inflammation. Normal and inflamed DPCs were further treated with 0.14 mg/mL Biodentine or 0.13 mg/mL MTA for different periods. mRNA expression level of IL-6, IL-8 and ALP were analysed by qPCR. DSPP protein expression was detected by western blot. The data were analysed by the Mann-Whitney test, unpaired t test or two-way ANOVA. RESULTS: After treatment for different times and with different concentrations of LPS, different severity of pulp inflammation was revealed by the expressions of IL-6 and IL-8. Higher concentrations of LPS induced higher IL-6 and IL-8 expressions, and these expressions first increased and then decreased (p < 0.0001). At 96 and 192 h, Biodentine significantly suppressed IL-6 expression in both normal and inflamed DPCs (p < 0.05). At 48 and 96 h, Biodentine suppressed ALP expression in both normal and inflamed DPCs (p < 0.05). At 48 and 96 h, Biodentine induced DSPP expressions in both normal and inflamed DPCs (p < 0.05). CONCLUSION: Biodentine enhanced more DSPP differentiation of both normal and inflamed DPCs under different treatment durations than MTA. CLINICAL RELEVANCE: The prognosis of vital pulp therapy may depend on the severity of pulp inflammation which is difficult to be determined in clinical settings. Therefore, Biodentine may enhance odontogenic differentiation in different severity of pulp inflammation imply its clinical indications.

miR-187* Enhances SiHa Cervical Cancer Cell Oncogenicity Via Suppression of WWOX.

BACKGROUND/AIM: Cervical cancer is one of the most common cancers worldwide and a major cause of cancer-related mortality among women. Previous studies have reported that microRNA-miR-187*, which is one of the non-coding parts of the genome producing small conserved ribonucleic acids, is associated with various cancers. In this study, we explored the function of miR-187* in cervical cancer cells. MATERIALS AND METHODS: miR-187* mimic, WWOX reporter constructs, siRNA and overexpression constructs were transfected into SiHa cells to investigate the function and regulatory mechanisms of miR-187*. RESULTS: Exogenous miR-187* was found to increase the oncogenic phenotypes of SiHa cells. The tumor suppressor gene WWOX is a novel target of miR-187* in SiHa cells. WWOX siRNA suppressed endogenous WWOX expression and increased the oncogenic phenotypes of SiHa cells. Exogenous WWOX expression was able to suppress the oncogenic phenotypes of SiHa cells and rescue cells from miR-187*-induced migration. CONCLUSION: miR-187* seems to enhance SiHa cervical cancer cell oncogenicity via suppression of the WWOX pathway.

miR-376c promotes carcinogenesis and serves as a plasma marker for gastric carcinoma.

Gastric carcinoma is highly prevalent throughout the world. Understanding the pathogenesis of this disease will benefit diagnosis and resolution. Studies show that miRNAs are involved in the tumorigenesis of gastric carcinoma. An initial screening followed by subsequent validation identified that miR-376c is up-regulated in gastric carcinoma tissue and the plasma of patients with the disease. In addition, the urinary level of miR-376c is also significantly increased in gastric carcinoma patients. The plasma miR-376c level was validated as a biomarker for gastric carcinoma, including early stage tumors. The induction of miR-376c was found to enrich the proliferation, migration and anchorage-independent growth of carcinoma cells and, furthermore, the repression of the expression of endogenous miR-376c was able to reduce such oncogenic phenotypes. ARID4A gene is a direct target of miR-376c. Knockdown of endogenous ARID4A increased the oncogenicity of carcinoma cells, while ARID4A was found to be drastically down-regulated in tumor tissue. Thus, expression levels of miR-376c and ARID4A mRNA tended to be opposing in tumor tissue. Our results demonstrate that miR-376c functions by suppressing ARID4A expression, which in turn enhances the oncogenicity of gastric carcinoma cells. It seems likely that the level of miR-376c in plasma and urine could act as invaluable markers for the detection of gastric carcinoma.

miR-31 is upregulated in oral premalignant epithelium and contributes to the immortalization of normal oral keratinocytes.

Oral squamous cell carcinoma (OSCC) is a prevalent malignancy worldwide. MicroRNAs are short non-coding RNAs that regulate gene expression and are crucial for tumorigenesis. Previously, we have identified that miR-31 is frequently upregulated in OSCC and that this miR-31 increase, together with downstream effector modulation, enhances oral carcinogenesis. We have identified higher levels of miR-31 expression in oral potential malignant disorder (OPMD) tissues compared with normal oral mucosa. Exogenous miR-31 and human telomerase reverse transcriptase (hTERT) expression were introduced into cultured normal oral keratinocytes (NOKs), which led to the immortalization; these lines were designated M31OK1 and M31OK3. These immortalized lines maintained the capability to undergo squamous differentiation. In addition, migration by both cell lines was attenuated by hTERT and miR-31 knockdown. M31OK1 carries a p53 gene mutation at codon 273. A serum-tolerant subline, M31OK1-D, exhibits potent anchorage-independent growth that is attenuated by knockdown of both hTERT and miR-31. miR-31-targeted factors inhibiting HIF (FIH), which upregulated vascular endothelial growth factor (VEGF), was found crucial for oral tumorigenesis. The proliferation, migration and epithelial-mesenchymal transition of M31OK1-D are associated with downregulation of FIH and upregulation of VEGF, which require miR-31 expression. High miR-31 expression is correlated with higher VEGF expression and lower E-cadherin expression in OPMD tissue. It can be concluded that miR-31 collaborates with hTERT to immortalize NOKs and that this may contribute to early stage oral carcinogenesis. The targeting of downstream factors by miR-31 may further advance the neoplastic progression of immortalized NOKs, allowing them to become malignant.

Lipopolysaccharide induces the migration of human dental pulp cells by up-regulating miR-146a.

INTRODUCTION: MicroRNAs are small noncoding RNAs that play crucial roles in regulating normal and pathologic functions. Bacterial lipopolysaccharide (LPS) is one of the key regulators of pulpal pathogenesis. This study investigated how LPS regulates microRNA expression and affects the phenotype of human dental pulp cells (DPCs). METHODS: Primary DPCs were established and immortalized to achieve immortalized DPCs (I-DPCs). DPCs and I-DPCs were treated with LPS and examined to identify changes in microRNA expression, cell proliferation, and cell migration. Quantitative reverse-transcriptase polymerase chain reaction was used to detect changes in gene expression. Exogenous miR-146a expression was performed transfection with pre-mir-146a mimic. Knockdown of interleukin receptor-associated kinase (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6) expression was performed by small interference oligonucleotide transfection. Western blot analysis was used to detect changes in the expression of the IRAK1 and TRAF6 proteins. RESULTS: The differentiation of DPCs was induced by osteogenic medium. I-DPCs had a higher level of human telomerase reverse transcriptase gene than the parental DPCs. Up-regulation of miR-146a expression and an increase in migration was induced by LPS treatment of DPCs and I-DPCs. Exogenous miR-146a expression increased the migration of DPCs and I-DPCs and down-regulated the expression of IRAK1 and TRAF6. Knockdown of IRAK1 and/or TRAF6 increased the migration of DPCs. CONCLUSIONS: The results suggested that LPS is able to increase the migration of DPCs by modulating the miR-146a-TRAF6/IRAK1 regulatory cascade.

miR-181 as a putative biomarker for lymph-node metastasis of oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is an important malignant disease around the world. Aberrant expression of MicroRNAs (miRNAs) has been implicated in carcinogenesis of various cancers. In previous studies, up-regulation of miR-181 was observed when OSCC progressed from leukoplakia, dysplasia to invasive carcinoma. However, the function of miR-181 in oral tumorigenesis remains unclear. MATERIALS AND METHODS: The expression levels of miR-181 in the tissue and plasma of OSCC patients were measured by quantitative RT-PCR. The correlation between miR-181 level and multiple clinical variables were then checked by Mann-Whitney test and Wilcoxon matched pairs test. To study the functional meaning of up-regulated miR-181, migration assay and invasion assay by transwells and colony forming assay were applied to analyze the tumorigenic phenotypes of OSCC cells with ectopical expression of miR-181. RESULTS: Among different clinical variables, over-expression of miR-181 was correlated with lymph-node metastasis, vascular invasion, and a poor survival. Functional assays revealed ectopically over-expressed miR-181 would enhance cell migration and invasion, but not the ability of anchorage-independent growth of OSCC cells. In addition, the up-regulation of miR-181 could be detected both in tumor tissues and plasma. CONCLUSION: miR-181 may enhance lymph-node metastasis through regulating migration, which could potentially be exploited as a putative biomarker for patients with OSCC.

miR-24 up-regulation in oral carcinoma: positive association from clinical and in vitro analysis.

MicroRNAs (miRNAs) play important roles in neoplastic process. miR-24 is localized on chromosome 9q22 and 19p13, regions frequently altered in oral squamous cell carcinoma (OSCC). This study showed that miR-24 was up-regulated in OSCC tissues relative to control samples. In addition, the plasma levels of miR-24 in OSCC patients were significantly higher than in control individuals. miR-24 expression was also higher in OSCC cell lines relative to normal oral keratinocytes. Experiments blocking miR-24 and using exogenous miR-24 expression indicated that miR-24 contributes to the growth of OSCC cells and that miR-24 may target p57. This study suggests that miR-24 is involved in the regulation of OSCC growth and that the miR-24's level in plasma may be validatable as a tumor marker for OSCC patients.

miR-31 ablates expression of the HIF regulatory factor FIH to activate the HIF pathway in head and neck carcinoma.

MicroRNAs (miRNA) are endogenously expressed noncoding RNAs with important biological and pathological functions that are yet to be fully defined. This study investigated alterations in miRNA expression in head and neck squamous cell carcinoma (HNSCC), the incidence of which is rising throughout the world. Initial screening and subsequent analysis identified a panel of aberrantly expressed miRNAs in HNSCC tissues, with miR-31 among the most markedly upregulated. Ectopic expression of miR-31 increased the oncogenic potential of HNSCC cells under normoxic conditions in cell culture or tumor xenografts. Conversely, blocking miR-31 expression reduced the growth of tumor xenografts. The in silico analysis suggested that miR-31 may target the 3' untranslated region (UTR) of factor-inhibiting hypoxia-inducible factor (FIH), a hypoxia-inducible factor (HIF) regulatory factor that inhibits the ability of HIF to act as a transcriptional regulator under normoxic conditions. In support of this likelihood, miR-31 expression repressed FIH expression and mutations within the predictive miR-31 target site in the FIH 3' UTR abrogated FIH repression. Furthermore, miR-31 expression increased HIF transactivation activity. We found that FIH suppressed oncogenic phenotypes under normoxic conditions and that this activity was abrogated by functional mutations. Lastly, increased miR-31 expression was correlated with decreased levels of FIH in tumor tissues. Our findings suggest that miR-31 contributes to the development of HNSCC by impeding FIH to activate HIF under normoxic conditions.

Curcumin upregulates insulin-like growth factor binding protein-5 (IGFBP-5) and C/EBPalpha during oral cancer suppression.

Curcumin is a common food ingredient derived from the plant Curcuma longa and is a potent drug against tumorigenesis. Both insulin-like growth factor binding protein-5 (IGFBP-5) and CCAAT/enhancer-binding protein alpha (C/EBPalpha) are suppressors of head and neck carcinogenesis. We identified curcumin as an inducer of IGFBP-5 expression in multiple types of oral keratinocytes; furthermore, curcumin induces IGFBP-5 promoter activity in SAS oral cancer cells. Promoter deletion mapping identified a region (nt -71 to nt -59 relative to the transcription start site) as containing a C/EBPalpha-binding element that is indispensable for curcumin-mediated IGFBP-5 upregulation. Chromatin immunoprecipitation assays revealed that in vivo binding of C/EBPalpha to this region was remarkably increased in the presence of curcumin. Curcumin increased nuclear C/EBPalpha expression and IGFBP-5 expression through p38 activation and this was abrogated by SB203580 treatment. Furthermore, MKK6 expression activated p38 and C/EBPalpha, increasing IGFBP-5 promoter activity and expression. Finally, curcumin-induced IGFBP-5 expression is associated with the suppression of xenograft tumorigenesis in mice due to oral cancer cells. We conclude that curcumin activates p38, which, in turn, activates the C/EBPalpha transactivator by interacting with binding elements in the IGFBP-5 promoter. The consequential upregulation of C/EBPalpha and IGFBP-5 by curcumin is crucial to the suppression of oral carcinogenesis.

Cytotoxicity of phenolic acid phenethyl esters on oral cancer cells.

Many phenolic acid phenethyl esters possess diverse biological effects including anti-cancer activity. A series of 14 derivatives were synthesized for the evaluation of their cytotoxic effect on oral cancer cells. These derivatives were tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric and trypan blue dye exclusion assay on the growth of oral squamous cell carcinoma (SAS), oral epidermoid carcinoma-Meng 1 (OEC-M1), and normal human oral fibroblast (NHOF) cells, respectively. Caffeic acid phenethyl esters, 3a (CAPE), and 3b, 3c, and 3d showed cytotoxic effects on the SAS and OEC-M1 cell lines, but not the NHOF cell line at a 5-100 microM dose range. Flow cytometric analysis showed that 3c caused OEC-M1 cell arrest at G2/M phase. Such differential effects on representative cancer and normal cells suggested these compounds might be useful in oral cancer chemotherapy.