口腔鳞状细胞癌预后相关基因标志物的分析

［摘要］ 目的 通过生物信息分析途径对口腔鳞状细胞癌患者与正常人群的差异基因进行分析,从分子水平探讨关键基因以及参与的信号通路,初步探索口腔鳞状细胞癌发生、发展的基因标志物。方法 从公共数据库基因表达数据库（GEO）中下载口腔鳞状细胞癌的相关芯片数据（GSE3524和GSE6631）,筛选出口腔鳞状细胞癌组织和对照组织表达有显著差异的基因。并对其功能及预后进行分析。结果 共筛选出129个差异表达基因,其中表达上调45个,下调84个,对其进行基因本体、京都基因与基因组百科全书和蛋白互作网络分析,发现分泌型焦磷酸蛋白（secreted phosphoprotein 1, SPP1）、金属基质蛋白酶（matrix metalloproteinase 1,MMP1）、丝氨酸蛋白酶抑制剂（serpin family E member 1,SERPINE1）、纤溶酶原激活剂（plasminogen activator urokinase,PLAU）处于基因核心节点位置。同时,根据这些关键基因表达水平的高低对口腔鳞状细胞癌患者进行分组,高表达组患者生存时间均低于低表达组,差异有统计学意义（PSPP1=0.045、PMMP1=0.046、PSERPINE1=0.0024和PPLAU=0.00049）。结论 基因组学分析方法筛选出的关键基因和信号通路有助于研究口腔鳞状细胞癌发生、发展的机制,也进一步为治疗靶点及预后分子标志物的选择提供了依据。     

A combinative analysis of gene expression profiles and microRNA expression profiles identifies critical genes and microRNAs in oral lichen planus

ObjectiveOral lichen planus (OLP) is a chronic inflammatory disease but aetiology and pathogenesis has not fully elucidated. To gain insight into the mechanism of OLP, bioinformatic analysis was performed in this study.DesignGSE38616 and GSE38615 were downloaded from GEO, including 7 cases of OLP and 7 healthy controls. Differentially expressed genes (DEGs) and miRNAs (DEMs) between OLP and control were screened with package Limma of R. Potential regulatory miRNAs were screened via gene set enrichment analysis. A protein–protein interaction network was constructed for the DEGs. KEGG pathways for DEGs were revealed using Gene Set Analysis Toolkit V2.ResultsAfter DEGs and DEMs were obtained, potential regulatory miRNAs of the DEGs were revealed and only miR-362 was differentially expressed in OLP compared with DEMs. Four targets of miR-362 were SLIT-ROBO Rho GTPase activating protein 2 (SRGAP2), vesicle-associated membrane protein 4 (VAMP4), leucine rich repeat transmembrane neuronal 4 (LRRTM4) and lysine (K)-specific demethylase5C (KDM5C). Identified DEGs were significantly enriched in olfactory transduction and ribosome pathways.ConclusionmiR-362, targeting SRGAP2 and VAMP4, may be a potential risk miRNA to regulate OLP. The findings may provide potential biomarkers for diagnosis or treatment of the disease.     

Effect of Myd88 deficiency on gene expression profiling in salivary glands of female non-obese diabetic (NOD) mice

ObjectivesSjögren's syndrome (SS) is a chronic autoimmune disease characterized by inflammatory lesions in the salivary and lacrimal glands, which are caused by distinct lymphocytic infiltrates. Female non-obese diabetic (NOD) mice spontaneously develop inflammatory lesions of the salivary glands with SS-like pathological features. Previous studies have shown that MyD88, a crucial adaptor protein that activates innate immune signaling, affects lymphocytic infiltration, but its detailed role remains unclear. In this study, we investigated the role of MyD88 through gene expression profiling in the early phase of pathogenesis in the salivary glands of female NOD mice.MethodsSubmandibular glands collected from 10-week-old female wild-type and Myd88-deficient NOD mice were used for RNA preparation, followed by microarray analysis. The microarray dataset was analyzed to identify Myd88-dependent differentially expressed genes (DEGs). Data generated were used for GO enrichment, KEGG pathway, STRING database, and INTERFEROME database analyses.ResultsMyd88 deficiency was found to affect 230 DEGs, including SS-associated genes, such as Cxcl9 and Bpifa2. Most of the DEGs were identified as being involved in immunological processes. KEGG pathway analysis indicated that the DEGs were putatively involved in autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. Furthermore, the DEGs included 149 interferon (IFN)-regulated genes.ConclusionsMyD88 is involved in the expression of specific genes associated with IFN-associated immunopathological processes in the salivary glands of NOD mice. Our findings are important for understanding the role of MyD88-dependent innate immune signaling in SS manifestation.     

Identification of potential therapeutic target genes,key miRNAs and mechanisms in oral lichen planus by bioinformatics analysis

The study aimed to identify the potential target genes and key miRNAs as well as to explore the underlying mechanisms in the pathogenesis of oral lichen planus (OLP) by bioinformatics analysis. The microarray data of GSE38617 were downloaded from Gene Expression Omnibus (GEO) database. A total of 7 OLP and 7 normal samples were used to identify the differentially expressed genes (DEGs) and miRNAs. The DEGs were then performed functional enrichment analyses. Furthermore, DEG-miRNA network and miRNA-function network were constructed by Cytoscape software. Total 1758 DEGs (598 up- and 1160 down-regulated genes) and 40 miRNAs (17 up- and 23 down-regulated miRNAs) were selected. The up-regulated genes were related to nuclear factor-Kappa B (NF-κB) signaling pathway, while down-regulated genes were mainly enriched in the function of ribosome. Tumor necrosis factor (TNF), caspase recruitment domain family, member 11 (CARD11) and mitochondrial ribosomal protein (MRP) genes were identified in these functions. In addition, miR-302 was a hub node in DEG-miRNA network and regulated cyclin D1 (CCND1). MiR-548a-2 was the key miRNA in miRNA-function network by regulating multiple functions including ribosomal function. The NF-κB signaling pathway and ribosome function may be the pathogenic mechanisms of OLP. The genes such as TNF, CARD11, MRP genes and CCND1 may be potential therapeutic target genes in OLP. MiR-548a-2 and miR-302 may play important roles in OLP development.     

Prognostic importance of DNA from human papillomavirus in patients with oral squamous cell carcinoma

Background Survival of patients with oral squamous cell carcinoma (OSCC) is generally low, with the likelihood of locoregional recurrence or disease progression (LR/DP). Knowledge of prognostic factors for survival is key to achieving an understanding and increased survival. The present study aimed to identify prognostic factors for patients with OSCC, especially the presence of DNA from human papillomavirus (HPV).Material and Methods Retrospective cohort study including 119 patients with OSCC treated at the National Cancer Institute in Mexico City (2009-2013). Clinical information was obtained from patient records including LR/DP. Formalin-fixed, paraffin-embedded tissues were obtained and used for detecting DNA from different types of HPV. Potential prognostic factors for Overall Survival (OS) were analyzed using the Cox proportional hazards model.Results After model adjustment, factors associated with longer OS were a pre-treatment platelet count above 400,000/mm3 (HR=0.09, p=0.026) and response to primary treatment (HR=0.26, p=0.001). HPV DNA was present in 23 (19.3%) of the patients and importantly, type 16 found in 19 of them. Although survival of HPV-positive patients was longer, difference was not significant. However, among patients with LR/DP, HPV positivity was significantly associated with increased survival (HR=0.23, p=0.034). Importantly, survival was significantly different for HPV-positive patients with LR/DP > 6 months (HR=0.20, p=0.002), had higher absolute lymphocyte count at start of treatment (HR=0.50, p=0.028) or had local rescue treatment (HR=0.24, p=0.019).Conclusions Although HPV positivity was not associated with a longer OS of OSCC patients, a better prognosis was significantly associated with HPV positivity and recurring or progressing disease, particularly with HPV type 16. Key words:HPV, human papillomavirus, HPV-16, oral squamous cell carcinoma, oral cancer, survival, locoregional recurrence, disease progression.     

Hypermethylation of Death-Associated Protein Kinase (DAPK1) and its association with oral carcinogenesis - An experimental and meta-analysis study

ObjectivesThe value of abnormal DNA methylation of DAPK1 promoter and its association with various cancers have been suggested in the literature. To establish the significance of DNA methylation of DAPK1 promoter in oral squamous cell carcinoma (OSCC), we a) performed a case-control study, b) evaluated published data for its utility in the diagnosis and prognosis of OSCC and c) identified the association of DAPK1 gene expression with promoter DNA methylation status.DesignBisulfite gene sequencing of DAPK1 promoter region was performed on non-malignant and malignant oral samples. Further, using a systematic search, 330 publications were retrieved from PubMed, Scopus, and Google Scholar and 11 relevant articles were identified.ResultsSignificant association of DAPK1 promoter methylation with OSCC (p < 0.0001) was observed in the case-control study. The studies chosen for meta-analysis showed prognostic and predictive significance of DAPK1 gene promoter, despite defined inconsistencies in few studies. Overall, we obtained a statistically significant (p-value < 0.001) association for both sensitivity and specificity of DAPK1 DNA promoter methylation in oral cancer cases, without publication bias.ConclusionDNA hypermethylation of DAPK1 gene promoter is a promising biomarker for OSCC prediction/prognostics and suggests further validation in large distinct cohorts to facilitate translation to clinics.     

A preoperative prognostic nutritional index is a prognostic indicator in oral squamous cell carcinoma patients undergoing radical surgery

The purpose of this study was to investigate the prognostic value of prognostic nutritional index (PNI) in oral squamous cell carcinoma (OSCC) patients and to undertake a comparative evaluation of the prognostic value of comparing PNI, neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and lymphocyte-to-monocyte ratio (LMR) in terms of prognostic utility. A retrospective study was conducted involving 203 consecutive patients with OSCC who were treated with radical surgery with curative intent. The PNI and systemic inflammatory response were developed, and their prognostic utility was evaluated. Kaplan–Meier curve analysis and log-rank testing showed that PNI (P< 0.001), NLR (P=0.011), PLR (P=0.013), and LMR (P=0.014) were significantly associated with overall survival. Multivariate analysis identified PNI as an independent prognostic factor for OSCC patients (P=0.029). In time-dependent receiver operating characteristic curve analysis, PNI was continuously superior to that of NLR, PLR, and LMR. In conclusion, this study suggested that PNI offered an independent prognostic biomarker in OSCC patients undergoing radical surgery. However, this study was small and retrospective, thus further investigations are needed to clarify the utility of PNI for tailor-made treatments in clinical settings.     

MicroRNA model that can predict the prognosis of oral squamous cell carcinoma based on bioinformatics analysis

目的 通过生物信息学筛选并建立与口腔鳞状细胞癌（OSCC）患者预后相关的微小RNA（miRNA）预后模型,以期对OSCC患者进行精准的分组,提高治疗的针对性。方法 通过癌症基因组图谱（TCGA）数据库下载OSCC患者的miRNA、mRNA表达谱和临床数据。采用单因素和多因素Cox风险回归分析筛选和建立miRNA预后模型。受试者工作特征曲线（ROC）和曲线下面积（AUC）检验预后模型的性能。预测6-miRNAs靶基因,与差异mRNA取交集后行基因本体论（GO）、京都基因与基因组百科全书（KEGG）信号通路富集分析。构建蛋白互作网络（PPI）筛选中枢基因。结果 通过单因素和多因素Cox回归分析得到基于6个miRNA的预后风险模型。train组、test组和所有样品组中预测5年生存率的ROC曲线下AUC值分别为0.757、0.673、0.724。单因素和多因素Cox回归分析显示,6-miRNAs预后模型可以作为一个独立的预后因素（P<0.001）。靶基因构建PPI网络中前10个中枢基因为CCNB1、EGF、KIF23、MCM10、ITGAV、MELK、PLK4、ADCY2、CENPF、TRIP13。其中EGF和ADCY2与生存预后相关（P<0.05）。结论 6-miRNAs可有效地作为OSCC患者一种新的独立的预后模型,或可成为指导OSCC精准治疗的新方法。     

PMS2: a potential prognostic protein marker in oral squamous cell carcinoma

Background An increase in oral squamous cell carcinoma (OSCC) cases was observed despite the reduction in exposure to classic risk factors. Although the exact cause of this trend remains unknown, epigenetic factors could be contributing to an increased occurrence of these tumors. This study aims to assess the influence of PMS2 protein immunoexpression on the prognosis of patients with OSCC. Material and Methods This study comprised 76 cases of OSCC treated between 2011 and 2016. Immunohistochemical staining for PMS2 was performed. For evaluation, 10 fields per histological section were photographed at a 400x magnification and positively-stained cells were counted with Image J. Mann-Whitney and Kruskal-Wallis tests were used to compare the immunolabeling pattern with the clinical-pathological and prognostic characteristics. Survival analysis was performed with Chi-square, Long-Rank Mantel-Cox and Cox regression tests (p<0.05). Results An overexpression of PMS2 was observed in N0/1 tumors and in oral cancers found in unusual locations. In patients ≤60 years of age, high levels of PMS2 ( >60%; p=0.041) were associated with low survival (p=0.029). In multivariate analysis, surgery combined with chemotherapy (p=0.030) and high PMS2 immunoexpression (p=0.042) significantly increased the risk of death for ≤60 years old patients. Conclusions The findings of this study indicate that PMS2 can be a potential prognostic protein marker in OSCC patients 60 years of age and younger. Key words:Squamous cell carcinoma, mouth neoplasms, mismatch repair endonuclease PMS2, survival.     

Hypovitaminosis D,oral potentially malignant disorders,and oral squamous cell carcinoma: a systematic review

Background Oral squamous cell carcinoma (OSCC) and potentially malignant oral disorders (OPMDs) could be associated with low levels of vitamin D. This systematic review aimed to determine the relationship between serum levels of vitamin D with OPMDs and OSCC.Material and Methods This review was conducted according to Cochrane guidelines (PROSPERO CRD42020207382) on literature retrieved from the PubMed, Cochrane, and Web of Science databases. The antecedents extracted were study design, methodology, sample (country, number of patients, age, and sex), oral manifestations (type of lesion, location, prevalence, and follow-up), serum vitamin D levels or use of vitamin D supplements, results, and conclusions.Results Twelve articles were selected. Some of the most relevant findings were alterations in vitamin D could favor the progress of OPMDs to OSCC. Higher levels of vitamin D can increase levels of anti-inflammatory mediators, CD4+ T lymphocytes and CD8+ T lymphocytes and CD3+ T lymphocytes in intratumoral tissue. The normalization of vitamin D levels in patients with OSCC can increased cytotoxic activity of natural killer cells, favoring antitumor immune response. Vitamin D supplemented can lower adverse effects associated with chemotherapy like mucositis and pain. Tobacco can increase risk of developing OSCC altering vitamin D levels.Conclusions Hypovitaminosis D could increase risk of developing OSCC from OPMDs, thus altering the immune response and it is associated with a lower survival rate in patients with OSCC, a greater recurrence of tumors in patients who underwent surgical treatment, and an increase in adverse reactions to chemotherapy. The use of vitamin D supplements can be a complement to primary therapy to prevent the recurrence of lesions and reduce adverse events associated with treatment. Key words:Oral squamous cell carcinoma (OSCC), Oral potentially malignant disorders (OPMDs), Vitamin D, Oral cancer.     

基于RNA-Seq分析挖掘唾液腺多形性腺瘤核心基因及通路

目的：筛选多形性腺瘤（pleomorphic adenoma,PA）与瘤旁唾液腺腺体间的差异表达基因,挖掘PA形成过程中的核心基因及通路。方法：采用RNA-Seq技术,检测5例PA患者的配对肿瘤与瘤旁唾液腺腺体,筛选2组间差异基因。运用蛋白互作数据库STRING分析预测差异基因所编码蛋白间的相互作用。筛选互作网络中的核心模块及核心基因,分析核心模块中激活的信号通路,预测上述模块与PLAG1可能的相互作用关系。RT-PCR验证核心基因在20例PA患者的配对肿瘤组织及瘤旁唾液腺中的表达。采用SPSS 20.0软件包对数据进行统计学分析。结果：共测得3810个差异基因,其中2021个下调,1789个上调。核心模块中存在PI3K-AKT信号通路、ER受体信号通路、Rap1信号通路、cGMP-PKG信号通路的激活。PLAG1可能通过PHLPP1、LRRK2、β-catenin蛋白与核心模块发生作用。RT-PCR显示,核心基因在20例PA样本及其瘤旁唾液腺组织中的差异趋势与测序结果一致,且具有统计学意义（P<0.0001）。结论：核心基因ADCY3、 ADCY5、 ADORA1、 APLN、 APP、 C5、 CCL28、 DRD2、 FPR3、 GABBR1可能在PA发生、发展过程中起着重要作用,可为预防PA复发及预后标志物筛选提供思路。PLAG1可能通过PHLPP1、LRRK2、β-catenin间接激活PI3K-AKT信号通路、ER受体信号通路、Rap1信号通路、cGMP-PKG信号通路,从而诱发PA形成。     

The role of components of the extracellular matrix and inflammation on oral squamous cell carcinoma metastasis

Objectives

Oral squamous cell carcinoma (OSCC) accounts for about 90% of malignant oral lesions, and is identified as the most frequently occurring malignant tumour of oral structures. We aimed to investigate the genes and pathways related with metastasis on Turkish OSCC patients.

Materials and methods

We performed whole genome expression profiling array on an Illumina platform. A total of 24 samples with 12 OSCC and 12-paired controls that had no tumour were included in the study. Hierarchic clustering and heat map were used for data visualisation and p-values assessed to identify differentially expressed genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Ingenuity Pathway Systems (IPA) analysis were performed to consider biologic meaning of differential expression of the genes between tumour and control groups.

Results

We identified 790 probe sets, corresponding to 648 genes that were effective in separating invasive and metastatic OSCC. Consequently, we found statistically relevant expression results on extracellular matrix members on MMPs such as MMP3, MMP10, MMP1 and MMP9; on laminin such as LAMC2, LAMA3 and LAMB3; several genes in the collagen family; and also on chemokines from the inflammation process.

Conclusion

Statistically relevant expression changes for MMPs, laminins, collagens, and chemokines, which are components of the extracellular matrix and inflammation process, may be considered as a molecular biomarker for early prediction. Further studies are necessary to determine and understand the molecular mechanisms that underlie OSCC metastasis.     

Insulin-like growth factor-II mRNA binding protein-3 and podoplanin expression are associated with bone invasion and prognosis in oral squamous cell carcinoma

ObjectiveThis study aimed to evaluate the prognostic implications of insulin-like growth factor-II mRNA binding protein-3 (IMP3) and podoplanin (PDPN) as therapeutic targets against oral squamous cell carcinoma (OSCC) with bone invasion.Study designWe elucidated the correlation of IMP3 and PDPN expression with bone invasion in 160 OSCC tissue specimens, and assessed a mouse calvarium xenograft model using an IMP3- and PDPN-depleted OSCC cell line.ResultsThe retrospective analysis revealed that the expression of IMP3 and PDPN is significantly correlated with T stage, lymph node metastasis, and the overall survival of OSCC patients. In addition, the dual expression of IMP3 and PDPN but not the single expression of either IMP3 or PDPN was associated with bone invasion and the number of osteoclasts in patients with OSCC. In support of these findings, IMP3 or PDPN depletion inhibited the invasive capacity of OSCC cells in a three-dimensional culture system, tumorigenesis, and regional bone destruction in a xenograft mouse model. In addition, IMP3 or PDPN depletion inhibited the expression of interleukin (IL)-6 and IL-8 in OSCC cells, and decreased the expression of receptor activator of NF-κB ligand (RANKL) in xenograft tumor tissues of OSCC.ConclusionsThese results suggest that IMP3 and PDPN may have strong influence on the pathogenesis of OSCC, especially in bone invasion, and may serve as novel therapeutic targets with prognostic implications for bone-invasive OSCC.     

High percentage of oral lichen planus and lichenoid lesion in oral squamous cell carcinomas

Objective: Oral lichen planus (OLP) and lichenoid lesions (OLL) are regarded as precursor lesions of oral squamous cell carcinoma (OSCC) with potential for malignant transformation. This potential is not clear due to difficulties in diagnosis of OLP and OLL. Our aim was therefore to evaluate previously identified OLP and OLL as precursor lesions in OSCC and to identify cancer related etiological factors such as smoking and alcohol consumption.

Material and methods: We retrospectively reviewed all cases (total 323, comprising 164 females and 159 males) with OSCC treated at the Department of Oral and Maxillofacial Diseases and Surgery, Helsinki University Hospital during 2015. Confirmed by histopathological biopsy, 58 (17.9%) had OLP and 13 had OLL (4.0%) as precursor lesion.

Results: Patients with OLP were slightly older than those without it. OLP was more common in females than in males (p?p?=?.006) and cancer relapses less common (p?=?.005). Smoking was less frequent in patients with OLP and OLL (p?p?Conclusion: Our findings confirm the importance of active follow-up of all patients with OLP and OLL even in patients who do not fit a traditional high-risk category for OSCC.     

Evaluation of survivin as a prognostic marker in oral squamous cell carcinoma

J Oral Pathol Med (2010) 39 : 368–375 Background: Poor prognosis of oral squamous cell carcinoma (OSCC) is partly attributed to the lack of significant tumor marker for accurate staging and prognostication. We have evaluated survivin, which is a member of the inhibitor of apoptosis family as a cancer marker associated with proliferation, angiogenesis, oral carcinogenesis, and OSCC patient survival, as we reported a prognostic significance of survivin expression in lymph node previously. Methods: To evaluate survivin expression in six OSCC cell lines, Western blotting was performed. Hamster oral carcinogenesis model was used to observe changes of survivin expression in oral carcinogenesis. Finally, we assessed the diagnostic and prognostic significance of survivin in a series of 38 primary OSCC through immunohistochemistry (CD31, PCNA) and Kaplan–Meier’s test. Results: Survivin expression was detected in all OSCC cell lines at a varying level but not observed in normal gingival keratinocyte cells. In hamster model, survivin expression was observed from 8 weeks through 16 weeks and the intensity of expression became strong until 16 weeks. Clinicopathological analysis revealed a significant correlation between survivin expression and lymph node metastasis (P = 0.006) and proliferation (P < 0.001). However, there was no significant relationship with differentiation, micro vessel density, and cancer stage based on TNM. Survivin overexpression had a significant negative effect on survival of patients. Conclusions: These results demonstrate the significant relationship between survivin expression and oral carcinogenesis and aggressiveness of OSCC including survival rate of patient. Survivin therefore may be used as a significant cancer marker to gain prognostic information of OSCC.     

Shared epigenetic alterations between oral cancer and periodontitis: A preliminary study

Introduction

We recently developed a non-invasive sampling procedure for oral squamous cell carcinoma (OSCC) detection based on DNA methylation analysis of a panel of 13 genes. Oral cancer, as well as acute and chronic inflammatory diseases, may influence the methylation level of several genes in the oral cavity. In the present study, we evaluated the presence of periodontal disease (PD) and the methylation status using our 13-gene panel.

Methods

Oral brushing specimens were collected from three different patient groups: 23 gingival OSCC patients, 15 patients affected by PD, and 15 healthy volunteers lacking evidence of PD. DNA methylation analysis was performed and each sample was determined to be positive or negative based on a predefined cut-off value.

Results

Positive results were found for 23/23 OSCC patients, 3/15 PD patients, and 0/15 samples from healthy volunteers. The GP1BB and MIR193 genes in the PD group exhibited mean methylation levels similar to OSCC patients. ZAP70 showed different methylation levels among three groups.

Conclusion

Preliminary data identified shared epigenetic alterations between PD and OSCC patients in two inflammatory genes (GP1BB and MIR193). This study may help to identify potential links between the two diseases and serve as a starting point for the future research focused on pathogenesis.