👤 Li-Jane Shih

Atherosclerotic vascular diseases remain the leading cause of death despite the use of lipid-lowering drugs. The development of more efficacious therapies targeting endothelial inflammation and endothelial-to-mesenchymal transition (EndMT) is an essential endeavor, aiming for better treatment outcomes. The increased mutation frequency of the The results of liquid chromatography-mass spectrometry, immunostaining, RNA sequencing, and Western blot in mouse and human arteries with atherosclerotic plaques identified TBK1 as one of the key mediators of EndMT and atherogenesis. Its role was then investigated in endothelium-specific TBK1 knockdown An increased expression of TBK1 was observed by liquid chromatography-mass spectrometry analysis in the aortas of The interaction between activated TBK1 and PAK1IP1 inhibits the binding of PAK1IP1 to PAK1, which, in turn, increases the phosphorylation of PAK1 and ERK1/2 in endothelial cells. This process drives EndMT. Endothelium-specific TBK1 knockdown or GSK8612 treatment inhibits EndMT and plaque formation. Safe TBK1 inhibitors could be developed into effective agents for the treatment of atherosclerotic vascular disease. Show less

BackgroundAmyloid accumulation and degeneration of the cholinergic white matter pathways are key factors in early Alzheimer's disease pathogenesis and progression. However, the relationship between them remains unclear.ObjectiveTo investigate the association between amyloid accumulation, the integrity of cholinergic white matter pathways, and cognitive performance.MethodsThis cross-sectional study recruited 109 individuals, including 37 controls with normal cognition and 72 patients with early Alzheimer's disease. All participants underwent neuropsychological testing: the Mini-Mental Status Examination (MMSE), Clinical Dementia Rating scale with sum of box (CDR-SB), and verbal fluency tests. Cholinergic white matter integrity and amyloid burden were assessed through diffusion tensor imaging study (DTI) and amyloid positron emission tomography (PET). Stepwise linear regression analyses were performed. Partial correlations between amyloid burden and cholinergic integrity were also evaluated according to apolipoprotein E4 ( Show less

Alzheimer's Disease (AD) incidence and prevalence increase every year, commonly related to neuron inflammation and degeneration conditions. Tempeh, a traditional fermented product from Indonesia, was reported to have anti-inflammatory, antioxidant, and anti-Alzheimer properties. However, anti-Alzheimer properties of tempeh peptide extracts have not been extensively examined. This research studied the effect of the extracted peptide from tempeh in preventing and delaying Alzheimer's disease. Tempeh peptide was extracted using water maceration and quantified using HPLC and spectrophotometry. Anti-Alzheimer properties of tempeh were analyzed with Ellman's assay of anticholinesterase and in vitro gene expression analysis using LPS-induced neural Schwann cells. As a result, tempeh contained 19.27% of GABA, which is reported to have anti-Alzheimer properties, and other amino acids. Tempeh peptide extract at 12.5 µg/mL had strong inhibition activity toward acetylcholinesterase at 12.61%, and 100 µg/mL of tempeh peptide extract had 8.97% butyrylcholinesterase inhibition activity. Tempeh peptides extract also altered the expression of various genes related to Alzheimer's disease, such as TNF-α, BACE 1, Ntrk 1, BDNF 2, and APP. This research proved that various peptides from tempeh have anti-Alzheimer properties. Show less

Epidermal growth factor receptor (EGFR) over-expression is commonly observed in advanced head and neck squamous cell carcinoma (HNSCC) and is correlated with poor patient outcomes. However, the role of dual-specificity phosphatase 6 (DUSP6) in EGFR-associated HNSCC progression remains poorly understood. This study aimed to investigate the correlation between DUSP6 expression and EGFR signaling in malignant HNSCC tissues. Data mining and in vitro assays were employed to assess DUSP6 expression levels in HNSCC tissues compared to normal tissues. Additionally, the correlation between DUSP6 and EGFR expression was examined. Functional assays were conducted to investigate the modulation of DUSP6 expression by EGFR signaling and its involvement in EGF-induced cell migration and anoikis resistance. Our analysis revealed a significant elevation in DUSP6 expression in HNSCC tissues compared to normal tissues and a strong correlation between DUSP6 and EGFR expression. EGFR signaling modulated DUSP6 expression in a dose- and time-dependent manner, primarily through the extracellular signal-regulated kinase (ERK) pathway. Knockdown experiments demonstrated the functional role of DUSP6 in EGF-induced cell migration and anoikis resistance. The findings of this study elucidate the intricate signaling networks governing DUSP6 expression and its interplay with EGFR signaling in HNSCC. Moreover, the results provide insights into the potential role of DUSP6 as a therapeutic target and highlight the importance of personalized treatment strategies in HNSCC management. Show less

Clonal hematopoiesis (CH) is a common premalignant state in the blood and confers an increased risk of blood cancers and all-cause mortality. Identification of therapeutic targets in CH has been hindered by the lack of an ex vivo platform amenable for studying primary hematopoietic stem and progenitor cells (HSPCs). Here, we utilize an ex vivo co-culture system of HSPCs with bone marrow endothelial cells to perform CRISPR/Cas9 screens in mutant HSPCs. Our data reveal that loss of the histone demethylase family members Kdm3b and Jmjd1c specifically reduces the fitness of Idh2- and Tet2-mutant HSPCs. Kdm3b loss in mutant cells leads to decreased expression of critical cytokine receptors including Mpl, rendering mutant HSPCs preferentially susceptible to inhibition of downstream JAK2 signaling. Our study nominates an epigenetic regulator and an epigenetically regulated receptor signaling pathway as genotype-specific therapeutic targets and provides a scalable platform to identify genetic dependencies in mutant HSPCs. Significance: Given the broad prevalence, comorbidities, and risk of malignant transformation associated with CH, there is an unmet need to identify therapeutic targets. We develop an ex vivo platform to perform CRISPR/Cas9 screens in primary HSPCs. We identify KDM3B and downstream signaling components as genotype-specific dependencies in CH and myeloid malignancies. See related commentary by Khabusheva and Goodell, p. 1768. Show less

Precision medicine in paediatric cardiac channelopathy and cardiomyopathy has a rapid advancement over the past years. Compared to conventional gene panel and exome-based testing, whole genome sequencing (WGS) offers additional coverage at the promoter, intronic regions and the mitochondrial genome. However, the data on use of WGS to evaluate the genetic cause of these cardiovascular conditions in children and adolescents are limited. In a tertiary paediatric cardiology center, we recruited all patients diagnosed with cardiac channelopathy and cardiomyopathy between the ages of 0 and 18 years old, who had negative genetic findings with prior gene panel or exome-based testing. After genetic counselling, blood samples were collected from the subjects and both their parents for WGS analysis. A total of 31 patients (11 cardiac channelopathy and 20 cardiomyopathy) were recruited. Four intronic splice-site variants were identified in three cardiomyopathy patients, which were not identified in previous whole exome sequencing. These included a pathogenic variant in WGS plays a role in identifying additional intronic splice-site variants in paediatric patients with isolated cardiomyopathy. With the demonstrated low extra yield of WGS albeit its ability to provide potential clinically important information, WGS should be considered in selected paediatric cases of cardiac channelopathy and cardiomyopathy in a cost-effective manner. Show less

Phosphaturic mesenchymal tumors (PMT) are uncommon neoplasms that cause hypophosphatemia/osteomalacia mainly by secreting fibroblast growth factor 23. We previously identified FN1::FGFR1/FGF1 fusions in nearly half of the PMTs and frequent KL (Klotho or α-Klotho) overexpression in only those with no known fusion. Here, we studied a larger cohort of PMTs for KL expression and alterations. By FN1 break-apart fluorescence in situ hybridization (FISH) and reappraisal of previous RNA sequencing data, 6 tumors previously considered "fusion-negative" (defined by negative results of FISH for FN1::FGFR1 fusion and FGF1 break-apart and/or of RNA sequencing) were reclassified as fusion-positive PMTs, including 1 containing a novel FN1::ZACN fusion. The final cohort of fusion-negative PMTs included 33 tumors from 32 patients, which occurred in the bone (n = 18), soft tissue (n = 10), sinonasal tract (n = 4), and brain (n = 1). In combination with previous work, RNA sequencing, RNA in situ hybridization, and immunohistochemistry showed largely concordant results and demonstrated KL/α-Klotho overexpression in 17 of the 28 fusion-negative and none of the 10 fusion-positive PMTs studied. Prompted by a patient in this cohort harboring germline KL upstream translocation with systemic α-Klotho overexpression and multifocal PMTs, FISH was performed and revealed KL rearrangement in 16 of the 33 fusion-negative PMTs (one also with amplification), including 14 of the 17 cases with KL/α-Klotho overexpression and none of the 11 KL/α-Klotho-low fusion-negative and 11 fusion-positive cases studied. Whole genomic sequencing confirmed translocation and inversion in 2 FISH-positive cases involving the KL upstream region, warranting further investigation into the mechanism whereby these rearrangements may lead to KL upregulation. Methylated DNA immunoprecipitation and sequencing suggested no major role of promoter methylation in KL regulation in PMT. Interestingly, KL-high/-rearranged cases seemed to form a clinicopathologically homogeneous group, showing a predilection for skeletal/sinonasal locations and typically matrix-poor, cellular solitary fibrous tumor-like morphology. Importantly, FGFR1 signaling pathways were upregulated in fusion-negative PMTs regardless of the KL status compared with non-PMT mesenchymal tumors by gene set enrichment analysis, perhaps justifying FGFR1 inhibition in treating this subset of PMTs. Show less

The global coronavirus disease 2019 (COVID-19) pandemic has a detrimental impact on public health. COVID-19 usually manifests as pneumonia, which can progress into acute respiratory distress syndrome (ARDS) related to uncontrolled TH17 immune reaction. Currently, there is no effective therapeutic agent to manage COVID-19 with complications. The currently available anti-viral drug remdesivir has an effectiveness of 30% in SARS-CoV-2-induced severe complications. Thus, there is a need to identify effective agents to treat COVID-19 and the associated acute lung injury and other complications. The host immunological pathway against this virus typically involves the THαβ immune response. THαβ immunity is triggered by type 1 interferon and interleukin-27 (IL-27), and the main effector cells of the THαβ immune response are IL10-CD4 T cells, CD8 T cells, NK cells, and IgG1-producing B cells. In particular, IL-10 exerts a potent immunomodulatory or anti-inflammatory effect and is an anti-fibrotic agent for pulmonary fibrosis. Concurrently, IL-10 can ameliorate acute lung injury or ARDS, especially those caused by viruses. Owing to its anti-viral activity and anti-pro-inflammatory effects, in this review, IL-10 is suggested as a possible treatment agent for COVID-19. Show less

Low HDL-C (high-density lipoprotein cholesterol) is the most frequent dyslipidemia in Mexicans, but few studies have examined the underlying genetic basis. Our purpose was to identify genetic variants associated with HDL-C levels and cardiovascular risk in the Mexican population. A genome-wide association studies for HDL-C levels in 2335 Mexicans, identified four loci associated with genome-wide significance: CETP, ABCA1, LIPC, and SIDT2. The SIDT2 missense Val636Ile variant was associated with HDL-C levels and was replicated in 3 independent cohorts (P=5.9×10−18 in the conjoint analysis). The SIDT2/Val636Ile variant is more frequent in Native American and derived populations than in other ethnic groups. This variant was also associated with increased ApoA1 and glycerophospholipid serum levels, decreased LDL-C (low-density lipoprotein cholesterol) and ApoB levels, and a lower risk of premature CAD. Because SIDT2 was previously identified as a protein involved in sterol transport, we tested whether the SIDT2/Ile636 protein affected this function using an in vitro site-directed mutagenesis approach. The SIDT2/Ile636 protein showed increased uptake of the cholesterol analog dehydroergosterol, suggesting this variant affects function. Finally, liver transcriptome data from humans and the Hybrid Mouse Diversity Panel are consistent with the involvement of SIDT2 in lipid and lipoprotein metabolism. This is the first genome-wide association study for HDL-C levels seeking associations with coronary artery disease in the Mexican population. Our findings provide new insight into the genetic architecture of HDL-C and highlight SIDT2 as a new player in cholesterol and lipoprotein metabolism in humans. Show less

Therapeutic elevation of high-density lipoprotein (HDL) is thought to minimize atherogenesis in subjects with dyslipidemia. However, this is not the case in clinical practice. The function of HDL is not determined by its concentration in the plasma but by its specific structural components. We previously identified an index for the prediction of HDL functionality, relative HDL (rHDL) index, and preliminarily explored that dysfunctional HDL (rHDL index value > 2) failed to rescue the damage to endothelial progenitor cells (EPCs). To confirm the effectiveness of the rHDL index for predicting HDL functions, here we evaluated the effects of HDL from patients with different rHDL index values on the endothelial-mesenchymal transition (EndoMT) of EPCs. We also analyzed the lipid species in HDL with different rHDL index values and investigated the structural differences that affect HDL functions. The results indicate that HDL from healthy adults and subjects with an rHDL index value < 2 protected transforming growth factor (TGF)-β1-stimulated EndoMT by modulating Smad2/3 and Snail activation. HDL from subjects with an rHDL index value > 2 failed to restore the functionality of TGF-β1-treated EPCs. Lipidomic analysis demonstrated that HDL with different rHDL index values may differ in the composition of triglycerides, phosphatidylcholine, and phosphatidylinositol. In conclusion, we confirmed the applicability of the rHDL index value to predict HDL function and found structural differences that may affect the function of HDL, which warrants further in-depth studies. Show less

Malignant cancer may contain highly heterogeneous populations of cells, including stem-like cells which were resistant to chemotherapy agents, radiation, mechanical stress, and immune surveillance. The characterization of these specific subpopulations might be critical to develop novel strategy to remove malignant tumors. We selected and enriched small population of human melanoma A2058 cells by repetitive selection cycles (selection, restoration, and amplification). These subpopulation of melanoma cells persisted the characteristics of slower cell proliferation, enhanced drug-resistance, elevated percentage of side population as analyzed by Hoechst33342 exclusion, Show less

Lipid metabolic disorders play critical roles in atherogenesis. Traditionally, it has been suggested that reduced high density lipoprotein (HDL) levels might be an important morbidity indicator for cardiovascular diseases. Therefore, it has been argued that therapeutically raising HDL levels may reduce atherogenesis in patients with dyslipidemia. However, recent clinical trials to elevate serum HDL levels by pharmacologic approaches failed to demonstrate clinical efficacy. Thus, to investigate the functionality of HDL and to explore the possible clinical relevance as well as to define an effective indicator that can represent HDL function may provide another key and reference to disclose the clinical treatment of dyslipidemia. We analyzed the association between the data of dichlorofluorescein assay (assay the functionality of HDL), the effect of HDL on oxidized low density lipoprotein (oxLDL)-stimulated endothelial progenitor cells (EPCs) in vitro, levels of circulating EPCs, and ex vitro EPC colony forming units of each case, we defined the indicator (relative HDL index (RHDL index) = dichlorofluorescein assay result of each subject/dichlorofluorescein assay reading of our young healthy controls) that may represent functionality of HDL. HDL from healthy adults protected oxLDL-treated EPCs by modulating p38 mitogen-activated protein kinase and Rho activation and by promoting nitric oxide production. HDL from subject with RHDL index ≧2 also failed to restore the functionality of oxLDL-treated EPCs via cell-signaling pathways in vitro. The RHDL index significantly correlated with patients' circulating EPC number or EPC colony forming units ex vivo. In conclusions, we explored the RHDL index as a score to predict a patient's EPC functions in vivo and ex vitro. Show less

Atherosclerosis is a process of imbalanced lipid metabolism in the vascular walls. The underlying pathology mainly involves the deposition of oxidized lipids in the endothelium and the accumulation of cholesterol in macrophages. Macrophages export excessive cholesterol (cholesterol efflux) through ATP-binding cassette transporter A1 (ABCA1) to counter the progression of atherosclerosis. We synthesized novel chalcone derivatives and assessed their effects and the underlying mechanisms on ABCA1 expression in macrophages. Human THP-1 macrophages were treated with synthetic chalcone derivatives for 24 h. In Western blot and flow cytometry analyses, a chalcone derivative, ( Show less

Optimal molecular markers for detecting colorectal cancer (CRC) in a blood-based assay were evaluated. A matched (by variables of age and sex) case-control design (111 CRC and 227 non-cancer samples) was applied. Total RNAs isolated from the 338 blood samples were reverse-transcribed, and the relative transcript levels of candidate genes were analyzed. The training set was made of 162 random samples of the total 338 samples. A logistic regression analysis was performed, and odds ratios for each gene were determined between CRC and non-cancer. The samples (n = 176) in the testing set were used to validate the logistic model, and an inferred performance (generality) was verified. By pooling 12 public microarray datasets(GSE 4107, 4183, 8671, 9348, 10961, 13067, 13294, 13471, 14333, 15960, 17538, and 18105), which included 519 cases of adenocarcinoma and 88 controls of normal mucosa, we were able to verify the selected genes from logistic models and estimate their external generality. The logistic regression analysis resulted in the selection of five significant genes (P < 0.05; MDM2, DUSP6, CPEB4, MMD, and EIF2S3), with odds ratios of 2.978, 6.029, 3.776, 0.538 and 0.138, respectively. The five-gene model performed stably for the discrimination of CRC cases from controls in the training set, with accuracies ranging from 73.9% to 87.0%, a sensitivity of 95% and a specificity of 95%. In addition, a good performance in the test set was obtained using the discrimination model, providing 83.5% accuracy, 66.0% sensitivity, 92.0% specificity, a positive predictive value of 89.2% and a negative predictive value of 73.0%. Multivariate logistic regressions analyzed 12 pooled public microarray data sets as an external validation. Models that provided similar expected and observed event rates in subgroups were termed well calibrated. A model in which MDM2, DUSP6, CPEB4, MMD, and EIF2S3 were selected showed the result in logistic regression analysis (H-L P = 0.460, R2= 0.853, AUC = 0.978, accuracy = 0.949, specificity = 0.818 and sensitivity = 0.971). A novel gene expression profile was associated with CRC and can potentially be applied to blood-based detection assays. Show less

The Notch3 signaling pathway is thought to play a critical role in cancer development, as evidenced by the Notch3 amplification and rearrangement observed in human cancers. However, the molecular mechanism by which Notch3 signaling contributes to tumorigenesis is largely unknown. In an effort to identify the molecular modulators of the Notch3 signaling pathway, we screened for Notch3-intracellular domain (N3-ICD) interacting proteins using a human proteome microarray. Pathway analysis of the Notch3 interactome demonstrated that ubiquitin C was the molecular hub of the top functional network, suggesting the involvement of ubiquitination in modulating Notch3 signaling. Thereby, we focused on functional characterization of an E3 ubiquitin-protein ligase, WWP2, a top candidate in the Notch3 interactome list. Co-immunoprecipitation experiments showed that WWP2 interacted with N3-ICD but not with intracellular domains from other Notch receptors. Wild-type WWP2 but not ligase-deficient mutant WWP2 increases mono-ubiquitination of the membrane-tethered Notch3 fragment, therefore attenuating Notch3 pathway activity in cancer cells and leading to cell cycle arrest. The mono-ubiquitination by WWP2 may target an endosomal/lysosomal degradation fate for Notch3 as suggested by the fact that the process could be suppressed by the endosomal/lysosomal inhibitor. Analysis of The Cancer Genome Atlas dataset showed that the majority of ovarian carcinomas harbored homozygous or heterozygous deletions in WWP2 locus, and there was an inverse correlation in the expression levels between WWP2 and Notch3 in ovarian carcinomas. Furthermore, ectopic expression of WWP2 decreased tumor development in a mouse xenograft model and suppressed the Notch3-induced phenotypes including increase in cancer stem cell-like cell population and platinum resistance. Taken together, our results provide evidence that WWP2 serves as a tumor suppressor by negatively regulating Notch3 signaling in ovarian cancer. Show less

High-throughput short-read sequencing of exomes and whole cancer genomes in multiple human hepatocellular carcinoma (HCC) cohorts confirmed previously identified frequently mutated somatic genes, such as TP53, CTNNB1 and AXIN1, and identified several novel genes with moderate mutation frequencies, including ARID1A, ARID2, MLL, MLL2, MLL3, MLL4, IRF2, ATM, CDKN2A, FGF19, PIK3CA, RPS6KA3, JAK1, KEAP1, NFE2L2, C16orf62, LEPR, RAC2, and IL6ST. Functional classification of these mutated genes suggested that alterations in pathways participating in chromatin remodeling, Wnt/β-catenin signaling, JAK/STAT signaling, and oxidative stress play critical roles in HCC tumorigenesis. Nevertheless, because there are few druggable genes used in HCC therapy, the identification of new therapeutic targets through integrated genomic approaches remains an important task. Because a large amount of HCC genomic data genotyped by high density single nucleotide polymorphism arrays is deposited in the public domain, copy number alteration (CNA) analyses of these arrays is a cost-effective way to reveal target genes through profiling of recurrent and overlapping amplicons, homozygous deletions and potentially unbalanced chromosomal translocations accumulated during HCC progression. Moreover, integration of CNAs with other high-throughput genomic data, such as aberrantly coding transcriptomes and non-coding gene expression in human HCC tissues and rodent HCC models, provides lines of evidence that can be used to facilitate the identification of novel HCC target genes with the potential of improving the survival of HCC patients. Show less

As the result of a rhJNK1 HTS, the imidazo[1,2-a]quinoxaline 1 was identified as a 1.6 μM rhJNK1 inhibitor. Optimization of this compound lead to AX13587 (rhJNK1 IC50=160 nM) which was co-crystallized with JNK1 to identify key molecular interactions. Kinase profiling against 125+ kinases revealed AX13587 was an inhibitor of JNK, MAST3, and MAST4 whereas its methylene homolog AX14373 (native JNK1 IC50=47 nM) was a highly specific JNK inhibitor. Show less

Oncogenic N-/KRAS mutations were frequently associated with MLL/AF10 in acute myeloid leukemia with myeloid sarcoma (MS). To study the cooperating leukemogenesis by MLL/AF10 and KRAS mutation, we retrovirally transduced MLL/AF10(OM-LZ) and KRASG12C into mouse bone marrow cells and generated two immortalized cell lines. The cells carrying cooperating MLL/AF10(OM-LZ) and KRASG12C had immature myelomonocytic phenotypes. Compared to a previously established cell line carrying MLL/AF10(OM-LZ) alone, cooperation of MLL/AF10(OM-LZ) with KRASG12C blocked the cells at a more immature myelomonocytic stage with reduced expression of monocyte/macrophage markers. The mice transplanted with the cells carrying cooperating MLL/AF10(OM-LZ) and KRASG12C, liked those transplanted with the cells carrying MLL/AF10(OM-LZ) alone, induced myeloproliferative disease-like myeloid leukemia, but in a shorter latency and formed multiple MS at the adipose tissues of skin, peritoneum and intraperitoneal cavity. Cooperation of MLL/AF10(OM-LZ) with KRASG12C increased cell adhesion via upregulation of an adhesion G-protein-coupled receptor Gpr125. Knockdown of Gpr125 in the cells by short hairpin RNA reduced cell aggregation and diminished MS formation in the transplanted mice. Our results indicated that upregulation of Gpr125 by cooperating MLL/AF10(OM-LZ) and KRASG12C promoted cell adhesion and contributed to the MS formation. Show less

The molecular mechanism involved in the hypertrophy of the orbital fat in patients with Graves' ophthalmopathy or thyroid eye disease (TED) remains unclear. Comparison of genome-wide expression profiles may help in the search for the gene sets involved in TED. Twenty-five orbital adipose tissue specimens were obtained, from which the RNA was isolated. Four of the tissue specimens (from four individuals, two with TED and two control subjects) were subjected to cDNA microarray analysis. The data were analyzed by the gene set enrichment analysis (GSEA) to survey the biological pathways involved in the pathogenesis of TED. Messenger RNA levels of some top-ranked genes in GSEA-selected pathways are validated by quantitative PCR (QPCR). The expression of specific gene sets related to lytic vacuoles, lysosomes, and vacuoles were different between the specimens obtained from patients with TED and control subjects (P < 0.001). These three gene sets overlapped. For QPCR, four top-ranked genes were selected from these overlapping gene sets and another one that related to visual failure, using 21 independent samples of patients with TED (n = 15) and control subjects (n = 6). The results showed that ceroid-lipofuscinosis, neuronal 2, late infantile (CLN2; P = 0.044) and ceroid-lipofuscinosis, neuronal 3, juvenile (CLN3, which related to visual failure; P = 0.012) were significantly downregulated in the orbital fat of patients with TED. The expression of the beta subunit of hexosaminidase A (HEXB) was reduced as well, but the change did not reach statistical significance (P = 0.058). Lysosome-related genes, such as CLN2, CLN3, and HEXB, may be involved in the pathogenesis of adipose tissue hypertrophy in TED. Show less

The components of the Wnt-signaling pathway are reported to be mutated in human cancer cells, but the relationship between the components and oral squamous carcinoma (SCC) is still unknown. In this study, we analyzed the epigenetic changes and expression patterns of four member proteins of the Wnt-signaling pathway and analyzed the mutations of beta-catenin and AXIN 1 genes, in order to explore the roles of the pathway in the development of oral cancer. The results showed that there are no beta-catenin and AXIN 1 gene mutations and no methylation of the CpG island of beta-catenin, AXIN I and GSK3beta genes in oral cancer cells; methylation of the CpG island of APC occurs in the precancerous stage and it is a dynamic change; the aberrant expressions or abnormal localization of the Wnt-signaling pathway proteins have no relationship with methylation status or mutation. From our results, we suggest that the Wnt pathway related genes play a very limited role in the development of oral SCC. Show less