|Year : 2022 | Volume
| Issue : 2 | Page : 228-235
Leaving no stone unturned: Role of profibrotic genes in oral submucous fibrosis – A systematic review
Amritha James1, Lekshmy Jayan1, Ramya Ramadoss2, Preethi Arunachalam1
1 Department of Oral Pathology and Microbiology, SRM Dental College, Chennai, Tamil Nadu, India
2 Department of Oral Pathology and Microbiology, Saveetha Dental College, Chennai, Tamil Nadu, India
|Date of Submission||31-Mar-2021|
|Date of Acceptance||05-Sep-2021|
|Date of Web Publication||28-Jun-2022|
SRM Dental College, Ramapuram, Chennai - 600 089, Tamil Nadu
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Introduction: Understanding the molecular pathogenesis of an entity helps in devising the mode of progression as well as mode of therapy. Even with years of research to claim the understanding of the molecular pathogenesis of oral submucous fibrosis (OSMF) is limited. More deeper knowledge of the genes responsible for this will help in understanding and managing this disease better.
Materials and Methods: The articles published during a time period of 1990–2020 were chosen in accordance with the inclusion and exclusion criteria according to the PRISMA guidelines.
Results: From a total of 80 articles obtained from both electronic search of PUBMED, EMBASE, MEDLINE and Cochrane registry as well as the manual search only 21 articles were selected and analyzed.
Conclusion: Careful analysis of the samples revealed that transforming growth factor-beta may be a potential biomarker or a candidate for targeted therapy in OSMF.
Keywords: Oral cancer, oral submucous fibrosis, profibrotic genes, SMAD, transforming growth factor-beta
|How to cite this article:|
James A, Jayan L, Ramadoss R, Arunachalam P. Leaving no stone unturned: Role of profibrotic genes in oral submucous fibrosis – A systematic review. J Oral Maxillofac Pathol 2022;26:228-35
|How to cite this URL:|
James A, Jayan L, Ramadoss R, Arunachalam P. Leaving no stone unturned: Role of profibrotic genes in oral submucous fibrosis – A systematic review. J Oral Maxillofac Pathol [serial online] 2022 [cited 2022 Aug 14];26:228-35. Available from: https://www.jomfp.in/text.asp?2022/26/2/228/348732
| Introduction|| |
Genes play a vital role in determining the course of fibrosis. Numerous genes are either upregulated or downregulated during the fibrotic disease process. Pro-fibrotic genes propel the disease toward fibrosis and include growth factors such as transforming growth factor-beta (TGFβ) and fibroblast growth factor (FGF2), collagen genes like collagen type I alpha 1 (COL1A1), COL1A2, tumor necrosis factor (TNF), connective tissue growth factor (CTGF), lysyl oxidase, tissue inhibitors of matrix metalloproteinases (TIMP) and matrix metalloproteinases (MMP11, MMP12, MMP19 and MMP23).,
Oral submucous fibrosis (OSMF) is a potentially malignant disorder with an increased prevalence in Asian countries, especially India. OSMF is a chronic, insidious disease that affects the lamina propria of the oral mucosa, and as the disease advances, it involves tissues deeper in the submucosa of the oral cavity with resulting loss of fibroelasticity. This disease is attributed to the use of areca nut and slaked lime. Earlier many factors such as chili, infections, autoimmunity were all considered but at present, arecoline is identified as the sole causative trigger in the development of OSMF. With decades of studies, still the pathogenesis of this condition is like a deep pit which refuses to be filled. Recently, many researchers have given their valuable time to try and fill this void. In this review, our aim is to analyze the literature to assess the role of profibrotic antifibrotic genes in the pathogenesis of OSMF.
| Materials and Methods|| |
Criteria used for selection of studies
Types of studies
Original researches (randomized control trials, case control, cohort, etc.,) evaluating the role of profibrotic genes in the pathogenesis of OSMF were included, whereas overviews, narrative reviews, letter to editors, short communications, case reports and case series were excluded from the study.
Types of participants
Both in vitro and in vivo studies were analyzed in the existing literature for inclusion in this systematic review.
Outcomes of the study
Effect of profibrotic genes in the pathogenesis of OSMF.
- Evaluation of the role of these genes in malignant transformation of OSMF
- Evaluation of the ability of these genes to act as potential targets in the management of OSMF.
Systematic review is the gold standard for answering any medicine-related question, for assessing association between a disease and cause or intervention or outcome. Detailed search strategies were developed for inclusion of studies for this review in accordance with the PRISMA checklist for systematic review as well as the Cochrane Highly Sensitive Search Strategy. The databases PUBMED, MEDLINE, Embase, LILAC and Cochrane Library were searched from 1990 to September 2020. The computer search strategy included two components where the first component focused on identifying the disease (OSMF) and the second component for the pathogenic factor (profibrotic genes). The search strategy was modified in accordance with the database that is searched to account for the difference in the vocabulary as well as the syntax rules. The first strategy utilizes the keywords fibrosis, OSMF, oral fibrosis or combination of the above for identifying the studies done on these conditions. The second strategy identified studies utilizing the profibrotic genes using the keywords profibrotic genes, CTGF, TGF-beta, SMAD, hypoxia-inducible factor 1 (HIF)-alpha, MMP, TIMP TNF, etc., either alone or in various combinations. Broad search pathway was ensured by not including any keywords related to outcomes as well as study design. Manual search for researches was also performed for identifying relevant researches.
Databases searched for the samples
The electronic databases reviewed for the selection of the studies were the Cochrane Oral Health Group Trials Register, the Cochrane Central Register of Controlled Trials, MEDLINE through OVID, EMBASE through OVID, PUBMED CENTRAL, LILAC and Cochrane Library. In addition, manual search of articles was also conducted. Only articles published in English languages were considered. All articles published from 1990 to September 2020 were included in this review.
Articles published in languages other than English were excluded. Articles that evaluated other pathogenic factors of fibrosis were excluded. Articles that included studies on other pathological conditions using these gene molecules were excluded.
Collection and analysis of data from the study samples
Two individual reviewers evaluated the title and abstract of each of the articles obtained from the electronic search engines to assess the eligibility. A third reviewer was included in case of disagreement in the eligibility of a study by the two reviewers. Full-text copies of all the eligible as well as potentially eligible studies were further evaluated next by all the reviewers. From this, the studies which did not meet the inclusion criteria were excluded. Any disagreement was resolved by discussion among all the authors.
Data extraction and management
The data were independently extracted by the two review authors. The reviewers were not blinded to the details of the study and its authors. All the data were extracted in accordance with the guidelines provided by the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2011). For clarification or requirement of additional details, the authors of the studies were contacted through E-mail.
Assessment of risk of bias in included studies
Four main categories of bias are commonly encountered in a systematic review
- Selection bias
- Performance bias
- Attrition bias
- Reporting bias.
Risk was then categorized as high, low and unclear.
| Results|| |
The search strategy using the combination of keywords yielded a total of 80 articles, from this, 59 articles were excluded in accordance with the PRISMA guidelines which included a series of systematic screening of the articles [identification, screening, eligibility and inclusion] giving a final sample size of 21 articles which satisfied all the inclusion and exclusion criterion. The search strategy of inclusion and exclusion of articles is illustrated in [Figure 1]. The specific study characteristics are recorded in [Table 1] which depicts the evidence level of the studies included in the review. The profibrotic genes included in the current study are illustrated in the form of a pie chart [Figure 2].
|Figure 1: Diagrammatic representation of selection of articles for the systematic review using PRISMA checklist|
Click here to view
| Discussion|| |
OSMF is one of the most researched potentially malignant disorders as it has high malignant potential and even with years of research, there are still many stones left unturned which has impeded the understanding of this entity. Many esteemed researchers have made many enlightening breakthroughs in the molecular pathogenesis of OSMF. At present, profibrotic and antifibrotic genes have gained a lot of attention in the pathogenesis of OSMF. In our study, we have cumulated the literature for researches that have analyzed the role of profibrotic genes in OSMF. Out of the 21 studies included in our systematic review, 10 studies were conducted using TGF-beta, 4 on MMP and TIMPs, 2 on SMAD including one study conducted in our institution and one each on TGF-alpha, TNF-alpha, HIF-1 alpha, FGF.
Transforming growth factor-beta
TGF-beta is a multifunctional cytokine of the TGF superfamily which includes three isoforms 1, 2, 3. It is produced by all white blood cells. It is the most important cytokine implicated in the pathogenesis of OSMF. Research has shown that it is produced by the epithelium in the early stages of the disease and later, the stroma begins to produce with disease progression, possibly by the epithelial-mesenchymal interactions mediated by the signaling molecules seen in normal as well as the lesional mucosa. In our review, 10 studies were conducted to evaluate either the role of TGF-beta in the pathogenesis or as a potential biomarker in the detection of the condition.
Sukumaran et al. from their study to analyze the TGF-beta polymorphisms in OSMF inferred that polymorphism of 5′UTR C-T in TGF-beta has a significant association in OSMF. They also suggested that although the exact role of this region in the progression or presentation of the disease is uncertain, at the same time is an area with potential for further research. Khan et al. used tissue microarray to identify and validate the genes expressed in OSMF. They also attempted to show the regulation of some of these genes by TGF-b and arecoline in keratinocytes and fibroblast cells. They showed that there is upregulation of TGF-b1, TGFBIp, THBS1, SPP1 and TIG1 and downregulation of BMP7. According to them, upregulation of profibrotic genes or cytokines and downregulation of antifibrotic molecules by TGF-beta may be the possible mechanism by which OSMF develops. The same authors published another paper in 2012, studying the gene expression profile in epithelial cells and fibroblasts following treatment with areca nut extract. They concluded that the expression of the profibrotic genes, especially TGF-beta induced by the polyphenols and alkaloids in areca nut, had little influence on the profibrotic molecule expression from fibroblast but induced increase expression in the epithelium. They proposed that the areca nut has a causative role in triggering profibrotic genes in the epithelial cells which further influence the underlying stroma to elicit the fibrotic response. Kale et al. evaluated the expression of TGF-beta in OSMF and its role in reduction of adipose tissue seen in this condition. They showed that the TGF-beta promotes lipodystrophy and inhibits angiogenesis. It is suggested that the reduction of adipose tissue may also contribute to the stiffness of mucosa and sunken appearance of the cheeks. Kamath et al. on correlating the levels of TGF-beta with different stages and grades of OSMF using IHC found a progressive increase TGF-beta with advancing grades of OSMF and also noted a substantial increase in scar tissue. This suggested that the fibrotic change in OSMF may be a reparative process in response to the injury by the noxious agents in areca nut, and thus, targeting this molecule may help in controlling the progression of the disease. Pant et al. showed in their study that Maria et al. injected Sprague-Dawley mice with extracts of pan masala and areca nut, producing OSMF like lesions with similar clinical and histopathological features. The levels of TGF-beta were analyzed using quantitative real-time PCR which showed a significant increase in the levels, confirming that areca nut and pan masala induce the production of TGF-beta which induces the fibrotic change in OSMF., Iyengar et al. aimed at evaluating the role of TGF-beta and COX-2 in the pathogenesis of OSMF to analyze potential targeted therapeutic applications. The levels of COX-2 were found to be higher in the early and moderate stages and grades of the disease progression whereas the levels of TGF-beta showed that progressive increase is noted with the progression of the disease.
Rai et al. conducted a case–control study evaluating the expression of TGF-beta in OSMF to understand its role in the molecular pathogenesis of OSMF by RT-PCR which was confirmed with IHC evaluation. They inferred that TGF-beta 1 was most expressed isoform of the molecule and that the receptors 1, 2 were also elevated. Increase in the mRNA for all three isoforms was also noticed, confirming that it plays a major role in the molecular pathogenesis of OSMF.
Connective tissue growth factor
CTGF or CCN2 is a matricellular associated heparin-binding protein. It plays important role in cell adhesion, migration, proliferation, angiogenesis, wound healing and skeletal development. Studies conducted on various fibrotic lesions revealed active participation of this gene, but this gene is not expressed in oral cavity under normal conditions. OSMF is the only condition where this gene is expressed and this may be a potential target for therapy. In this current review, only one study was included. Patil et al. estimated the serum levels of CTGF in OSMF, correlating the level of this gene with the different grades of the condition. It was inferred that the levels of CTGF levels were increased progressively from Grade 1 to Grade 3. Blocking the activation of this profibrotic gene may pave a way in managing this condition better.
It comprises a family of structurally similar proteins which act as signal transducers for receptors of the TGF-beta, regulating cell development and growth. Two researches were included in our current review. Hu et al. investigate the expression and function of SMAD7 in the progression of OSMF and OSCC. The SMAD7 levels were consistently upregulated in OSMF and OSCC, and the subsequent bioinformatics evaluation revealed that there was no mutation in the SMAD7 protein encountered in HNSCC and was elevated in OSCC. Another study conduction in our instituition by Zagabathina et al. in 2020 revealed that the levels of SMAD2 were more in OSMF than in reactive lesions or normal tissues. As mentioned before, SMAD2 being the initiator of transcription of TGF-beta further emphasizes the role of this cytokine if fibrogenesis in OSMF.
Matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases
Matrix metalloproteinase or collagenase is a group of enzymes produced by fibroblasts for degrading the extracellular matrix, and TIMPs are the enzymes responsible for inhibiting the action of MMPs. Chaudhary et al. analyzed the role of functional polymorphism of MMP-2 and 9 promoters in OSMF and HNSCC. The results concluded that SNPs in MMP-2 (-1306 C/T) and MMP-9 (-1562 C/T) promoter region may be associated with susceptibility to HNSCC, and addiction habits such as areca nut chewing and tobacco smoking may enhance the polymorphic association of C/T allele of the MMP-2 and MMP-9 gene polymorphisms in an Indian population. This polymorphism could be a prognostic maker in head-and-neck cancer. Katarkar et al. concluded that a definitive role of MMP-9 coding SNPs has predictive and prognostic value in determining OSMF and can promote to the basement membrane degradation and epithelial atrophy. Mishra et al. evaluated the role of collagenase 1 in OSMF. The intensity of MMP1 expression was decreased as the grade of the disease progressed.
Fibroblast growth factor
FGF belongs to a family of cell signaling proteins necessary for normal development. One study conducted by Bishen et al. in 2008 was included in this review. The study evaluated the role of bFGF in the progression of OSMF and to study the changes in the stroma with increase in the severity of OSMF. The increase in bFGF expression in early stages of OSMF is parallel to the stage of injury caused by areca nut consumption. This might be a contributory event or element in the molecular alteration at a cellular event in OSMF.
Tumor necrosis factor-alpha
TNF alpha is a cytokine which plays an important role in mediating inflammatory reactions in the body. Sodhi et al. evaluated the levels of TNF alpha with increase in disease severity. They found that the levels of TNF alpha increased with the severity of the disease.
Hypoxia inducible factor 1 alpha
This gene is located on chromosome14 and codes for a transcription factor that controls cellular responses to reduced oxygen concentrations within tissues. HIF-1α regulates numerous profibrotic mediators and contributes to fibrosis. TGF-β1 induces HIF1A stabilization in fibroblasts even without prominent hypoxic conditions. Tilkaratne et al. tested the role of hypoxia in the progression as well as malignant transformation of OSMF. They found that as the grade of dysplasia increased, the mRNA as well as the protein levels of HIF-1 alpha also increased. It may act as a potential marker for malignant transformation in OSMF.
In our study, we focused on the literature which has been done to evaluate the role of profibrotic genes in OSMF. We had collected 21 articles including one conducted in our institution and reviewed all of the samples. Out of these 21 articles, 10 were conducted using TGF-beta, all of the studies evaluated showed an increase in the molecule as the disease progresses. All the studies conducted had shown a positive response with each of these molecules showing elevation with progression of the disease. Since the research obtained for the other genes was scanty, the available literature supports the statement that TGF-beta as plays an important role in the promotion of the disease. The role of these profibrotic genes has potential as both a promoter and a potential biomarker in OSMF. The expression of the genes when closely monitored may also be useful in analyzing the progression to a malignancy. The exact inference was difficult to obtain as there is no standardized procedure used, and many studies used IHC to study the molecules whereas others utilized PCR for the quantification of these genes. In addition to all this, if the exact function of these genes can be blocked, it may help in stopping the progression of the disease. Thus, profibrotic genes may be the future of research in OSMF as it may provide answer to the dilemma of the management of OSMF.
| Conclusion|| |
TGF-beta is a key mediator of the fibrotic cascade in OSMF. Careful analysis of the samples revealed that TGF-beta may be used as a potential biomarker or a candidate for targeted therapy and could aid in early diagnosis as well as predict the malignant transformation in OSMF.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]