Exploring protein profiles and hub genes in ameloblastoma.

Ameloblastoma (AM) is a prominent benign odontogenic tumor characterized by aggressiveness, likely originating from tooth-generating tissue or the dental follicle (DF). However, proteomic distinctions between AM and DF remain unclear. In the present study, the aim was to identify the distinction between AM and DF in terms of their proteome and to determine the associated hub genes. Shotgun proteomics was used to compare the proteomes of seven fresh-frozen AM tissues and five DF tissues. Differentially expressed proteins (DEPs) were quantified and subsequently analyzed through Gene Ontology-based functional analysis, protein-protein interaction (PPI) analysis and hub gene identification. Among 7,550 DEPs, 520 and 216 were exclusive to AM and DF, respectively. Significant biological pathways included histone H2A monoubiquitination and actin filament-based movement in AM, as well as pro-B cell differentiation in DF. According to PPI analysis, the top-ranked upregulated hub genes were ubiquitin C (UBC), breast cancer gene 1 (BRCA1), lymphocyte cell-specific protein-tyrosine kinase (LCK), Janus kinase 1 and ATR serine/threonine kinase, whereas the top-ranked downregulated hub genes were UBC, protein kinase, DNA-activated, catalytic subunit (PRKDC), V-Myc avian myelocytomatosis viral oncogene homolog (MYC), tumor protein P53 and P21 (RAC1) activated kinase 1. When combining upregulated and downregulated genes, UBC exhibited the highest degree and betweenness values, followed by MYC, BRCA1, PRKDC, embryonic lethal, abnormal vision, Drosophila, homolog-like 1, myosin heavy chain 9, amyloid beta precursor protein, telomeric repeat binding factor 2, LCK and filamin A. In summary, these findings contributed to the knowledge on AM protein profiles, potentially aiding future research regarding AM etiopathogenesis and leading to AM prevention and treatment.

Immunohistochemical expression of SPARC in odontogenic keratocysts: a comparative study with other odontogenic cysts.

BACKGROUND: Secreted protein acidic and rich in cysteine (SPARC) has been shown to modulate aggressive behavior in several benign and malignant tumors. Little is known about SPARC expression in odontogenic keratocyst (OKC), an odontogenic cyst with an aggressive nature. To the best of our knowledge, only one study has been investigated the expression of this protein in OKCs. This study aimed to characterize SPARC expression in OKCs. Additionally, to determine whether SPARC is associated with aggressive behavior in OKCs, SPARC expression in OKCs was compared with radicular cysts (RCs), dentigerous cysts (DCs) and calcifying odontogenic cysts (COCs). These odontogenic cysts showed no or less aggressive behavior. METHODS: SPARC expression was evaluated in 38 OKCs, 39 RCs, 35 DCs and 14 COCs using immunohistochemistry. The percentages of positive cells and the intensities of immunostaining in the epithelial lining and the cystic wall were evaluated and scored. RESULTS: Generally, OKCs showed similar staining patterns to RCs, DCs and COCs. In the epithelial lining, SPARC was not detected, except for ghost cells in all COCs. In the cystic wall, the majority of positive cells were fibroblasts. Compared between 4 groups of odontogenic cysts, SPARC expression in OKCs was significantly higher than those of RCs (P < 0.001), DCs (P < 0.001) and COCs (P = 0.001). CONCLUSIONS: A significant increase of SPARC expression in OKCs compared with RCs, DCs and COCs suggests that SPARC may play a role in the aggressive behavior of OKCs.

The Association of pre-miR27a Gene Polymorphism and Clinicopathological Data in Thai Breast Cancer Patients.

BACKGROUND: MiR27a plays an important role in carcinogenesis, cell proliferation, apoptosis, invasion, migration and angiogenesis. Several studies have identified an important role of pre-miR27a (rs895819) A>G polymorphism in several types of cancer. This research aims to investigate the association of pre-miR27a (rs895819) A>G and breast cancer susceptibility, clinicopathological data and survival. Blood DNA samples of 143 Thai breast cancer patients and 100 healthy Thai women were studied for pre-miR27a (rs895819) A>G polymorphism using polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP). RESULTS: The results revealed that the frequency of pre-miR27a (rs895819) A>G genotypes was not statistically significant different between breast cancer patient and normal control subjects. The rs895819 A>G genotype was significantly associated with clinicopathological parameter of grade III differentiation (P = 0.006), progesterone receptor (P = 0.011) and triple negative (P = 0.031) in breast cancer patients, but not with breast cancer susceptibility. CONCLUSION: The pre-miR27a (rs895819) A>G genotype was significantly associated with poorly differentiated, progesterone receptor and triple-negative in breast cancer patients. Therefore, pre-miR27a (rs895819) A>G may be used as a biomarker for poor prognosis.

Epithelial and fibroblast SPARC expression patterns in oral leukoplakia and oral squamous cell carcinoma.

OBJECTIVE: This study evaluated and compared the expression of secreted protein acidic and rich in cysteine (SPARC) in epithelial cells and fibroblasts of oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) using normal oral mucosa as a control. STUDY DESIGN: The expression of SPARC was determined in samples of normal oral mucosa (n = 12), OL without dysplasia (n = 31), OL with dysplasia (n = 54), and OSCC (n = 69) using immunohistochemistry. The percentage of positive cells in epithelial cells and fibroblasts was independently evaluated. RESULTS: Epithelial SPARC was found in 33.3%, 35.5%, 25.9%, and 66.7% of normal oral mucosa, OL without dysplasia, OL with dysplasia, and OSCC, respectively. Fibroblast SPARC was found in 50.0%, 29.0%, 46.3%, and 84.1% of normal oral mucosa, OL without dysplasia, OL with dysplasia, and OSCC, respectively. OSCC had higher epithelial and fibroblast SPARC expression than normal oral mucosa, OL without dysplasia, and OL with dysplasia (P < .05). No significant differences were observed in epithelial and fibroblast SPARC among normal oral mucosa or OL with and without dysplasia. CONCLUSION: Overexpression of epithelial and fibroblast SPARC was observed in OSCC but not in OL, suggesting that SPARC is involved in the late stage of oral carcinogenesis.

Diagnostic Value of Cytokeratin 17 during Oral Carcinogenesis: An Immunohistochemical Study.

BACKGROUND: Little is known about the role of cytokeratin 17 (CK17) during oral carcinogenesis. CK17 expression in oral leukoplakia (OL), the most encountered oral potentially malignant disorders and oral squamous cell carcinoma (OSCC), remains very limited. To determine the role of CK17 during oral carcinogenesis and its potential diagnostic marker in oral premalignant and malignant lesions, this study evaluated CK17 expression in OL without dysplasia, OL with dysplasia, and OSCC. CK17 expression in these tissues was compared with those of normal oral mucosa (NOM). Additionally, the relationship between CK17 expression and clinicopathologic factors of OSCC was investigated. METHODS: CK17 expression was evaluated in 186 samples consisting of 12 NOM, 33 OL without dysplasia, 58 OL with dysplasia, and 83 OSCC using immunohistochemistry. The proportion of positively immunostained cells was evaluated and scored. RESULTS: CK17 was expressed in 8.3%, 54.5%, 74.1%, and 90.4% of NOM, OL without dysplasia, OL with dysplasia, and OSCC, respectively. NOM had a significantly lower CK17 score than OL with dysplasia (p=0.0003) and OSCC (p < 0.0001). A significant association between CK17 expression and histopathologic differentiation of OSCC was found. Tumors with well differentiation had high CK17 expression compared with those of moderate and poor differentiation. CONCLUSION: CK17 was overexpressed in OL with dysplasia and OSCC, suggesting that CK17 plays a pivotal role in the development of premalignant lesions and OSCC. Of clinical significance, CK17 may be a good diagnostic marker for oral premalignant lesions and OSCC. Additionally, CK17 could be used as an objective tool to classify histopathologic grade in OSCC. The findings that CK17 expression is high in OSCC but low in NOM imply that CK17 may serve as a potential therapeutic target for OSCC.

An integrative analysis of genome-wide methylation and expression in ameloblastoma: A pilot study.

OBJECTIVE: DNA methylation regulates the expression of various genes involved in tumorigenesis. Ameloblastoma is a benign odontogenic jaw tumor. It is locally aggressive with a high level of recurrence. A delay in treatment can lead to severe facial disfigurement. To the best of our knowledge, this is the first integrated analysis of DNA methylation and gene expression in ameloblastoma with the aim to identify genes that may be regulated by DNA methylation. MATERIALS AND METHODS: We used an Infinium MethylationEPIC array to measure genome-wide methylation and the Illumina HiSeq platform to obtain gene expression data in ameloblastoma tissues from five patients and dental follicles from three healthy subjects. An integration analysis was performed using City of Hope CpG Island Analysis Pipeline software. RESULTS: We identified 25,255 differentially methylated CpG sites and 17 differentially methylated CpG islands; six of the islands were negatively correlated with the expression of BAIAP2, DUSP6, FGFR2, FOXF2, NID2, and PAK6. Pyrosequencing and immunostaining techniques were further used to validate FGFR2, NID2, and PAK6. CONCLUSIONS: This analysis identifies a group of novel genes that may be regulated by DNA methylation and will possibly lead to new insights into the pathology and invasion mechanism of ameloblastoma.

Identification of Novel Candidate Biomarkers for Oral Squamous Cell Carcinoma Based on Whole Gene Expression Profiling.

This study aimed to determine the whole gene expression profiles and to ascertain potential biomarkers for 22 oral squamous cell carcinoma (OSCC) among Thai patients using the Illumina Human HT-12, V4.0 Expression BeadChip array. Result indicated 2,724 differential expressed genes composed of 1,560 up-regulated and 1,164 down-regulated genes (unpaired t-test, p-value <0.05; fold change ≥2.0 and ≤2.0). The top 9 up-regulated genes were validated in 39 OSCC cases using TaqMan real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay. Among these, the up-regulation of peptidase inhibitor 3 (PI3) and keratin 17 (KRT17) genes was harbored in all 39 OSCC patients (100%). Likewise, statistical analysis indicated that gene expression in 8 selective genes including keratin 16 (KRT16), keratin 14 (KRT14), keratinocyte differentiation-associated protein (KRTDAP), keratin 6B (KRT6B), PI3, S100 calcium binding protein A7 (S100A7), stratifin (SFN) and keratin 5 (KRT5) was significantly associated with well differentiated OSCC (p-value <0.05). Moreover, high level of KRT17 protein was significantly associated with well differentiated OSCC compared to moderately OSCC (p-value = 0.041). Notably, using nested-PCR analysis indicated all OSCC cases in this study were HPV-free. Especially, KRTDAP, PI3, SFN mRNA expression were first reported among patients with OSCC. Conclusion, the whole transcript expression study and TaqMan real-time qRT-PCR assay were relevant regarding the increase in gene expression in OSCC. In addition, the up-regulation of PI3 and KRT17 might constitute potential candidate molecular biomarkers to diagnose patients with OSCC.

Down-regulation of mitochondrial NADH and cytochrome b gene associated with high tumor stages in head and neck squamous cell carcinoma.

OBJECTIVE: This study aimed to determine mitochondrial mRNA expression levels and the relationships between these expression levels and various adverse clinicopathological characteristics. METHODS: The mRNA expression levels of all 12 genes encoded protein, located on the heavy-strand of mitochondrial DNA including cytochrome b, NADH1, NADH2, NADH3, NADH4, NADH4L, NADH5, ATPase6, ATPase8, cytochrome c oxidase subunit 1, cytochrome c oxidase subunit 2, cytochrome c oxidase subunit 3 were analyzed in 30 head and neck squamous cell carcinoma (HNSCC) and the corresponding normal tissues using reverse transcriptase quantitative real time PCR. Pearson Chi-square test was used to determine the relationships between these expression levels and categorical parameters. RESULTS: The expression levels of 12 mitochondrial mRNAs were observed in all 30 HNSCC patients with down-regulation, ranging from 43.3% to 76.7% and up-regulation, ranging from 10.0% to 36.7%. Furthermore, the number of cases with down-regulations in all 6 NADH and cytochrome b mRNA with TMN stages III and IV were significantly higher than that in stages I and II (p=0.049 and 0.007, respectively). CONCLUSION: Down-regulation of all mitochondrial NADH mRNA as well as mitochondrial cytochrome b mRNA was associated with high tumor stage among HNSCC patients.

Increased Risk of Thai Childhood Acute Lymphoblastic Leukemia with the MiR196a2 T>C Polymorphism

Objectives: This study assessed associations of the miR196a2 (rs11614913) T>C polymorphism withsusceptibility to childhood acute lymphoblastic leukemia (ALL) and clinical outcomes. Materials and Methods: Blood DNA samples from 104 childhood ALL patients and 180 healthy children were studied for the miR-196a2 (rs11614913) polymorphism using a polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) approach. Results: The frequency of the miR-196a2 (rs11614913) T allele in controls was 0.51 compared with 0.33 in ALL cases. In this study, CC, TC heterozygote and CC/TC genotypes were significantly associated with increase childhood ALL susceptibility compared with the TT wild type (OR =4.321, 95% CI = 2.091-8.930 p=0.000, OR = 2.248, 95% CI =1.103-4.579, p=0.024, OR = 2.921, 95% CI = 1.504-5.673 p=0.001, respectively). However, the miR-196a2 (rs11614913) T>C polymorphism was not associated with demographic data or clinico-pathological data in ALL cases. Conclusion: CC, TC and CC+TC genotypes of miR-196a2 (rs11614913) was significantly associated with increased susceptibility in Thai childhood ALL but not with clinical variables.

Expression of cdk6 in head and neck squamous cell carcinoma.

OBJECTIVE: Cdk6 is a key regulator during the G1/S cell cycle transition. Aberrant expression of cdk6 protein has been observed in many cancer types. However, little is known about the expression of cdk6 in head and neck squamous cell carcinoma (HNSCC) and its clinical significance. This study evaluated the expression of cdk6 in HNSCC and analyzed the relationship between cdk6 expression and clinicopathological parameters of HNSCC. MATERIALS AND METHODS: Expression of cdk6 was immunohistochemically investigated in 98 HNSCCs. Nuclear and cytoplasmic positive cells were counted separately. Data were presented as the percentage of positive cells. The correlation between the percentage of positive cells and clinicopathological factors was determined. RESULTS: Nuclear and cytoplasmic staining for cdk6 were detected in 91 cases and 97 cases, respectively. A significant correlation was found only between the percentage of nuclear positive cells and T classification (p value = 0.0410). Tumors with high nuclear cdk6-positive cells showed a linear trend toward advanced tumor status (p value = 0.0064). CONCLUSIONS: Cdk6 was highly expressed in HNSCC. Tumors with high nuclear cdk6 expression tended to have advanced tumor status. These results suggest that cdk6 plays a vital role in HNSCC and is involved in tumor progression of this cancer. CLINICAL RELEVANCE: An increased nuclear cdk6 expression is an unfavorable factor for HNSCC. Cdk6 may serve as a therapeutic target in this cancer.

Genetic alteration in oral squamous cell carcinoma detected by arbitrarily primed polymerase chain reaction.

Oral cancer ranks as one of the top ten cancers in Thailand. Molecular carcinogenesis of this disease remains unknown. The purpose of this report was to identify the genetic alteration profile in Thai oral squamous cell carcinoma (OSCC) patients using arbitrarily primed PCR and to determine the association between genetic alterations and clinico-pathological characteristics. Band alteration profiles in the 32 OSCC tissues were compared with corresponding normal tissues amplified from 60 arbitrary primers using arbitrarily primed polymerase chain reaction (AP-PCR) were identified with 12 primers. Among these, 45 band patterns presented the alteration ranged from 36% to 88%. Primer AD15 at 750 base pairs (AD15-750 bp) was found to have both the highest band alteration (88%) and the highest band loss (37%). The highest DNA band amplification was found in primer AX11-1300 bp (56%). Primer AX-11 at 1300 base pairs at the altered frequency of 78% was significantly associated with smoking (p=0.007), and primer N20 at 800 base pairs showed association with low grade tumors (p=0.030). Our results indicate that AP-PCR is a useful technique for detect genetic alteration in oral squamous cell carcinoma and provide various genetic alternative data.

Expression of p16 in oral cancer and premalignant lesions.

BACKGROUND: Expression of p16 has been proposed as a marker for malignant transformation. This study aimed to evaluate p16 expression in oral squamous cell carcinoma (OSCC) and premalignant lesions including oral leukoplakia (OL) with and without dysplasia. METHODS: Expression of p16 was investigated in 56 samples including OSCC, OL with and without dysplasia, and normal oral mucosa. Expression of p16 was identified by immunohistochemistry, using the CINtecTM p16INK4a Histology Kit. Both nuclear and/or cytoplasmic staining of the keratinocytes were considered to be positive and the percentage of positive cells was calculated. RESULTS: Expression of p16 was detected in 3/16 (18.75%) cases of OSCC, in 4/15 (26.7%) cases of OL without dysplasia, and in none of OL with dysplasia and normal mucosa. No significant differences in p16 expression prevalence were found among OSCC, OL with and without dysplasia and normal mucosa. The percentages of positive cells in OSCC and OL without dysplasia were 0.89 and 0.17, respectively. No significant difference in the percentage of positive keratinocytes was found. CONCLUSION: As a marker, p16 is not reliable for oral mucosal dysplasia and malignant transformation.

Detection of human papillomavirus in oral squamous cell carcinoma, leukoplakia and lichen planus in Thai patients.

Although tobacco, alcohol abuse and betel nut chewing habit are well recognized risk factors for oral squamous cell carcinoma (OSCC), there is evidence to indicate that human papillomavirus (HPV) may also play some inducing role. The purpose of this study was to investigate the presence of HPV in Thai patients with oral squamous cell carcinoma, leukoplakia and lichen planus using the polymerase chain reaction (PCR). Biopsies of oral squamous cell carcinoma, leukoplakia and lichen planus were obtained from 65 patients, 15 males and 50 females, aged between 30- 88 years old. Extracted DNA was evaluated for HPV infections by PCR analysis using consensus primers specific for L1 region of HPV. Only one sample (1.54%) was positive, suggesting that HPV may not play an important role in this group of Thai patients.