Low-dose arecoline regulates distinct core signaling pathways in oral submucous fibrosis and oral squamous cell carcinoma.

BACKGROUND: Betel nut chewing plays a role in the pathogenesis of oral submucous fibrosis (OSF) and oral squamous cell carcinoma (OSCC). As the major active ingredient of the betel nut, the effect of arecoline and its underlying mechanism to OSF and OSCC pathogenesis remain unclear. METHODS: Next-generation sequencing-based transcriptome and dRRBS analysis were performed on OSF and OSCC cells under low-dose arecoline exposure. Functional analyses were performed to compare the different roles of arecoline during OSF and OSCC pathogenesis, and key genes were identified. RESULTS: In this study, we identified that low-dose arecoline promoted cell proliferation of both NFs and OSCC cells via the acceleration of cell cycle progression, while high-dose arecoline was cytotoxic to both NFs and OSCC cells. We performed for the first time the transcriptome and methylome landscapes of NFs and OSCC cells under low-dose arecoline exposure. We found distinct transcriptome and methylome profiles mediated by low-dose arecoline in OSF and OSCC cells, as well as specific genes and signaling pathways associated with metabolic disorders induced by low-dose arecoline exposure. Additionally, low-dose arecoline displayed different functions at different stages, participating in the modulation of the extracellular matrix via Wnt signaling in NFs and epigenetic regulation in OSCC cells. After exposure to low-dose arecoline, the node roles of FMOD in NFs and histone gene clusters in OSCC cells were found. Meanwhile, some key methylated genes induced by arecoline were also identified, like PTPRM and FOXD3 in NFs, SALL3 and IRF8 in OSCC cells, indicating early molecular events mediated by arecoline during OSF and OSCC pathogenesis. CONCLUSIONS: This study elucidated the contribution of low-dose arecoline to OSF and OSCC pathogenesis and identified key molecular events that could be targeted for further functional studies and their potential as biomarkers.

UCHL1 promotes proliferation and metastasis in head and neck squamous cell carcinoma and could be a potential therapeutic target.

OBJECTIVE: The purpose of this study was to research the physiological roles of ubiquitin carboxyl-terminal esterase L1 (UCHL1) in head and neck squamous cell carcinoma (HNSCC). STUDY DESIGN: Ten HNSCC samples and matched normal oral mucosal tissues were collected. UCHL1 expression of these tissues was detected by the immunohistochemical staining and real-time quantitative polymerase chain reaction. The human HNSCC cell line HN6 UCHL1 knockout (UCHL1 KO) cell line was constructed using CRISPR/CAS9 gene editing and verified by western blotting. Wound healing assay, cell proliferation assay, cell invasion assay, and flow cytometric analysis of the cell cycle and apoptosis were applied to research the role of UCHL1 in HNSCC. Also, an RNAseq gene expression data set and HNSCC patient survival data from The Cancer Genome Atlas were analyzed. RESULTS: UCHL1 was highly expressed in HNSCC tissues compared with normal oral mucosal tissues (P = .032). A decreased proliferation (P < .0001), migration (P < .0001), and invasion (P = .0049) ability of HN6 cells was exhibited after knockout of UCHL1. However, HN6 UCHL1 KO cells showed no significant differences in the cell cycle or apoptosis. The progression, nodal metastasis status, and stage of HNSCC had a positive correlation with the expression of UCHL1. CONCLUSIONS: UCHL1 plays an important role in HNSCC, and we consider that targeting UCHL1 may be a feasible therapeutic strategy for HNSCC.

Exosome-derived long non-coding RNA ADAMTS9-AS2 suppresses progression of oral submucous fibrosis via AKT signalling pathway.

Oral submucosal fibrosis (OSF) is one of the pre-cancerous lesions of oral squamous cell carcinoma (OSCC). Its malignant rate is increasing, but the mechanism of malignancy is not clear. We previously have elucidated the long non-coding RNA (lncRNA) expression profile during OSF progression at the genome-wide level. However, the role of lncRNA ADAMTS9-AS2 in OSF progression via extracellular communication remains unclear. lncRNA ADAMTS9-AS2 is down-regulated in OSCC tissues compared with OSF and normal mucous tissues. Low ADAMTS9-AS2 expression is associated with poor overall survival. ADAMTS9-AS2 is frequently methylated in OSCC tissues, but not in normal oral mucous and OSF tissues, suggesting tumour-specific methylation. Functional studies reveal that exosomal ADAMTS9-AS2 suppresses OSCC cell growth, migration and invasion in vitro. Mechanistically, exosomal ADAMTS9-AS2 inhibits AKT signalling pathway and regulates epithelial-mesenchymal transition markers. Through profiling miRNA expression profile regulated by exosomal ADAMTS9-AS2, significantly enriched pathways include metabolic pathway, PI3K-Akt signalling pathway and pathways in cancer, indicating that exosomal ADAMTS9-AS2 exerts its functions through interacting with miRNAs during OSF progression. Thus, our findings highlight the crucial role of ADAMTS9-AS2 in the cell microenvironment during OSF carcinogenesis, which is expected to become a marker for early diagnosis of OSCC.

Long Non-Coding RNA Expression Profile Associated with Malignant Progression of Oral Submucous Fibrosis.

Oral submucous fibrosis (OSF) as one of the premalignant disorders endures a series of histopathological stages to invasive oral squamous cell carcinoma (OSCC) eventually. However, the role of long non-coding RNA (lncRNA) expression in OSF malignant progression still remains poorly understood. Through RNA-sequencing normal mucous, OSF and OSCC tissues, we found 687 lncRNA transcripts significantly and differentially expressed during OSF progression, including 231 upregulated lncRNAs and 456 downregulated lncRNAs, indicating that lncRNAs are involved in the regulation of different stages of OSF development. Further functional enrichment analysis showed these differentially expressed lncRNAs participated in inflammation signaling, Wnt signaling, angiogenesis, CCKR signaling, integrin signaling, PDGF signaling, p53 signaling, and EGF receptor (EGFR) signaling pathways, which contribute to inflammatory and fibroelastic pathogenetic changes of OSF and further malignant progression. Five novel lncRNAs were differentially expressed during OSF progression with varied expression levels, indicating the importance of these lncRNAs in OSF malignant development. Moreover, some lncRNAs have been previously identified to be associated with OSCC pathogenesis, including HCG22, RP11-397A16.1, LINC00271, CTD-3179P9.1, and ZNF667-AS1. Thus, our study firstly comprehensively elucidated lncRNAs expression profile of malignant procession from OSF premalignant lesion to OSCC, which will enlighten our understanding of the importance of lncRNA involved in OSF malignant development.

Effects of Postoperative Radiotherapy on Vascularized Nerve Graft for Facial Nerve Repair in a Rabbit Model.

PURPOSE: High fractional dose radiotherapy (RT) produces better radiobiological results. However, a concern always exists regarding radiation-induced damage to normal tissues, in particular, the peripheral nerves. In the present study, we assessed the effects of postoperative RT on vascularized facial nerve grafts in a rabbit model. MATERIALS AND METHODS: Surgical defects were created bilaterally on the upper buccal branches of the facial nerve in rabbits. One side received a vascularized nerve graft (VNG), and the other side received a free nerve graft (FNG). RT was planned in 1 group at 1 month postoperatively. The dose was equivalent to 60 Gy for each side. Evaluation of the facial performance, electrophysiologic monitoring, histologic studies, toluidine blue staining, and scanning electron microcopy were performed at 3 months after RT. RESULTS: In the RT group, the pathological changes included surrounding tissue fibrosis, nerve cell shrinkage, Schwann cell injury, and demyelination. Compared with the control group, postoperative RT had no obvious effect on the regeneration and functional recovery of the facial nerves. The functional recovery rate of the VNG was faster than that of the FNG in the RT group. In addition, the difference in the nerve conduction velocity and amplitude was statistically significant between the 2 groups. CONCLUSIONS: Postoperative RT influenced the functional recovery of facial nerves to a certain degree. The use of VNGs could alleviate the adverse effects of RT on facial nerve regeneration.

Risk factors for postoperative delirium in patients undergoing major head and neck cancer surgery: a meta-analysis.

OBJECTIVE: Postoperative delirium is common after extensive surgery. This study aimed to collate and synthesize published literature on risk factors for delirium in patients with head and neck cancer surgery. METHODS: Three databases were searched (MEDLINE, Embase, and Cochrane Library) between January 1987 and July 2016. The Newcastle Ottawa Scale (NOS) was adopted to evaluate the study quality. Pooled odds ratios or mean differences for individual risk factors were estimated using the Mantel-Haenszel and inverse-variance methods. RESULTS: They provided a total of 1940 patients (286 with delirium and 1654 without), and predominantly included patients undergoing head and neck cancer surgery. The incidence of postoperative delirium ranged from 11.50% to 36.11%. Ten statistically significant risk factors were identified in pooled analysis. Old age, age >70 years, male sex, duration of surgery, history of hypertension, blood transfusions, tracheotomy, American Society of Anesthesiologists physical status grade at least III, flap reconstruction and neck dissection were more likely to sustain delirium after head and neck cancer surgery. CONCLUSION: Delirium is common in patients undergoing major head neck cancer surgery. Several risk factors were consistently associated with postoperative delirium. These factors help to highlight patients at risk of developing delirium and are suitable for preventive action.

Expression pattern of DKK3, dickkopf WNT signaling pathway inhibitor 3, in the malignant progression of oral submucous fibrosis.

Oral squamous cell carcinoma (OSCC) is a common malignant neoplasm of the head and neck. Oral submucous fibrosis (OSF) is one of its pre-cancerous lesions; however, the key molecular events in the tumorigenesis of OSF remain elusive. Dickkopf WNT signaling pathway inhibitor 3 (DKK3) is one of the Wnt antagonists, and its downregulation and methylation have been reported in multiple malignancies, while no report of its expression in the carcinogenesis of OSF exists. In the present study, we investigated DKK3 expression at the protein and mRNA levels by immunochemical staining and semi­quantitative RT-PCR in normal oral, OSF and OSCC tissues. We found that DKK3 was readily expressed in normal oral mucous tissues, but was gradually increased in early, moderately advanced and advanced OSF tissues, and strongly expressed in OSCC tissues. DKK3 was localized in the cytoplasm during OSF progression. A rare mutation of DKK3 was observed in OSCC, along with increased copy numbers. Furthermore, through analysis of its co-expressed genes, DDK3 may deregulate Wnt signaling, p53 signaling, apoptosis, Ca2+ signaling and mitochondrial signaling pathways in OSCC pathogenesis. Thus, our results demonstrated that DKK3 is upregulated in the carcinogenesis of OSF, due to gain of copy number, which could be a potential tumor marker for the early detection of OSCC.

Low kilovoltage peak (kVp) with an adaptive statistical iterative reconstruction algorithm in computed tomography urography: evaluation of image quality and radiation dose.

PURPOSE: The purpose of this study was to evaluate the image quality and radiation dose in computed tomography urography (CTU) images acquired with a low kilovoltage peak (kVp) in combination with an adaptive statistical iterative reconstruction (ASiR) algorithm. METHODS: A total of 45 subjects (18 women, 27 men) who underwent CTU with kV assist software for automatic selection of the optimal kVp were included and divided into two groups (A and B) based on the kVp and image reconstruction algorithm: group A consisted of patients who underwent CTU with a 80 or 100 kVp and whose images were reconstructed with the 50% ASiR algorithm (n=32); group B consisted of patients who underwent CTU with a 120 kVp and whose images were reconstructed with the filtered back projection (FBP) algorithm (n=13). The images were separately reconstructed with volume rendering (VR) and maximum intensity projection (MIP). Finally, the image quality was evaluated using an image score, CT attenuation, image noise, the contrast-to-noise ratio (CNR) of the renal pelvis-to-abdominal visceral fat and the signal-to-noise ratio (SNR) of the renal pelvis. The radiation dose was assessed using volume CT dose index (CTDIvol), dose-length product (DLP) and effective dose (ED). RESULTS: For groups A and B, the subjective image scores for the VR reconstruction images were 3.9±0.4 and 3.8±0.4, respectively, while those for the MIP reconstruction images were 3.8±0.4 and 3.6±0.6, respectively. No significant difference was found (p>0.05) between the two groups' image scores for either the VR or MIP reconstruction images. Additionally, the inter-reviewer image scores did not significantly differ (p>0.05). The mean attenuation of the bilateral renal pelvis in group A was significantly higher than that in group B (271.4±57.6 vs. 221.8±35.3 HU, p<0.05), whereas the image noise in group A was significantly lower than that in group B (7.9±2.1 vs. 10.5±2.3 HU, p<0.05). The CNR and SNR in group A were both significantly higher than those in group B (53.61±24.74 vs. 32.30±6.52 for CNR; 38.13±19.86 vs. 21.76±4.85 for SNR; all p<0.05). The CTDIvol, DLP and ED in group A were significantly lower than those in group B (9.26±2.77 vs. 16.19±5.60 mGy for CTDIvol; 368.86±119.38 vs. 674.38±239.37 mGy×cm-1 for DLP; 5.53±1.79 vs. 10.12±3.59 mSv for ED; all p<0.05). CONCLUSIONS: The low kVp CTU images with 50% ASiR reconstruction exhibit sufficient image quality and facilitate up to a 44% radiation dose reduction.

Approaches of robot-assisted neck dissection for head and neck cancer: a review.

Robot-assisted surgery is being increasingly used by surgeons because of its enhancement of visualization, precision, and articulation compared with conventional minimally invasive techniques. In recent years, robot-assisted neck dissection (RAND) has begun to be used as an alternative method of neck dissection, one of the classic surgical procedures in the area of head and neck surgery. Currently, there are four kinds of approaches for RAND: (1) modified facelift or retroauricular incision, (2) combined transaxillary and retroauricular incision, (3) transaxillary incision, and (4) transoral incision. RAND may help perform minimally invasive surgery and achieve excellent cosmetic results as well as the desired oncologic outcomes, and this requires selecting an appropriate approach based on the different needs of neck dissections. Although experienced surgeons wishing to avoid large cervical incisions in patients can safely perform RAND, there are still quite a few limitations; in particular, surgical morbidity and oncologic outcomes should be verified by further prospective clinical trials with longer follow-up periods. Also, RAND needs to be standardized and its use disseminated. In this review, we introduce the applications of different approaches for RAND and their indications and determine whether RAND can be more beneficial compared with conventional surgeries.

Oropharyngeal reconstruction with a pedicled submandibular gland flap.

Locoregional flaps are widely used for reconstruction of small and medium defects in the oral cavity. The submandibular gland flap is a pedicled flap, which derives its blood supply from the facial artery, based on the submandibular gland. We describe the use of the flap in 20 patients who required oropharyngeal reconstruction with a pedicled submandibular gland flap after resection of a tumour between July 2012 and October 2014. Patients with squamous cell carcinoma were excluded. All flaps were pedicled on the facial vessels (inferiorly in 17 patients and superiorly in 3). The indications were: reconstruction of intraoral mucosal defects (n=13), filling the parapharyngeal dead space (n=6), and obliteration of the mastoid (n=1). All the flaps atrophied, but with no clinical effect. One patient developed partial loss of the flap, and one early leakage. There were no cases of xerostomia, and no signs of recurrence during the postoperative follow-up period of 3-26 months. The flap is useful, as it is simple and reliable for reconstruction of small to medium oropharyngeal defects in carefully selected cases, and gives good cosmetic and functional results.

Deregulation of secreted frizzled-related proteins is associated with aberrant ß-catenin activation in the carcinogenesis of oral submucous fibrosis.

Secreted frizzled-related proteins (SFRPs), the first identified Wnt antagonists, have been well recognized as tumor suppressors in multiple human cancers through suppressing the Wnt/ß-catenin pathway. To better elucidate the mechanisms of SFRPs involved in the carcinogenesis of oral submucous fibrosis (OSF), one of the precancerous lesions of oral squamous cell carcinoma (OSCC), we investigated expression and localization of SFRP1, SFRP5, and ß-catenin in normal oral epithelium, OSF, and OSCC tissues. We found that SFRP1 and SFRP5 were readily expressed in normal oral mucous tissues but gradually decreased in OSF early, moderately advanced, and advanced tissues and rarely expressed in OSCC tissues. We found the changes of SFRP1 localization and SFRP5 localization from nucleus to cytoplasm in the carcinogenesis of OSF. There is a significant association among reduced SFRP1, SFRP5, and cytoplasmic/nuclear ß-catenin expression, which is correlated with higher tumor grade and stage of OSCC. We further found that SFRP1 and SFRP5 were frequently methylated in OSCC cases with betel quid chewing habit but not in normal oral mucous and different stages of OSF tissues, suggesting that methylation of SFRP1 and SFRP5 is tumor specific in the carcinogenesis of OSF. Taken together, our data demonstrated that reduced SFRP1 and SFRP5 by promoter methylation could lead to cytoplasmic/nuclear accumulation of ß-catenin and tumor progression. The changes of SFRPs and ß-catenin localization, as well as SFRPs' methylation, could be useful predictors or biomarkers of OSF malignant progression and prognosis.

Expression and promoter methylation of Wnt inhibitory factor-1 in the development of oral submucous fibrosis.

Oral squamous cell carcinoma (OSCC) is a type of head and neck malignancy with a high mortality rate. Oral submucous fibrosis (OSF) is the pre-cancerous lesion of OSCC, whose molecular mechanisms in OSCC tumorigenesis remain largely unclear. Activation of the Wnt/ß-catenin signaling pathway plays an important role in oral mucous carcinogenesis, although rare mutations of Wnt signaling molecules are found in OSCC, suggesting an epigenetic mechanism mediating aberrant Wnt/ß­catenin signaling in OSCC. Wnt inhibitory factor-1 (WIF1) is an Wnt antagonist, and its downregulation and methylation have been reported in a number of malignancies. However, the expression and methylation of WIF1 in the development of OSF have yet to be reported. In the present study, we investigated the WIF1 expression level by immuno-histochemical staining and semi­quantitative RT-PCR in normal oral, OSF and OSCC tissues, as well as the methylation status by methylation-specific PCR and bisulfite genomic sequencing. The results showed that WIF1 was readily expressed in normal oral mucous tissues, but decreased gradually in OSF early, moderately advanced and advanced tissues, and was less expressed in OSCC tissues. Moreover, WIF1 was able to translocate from the nuclear to cytoplasm in OSF and OSCC tissues. Furthermore, WIF1 was frequently methylated in OSCC cases with betel quid chewing habit, but not in normal oral mucous and different stages of OSF tissues, suggesting WIF1 methylation is tumor-specific in the development of OSF. Thus, the results demonstrated that WIF1 is frequently downregulated or silenced by promoter methylation in the carcinogenesis of OSF, which serves as a potential epigenetic biomarker for the early detection of OSCC.

Vascularized versus nonvascularized facial nerve grafts using a new rabbit model.

BACKGROUND: The use of vascularized nerve graft models has been limited because of the complexity of the operation. The authors sought to develop a simple and effective rabbit model for facial nerve repair and evaluated its advantages over conventional nerve grafts. METHODS: Rabbits were divided into three groups consisting of six rabbits each. The central auricular nerve and its nutrient vessels were used as a vascularized graft. Rabbits were grafted with a vascularized facial nerve graft (vascularized nerve graft group), with a free nerve graft (free nerve graft group), or with a vascularized nerve graft and a free nerve graft on each side of the face (vascularized nerve graft/free nerve graft group). Four months after surgery, facial performance and electrophysiologic monitoring were evaluated. The rabbits were then killed to prepare the nerve specimens for histologic, immunohistochemical, and transmission electron microscope study. RESULTS: At 4 months after the facial nerve repair, the functional recovery of the facial nerve was observed and analyzed. The side grafted with vascularized nerve graft was superior to the side grafted with free nerve graft. Regenerated nerve fibers were observed in all groups, and rabbits grafted with vascularized nerve grafts had more regenerated axons than those that underwent free nerve grafting, although the regenerated nerves were not as good as the natural nerves. CONCLUSIONS: This study demonstrates that it is feasible to establish a vascularized nerve graft model in rabbits. The model offers the obvious advantages of operability and reliability. The vascularized nerve graft is demonstrated to have a superior value for facial nerve repair.

Gene expression profiling of craniofacial fibrous dysplasia reveals ADAMTS2 overexpression as a potential marker.

Fibrous dysplasia (FD) as an abnormal bone growth is one of the common fibro-osseous leasions (FOL) in oral and maxillofacial region, however, its etiology still remains unclear. Here, we performed gene expression profiling of FD using microarray analysis to explore the key molecule events in FD development, and develop potential diagnostic markers or therapeutic targets for FD. We found that 1,881 genes exhibited differential expression with more than two-fold changes in FD compared to normal bone tissues, including 1,200 upregulated genes and 681 downregulated genes. Pathway analysis indicated that obviously activated pathways are Ribosome and ECM-receptor interaction pathways; downregulated pathways are "Hepatitis C" and "cancer" signaling pathways. We further validated the expression of ADAMTS2, one of most differentiated expressed genes, by Immunohistochemistry (IHC) in 40 of FD cases. Results showed that ADAMTS2 was significantly overexpressed in FD tissues, but rarely expressed in normal bone tissues, suggesting that ADAMTS2 could be a potential biomarker for FD. Thus, this study uncovered differentially expressed candidate genes in FD, which provides pilot data for understanding FD pathogenesis, and developing novel biomarkers for diagnosis and targeting of FD.

[A retrospective study of oral squamous cell carcinomas originated from oral submucous fibrosis].

OBJECTIVE: To investigate the clinical characteristics, pathological and biological behaviors of oral squamous cell carcinomas (OSCC) originated from oral submucous fibrosis (OSF). METHODS: A total of 528 patients with OSCC treated at Department of Oral and Maxillofacial Surgery, Xiangya Hospital, Central South University from 2002 to 2010 were divided into two groups, OSCC originated from OSF and OSCC not originated from OSF. The clinical data regarding age, gender, location, pathological type, metastasis and recurrence rate of these patients were analyzed retrospectively. RESULTS: The mean age of the group of OSCC originated from OSF (45.8 years) was younger than that of the group of OSCC not originated from OSF (55.9 years). The male female ratio of the group of OSCC originated from OSF (32.3:1) was higher than that of the group of OSCC not originated from OSF (2.3:1). The metastasis and recurrence rate of the group of OSCC originated from OSF (13.5%, 39.1%) was higher than that of the group of OSCC not originated from OSF (7.6%, 27.8%.). CONCLUSIONS: OSCC originated from OSF occurs at younger age and more in male, and is clinically more invasive and metastatic.

Survivin as a potential early marker in the carcinogenesis of oral submucous fibrosis.

OBJECTIVE: Oral submucous fibrosis (OSF) is a chronic precancerous condition. Survivin is one of the inhibitors of apoptosis protein, and is focused on owing to its unique therapeutic and prognostic potential. STUDY DESIGN: To determine the potential involvement of survivin in the carcinogenesis of OSF, we analyzed the relationship between the survivin and clinical characteristic. RESULTS: Immunohistochemistry was used to show that survivin expression levels were significantly higher in the oral squamous cell carcinoma transformed from OSF group compared with normal group (P < .01) and OSF group (P < .01). In the different stages of OSF, survivin expression exhibited difference as well. Furthermore, Western blotting and reverse-transcription polymerase chain reaction analysis confirmed the increased expression of survivin in the carcinogenesis of OSF. CONCLUSION: These results suggest that survivin plays an important role during the malignant transformation of OSF and may provide an indication to early prevention and diagnosis in the progression of OSF.

Anthropometric measurement and analysis of the external nasal soft tissue in 119 young Han Chinese adults.

A random sample of 119 young, healthy Han Chinese adults (56 men and 63 women) between the age of 18 and 25 years (mean, 22.7 y) in PR China was obtained for this study. By the guidance of standard methods, based on Farkas's anthropometric measurements in craniofacial region, 12 nasal soft tissue landmarks and 12 linear and 3 angular measurements were chosen. The linear measurements were taken directly, whereas the angular measurements were taken by photogrammetric method. Eight nasal proportion indices were calculated according to the linear measurements. The application of the independent-samples t-test showed sex dimorphism in most parameters of the nasal region. All the linear measurements were larger in men than in women, whereas all the angular measurements were smaller in men than in women. The significant differences in partial parameters between men and women have been proved. Ten of 12 linear measurements, 1 of 3 angular measurements, and 3 of 8 nasal proportion indices showed significant sexual dimorphism (P < 0.01). Compared with other racial/ethnic groups, the nasal anthropometric measurements and proportion indices of Han Chinese adults were different, to some extent. This study could provide credible and objective reference material for plastic and maxillofacial surgeons for the external nasal soft tissue evaluation and planning of the cosmetic nasal surgery. Besides, these results could be a useful guidance for preoperative and postoperative evaluations of secondary rhinoplasty in nasal deformity associated with cleft lip and palate.

The phosphorylation of survivin Thr34 by p34cdc2 in carcinogenesis of oral submucous fibrosis.

Survivin is a crucial node molecule involved in apoptosis, cell division and drug discovery. Up-regulation of survivin in the tissues of oral submucous fibrosis (OSF) and oral squamous cell carcinoma (OSCC) originated from OSF has already been demonstrated. Survivin Thr34 phosphorylation is involved in the inhibition of apoptosis and cell division. To determine the potential involvement of survivin Thr34 phosphorylation in carcinogenesis of OSF, 40 OSFs, 42 OSCCs originated from OSF and 10 normal tissues from surgical specimens were studied. Immunohistochemistry showed that the positive staining rate of the survivin phosphorylation on Thr34 in OSCC originated from OSF group was significantly higher than that in OSF group (P<0.01), and none in the normal oral mucosa specimens. Survivin phosphorylation on Thr34 is predominantly located in the nucleus, which account for its function in apoptosis at cell division. Western blotting analysis showed increasing expression of survivin Thr34 phosphorylation, cyclin B1 and p34cdc2 in carcinogenesis of OSF. Furthermore, p34cdc2-cyclin B1 kinase was confirmed to phosphorylate survivin on Thr34 in carcinogenesis of OSF by immunoprecipitation and immunoblot. These results suggest that the phosphorylation of survivin on Thr34 critically regulate survivin and plays an important role during the malignant transformation of OSF, which will provide an indication to early diagnosis and therapy in carcinogenesis of OSF.

Gene expression profiling of oral submucous fibrosis using oligonucleotide microarray.

Although oral submucous fibrosis (OSF) is the most common precancerous lesion of the oral cavity in Southeast Asia where the habit of betel quid (BQ) chewing is popular, its molecular biological properties are largely unknown. The aim of this study was to identify the genes responsible for its pathogenesis and malignant transformation using oligonucleotide microarray. The expression profiles of 14,500 genes in human oral submucous fibrosis and normal control were analyzed using Affymetrix U133A 2.0 GeneChip arrays. The results revealed that 716 genes were upregulated and 149 genes were downregulated in OSF. Hierarchical clustering revealed that the gene expression profiles of normal and OSF were clearly distinct by these different expression genes. Gene Ontology (GO) and relevant bioinformatics tools identified a list of significant differentially expressed genes involved in immune response, inflammatory response and epithelial-mesenchymal transition (EMT) induced by TGF-beta signaling pathway. Five EMT-related genes including SFRP4, THBS1, MMP2, ZO-1, and CK18 were validated with RT-PCR. Our data suggested that gene abnormalities in immune response, inflammatory response and EMT induced by TGF-beta might play an important role in the pathogenesis and malignant transformation of OSF.

Epstein-Barr virus latent membrane protein 1 mediates serine 25 phosphorylation and nuclear entry of annexin A2 via PI-PLC-PKCalpha/PKCbeta pathway.

We have previously elucidated that Epstein-Barr-virus-encoded latent membrane protein 1 (LMP1) can increase the serine phosphorylation level of annexin A2 by activating the protein kinase C (PKC) signaling pathway and that LMP1 induces the nuclear entry of annexin A2 in an energy- and temperature-dependent manner. Here, we further confirm that LMP1 increases the serine phosphorylation level of annexin A2 by activating the phosphoinositide-specific phospholipase C (PI-PLC)-PKC alpha/PKC beta pathway, mainly through the activation of the PKCbeta pathway. Additionally, active recombinant PKC alpha, PKC beta I, and PKC beta II kinases are able to phosphorylate annexin A2 in vitro. Annexin A2 in the nucleus plays an important role in DNA synthesis and cell proliferation. By site-specific substitution of glutamic acid in the place of serine 11 and 25 in the N-terminus, we show that serine 25 phosphorylation of annexin A2 was associated with the nuclear entry of annexin A2, DNA synthesis and cell proliferation, whereas serine 11 has no obvious influence. We demonstrate for the first time that the PI-PLC-PKCalpha/PKCbeta pathway plays an important role in serine phosphorylation and in the nuclear entry of annexin A2 mediated by LMP1. In addition, we show that annexin A2 is the substrate protein of PKC alpha, PKC betaI, and PKC betaII kinases. Serine 25 phosphorylation of annexin A2 is shown to be associated with its nuclear entry, DNA synthesis, and cell proliferation.