Comparative molecular analysis of oral submucous fibrosis and other organ fibrosis based on weighted gene co-expression network analysis / 中南大学学报(医学版)

OBJECTIVES@#There is currently a lack of economic and suitable animal models that can accurately recapitulate the oral submucous fibrosis (OSF) disease state for indepth study. This is one of the primary reasons for the limited therapeutic methods available for OSF. Based on the underlying logic of pan-cancer analysis, this study systematically compares OSF and the other four types of organ fibrosis from the aspects of molecules, signaling pathways, biological processes, etc. A comprehensive analysis of the similarities and differences between OSF and other organ fibrosis is helpful for researchers to discover some general rules of fibrosis disease and may provide new ideas for studying OSF.@*METHODS@#Microarray data of the GSE64216, GSE76882, GSE171294, GSE92592, and GSE90051 datasets were downloaded from GEO. Differentially expressed mRNAs (DEmRNAs) of each type of fibrosis were identified by Limma package. Weighted gene co-expression network analysis (WGCNA) was used to identify each type of fibrosis-related module. The similarities and differences of each fibrosis-related-module genes were analyzed by function and pathway enrichment analysis.@*RESULTS@#A total of 6 057, 10 910, 27 990, 10 480, and 4 801 DEmRNAs were identified in OSF, kidney intestinal fibrosis (KIF), liver fibrosis (LF), idiopathic pulmonary fibrosis (IPF), and skin fibrosis (SF), respectively. By using WGCNA, each type of fibrosis-related module was identified. The co-expression networks for each type of fibrosis were constructed respectively. Except that KIF and LF have 5 common hub genes, other fibrotic diseases have no common hub genes with each other. The common pathways of OSF, KIF, LF, IPF, and SF mainly focus on immune-related pathways.@*CONCLUSIONS@#OSF and the other 4 types of fibrotic diseases are tissue- and organ-specific at the molecular level, but they share many common signaling pathways and biological processes, mainly in inflammation and immunity.

Evaluation of DNA damage in oral precancerous and squamous cell carcinoma patients by single cell gel electrophoresis.

Context: Single cell gel electrophoresis (SCGE) or "comet assay" is a rapid and very sensitive fluorescent microscopic method for detecting various forms of DNA damage at individual cell level. Aims: The aim of the present study was to detect the extent of DNA damage in oral cancer, oral submucous fibrosis (OSMF) and leukoplakia in comparison to normal individual. Settings and Design: A total of 44 consecutive patients with oral cancer (n=26), leukoplakia (n=12) and OSMF (n=6) and 10 healthy normal volunteers with normal oral epithelia (controls) were recruited from Dr. R. Ahmed Dental College and Hospital and were assessed for the extent of DNA damage using SCGE following clinical diagnosis. Materials and Methods: Peripheral blood was collected by venepuncture and comet assay was performed using SCGE. Mean tail length was compared between diagnostic groups and between different oral habit groups using t-tests and analysis of variance (ANOVA). Pearson's product moment correlation was used to examine the linear association between the extent of DNA damage and oral habit pack-years. Scheffe's pair-wise test was employed to adjust for multiple comparisons. Results: None of the controls were associated with any oral habits. Mean (±SD) tail lengths (in mm) for cancer (24.95 ± 5.09) and leukoplakia (12.96 ± 2.68) were significantly greater than in controls (8.54 ± 2.55, P<0.05). After adjustment, well-, moderately, and poorly differentiated carcinomas had significantly greater tail length than controls. Whereas the extent of DNA damage in cancer cases was significantly greater in leukoplakia than in compared to OSMF (11.03 ± 5.92), the DNA damage in latter was not different from controls. DNA damage for people with any oral habit (19.78 ± 7.77) was significantly greater than those with no habits (8.54 ± 2.55; P<0.0001). Conclusions: DNA damage measured by SCGE is greater in leukoplakia and squamous cell carcinoma, but not in OSMF. Deleterious oral habits are also associated with greater DNA damage.

Transforming growth factor-β-1 polymorphisms are infrequent but exist at selected loci in oral submucous fibrosis.

Background : Oral submucous fibrosis (OSF) may be considered a collagen metabolic disorder resulting from areca-nut alkaloid exposure and individual variation in collagen metabolism. Due to the complexity of OSF pathogenesis, it is important to elucidate independent and interactive effects of polymorphisms of collagen-related genes on OSF risk. Materials and Methods : This study is focused on seven polymorphisms (SNPs) of transforming growth factor-beta-1 (TGF-beta-1) gene in patients with oral submucous fibrosis (OSF), belonging to south Indian ethnic extraction. The mean age at presentation was 43.9 years, range 23-72 years (n=50, M:F ratio, 2.6:1). DNA samples from 50 subjects of the same ethnic group and comparable demographic features who have had practiced the habit of areca-chewing of almost equal duration, but remained free of disease constituted the controls. All DNA samples were collected progressively and purified from peripheral blood employing standard protocols and tested for SNPs. They included two polymorphisms in the promoter region (C-509T and G-800A), three polymorphisms in exon-1 (Arg25Pro(G915C), Leu10Pro(T869C), Glu47Gly(A979G) and two in 5 ͲUTR regions (C→T(rs13306708) and G→A (rs9282871). The extracted DNA samples along with the primers underwent PCR amplification and the genotypic and allelic frequencies were calculated. All calculations were performed using the SPSS software. The PCR products were purified and subsequently sequenced using Flour S™ multi-imager system (Biorad). The sequenced data were analyzed using the BioEdit sequence analysis software. Results : Out of the seven polymorphisms analyzed, six such as two in the promoter region, three in exon-1 and one in 5¢UTR were found to have a " P" value above 0.05 and hence were not significant. The C→T transition (rs13306708) in the 5¢UTR region recorded a " P" value of 0.03 on comparison and hence was found to be significant. The allelic frequencies for this C→T transition in patients were 68.7% C and 31.2% T (27CC, 15CT, 8TT) and that in controls were 89.5% C and 10.4% T (42CC, 6CT, 2TT). Conclusions : The polymorphism in 5¢UTR C-T in TGF beta 1 gene has a significant association with OSF, being a prime determinant in the pro-angiogenic pathway which has got direct bearing with the pathophysiology of the disease. The proximity of this polymorphism to the transcription site and the associated risk involved is discussed.

GST null genotype and antioxidants: Risk indicators for oral pre-cancer and cancer.

Objectives : This study was undertaken to detect the gene polymorphism of detoxification enzymes and estimate the antioxidant enzyme status in patients with oral cancer, oral leukoplakia and oral submucous fibrosis (OSF). Materials and Methods : The GSTM1 and GSTT1 gene polymorphism was evaluated using polymerase chain reaction; the antioxidant enzyme was estimated using biochemical methods. Statistical analyses were performed using student t-test and odds-ratio to estimate relative risk (RR). Results : The RR at 95% confidence interval (CI) for GSTM1 and GSTT1 was statistically significant for all groups. The mean values of glutathione were significantly raised in all groups. The mean values of ceruloplasmin and malonaldehyde was statistically significant among cancer and OSF patients but was insignificant in smokers and cases with leukoplakia. Conclusion : Several genes perform the same function which implies the need to test for several genetic polymorphisms to identify individuals at high risk. The level of antioxidant enzymes correlate with the degree of oxidative damage. The need for further studies is emphasised.

Familial occurrence of oral submucous fibrosis: report of eight families from northern Kerala, south India.

BACKGROUND: Oral submucous fibrosis (OSF) is an archetype of pathological fibrosis due to possibly its presence for centuries amongst people of Indian ethnic extraction. The WHO criteria for a precancerous condition accords well with this disease. The fact that the disease has been reported in at least few subjects who do not practice the habit of areca nut chewing and the inability to prove a dose-effect relationship in almost all cases, the question arose whether there is a predisposition for the disease. METHODS: A house- to-house survey was carried out in northern Kerala, South India, for the detection of early and advanced OSF from June 2003 through May 2004. An analysis of the family pedigrees of all patients with a positive history of fibrosis has been carried out. RESULTS: Preliminary analysis of the family survey revealed that OSF tends to aggregate in families. The pedigrees of eight (8) such families presented here and the concept of OSF having a genetic predisposition to its occurrence revisited. CONCLUSION: The view that OSF having a family predilection, at least in a proportion of cases, and its tendency for familial linkage seems to be apparent by this study. This rather denovo observation of a trend relationship between OSF risk and the number of putative high-risk genotypes should be separately evaluated for the collagen metabolizing and cross-linking pathways. Establishing familial linkage of the disease seems to be an initial step in deciphering further the molecular pathways involved in its causation and biologic progression.

Cytogenetic investigations on patients with oral submucous fibrosis.

Lymphocyte cultures were set up from venous blood samples collected from 23 patients of submucous fibrosis (SMF) and 10 normal controls. Slides, thus prepared, were processed and screened for G-, C-banding and sister chromatid exchange (SCE) frequency analysis. No gross chromosomal anomalies except that a few breaks and gaps were observed to be randomly distributed throughout the genome. However, a proportionate increase in SCE frequency in SMF patients as compared to the normal control individuals was observed. An attempt has been made to correlate the period of betel leaves, nuts, quid and tobacco chewing with the incidence of chromosomal anomalies and increase in SCE frequency and its sexwise distribution in these patients.