|Year : 2022 | Volume
| Issue : 3 | Page : 290-297
Expression of Sirtuin 3 in oral cancer and oral leukoplakia: A cross-sectional observational study
Nandhini Ramesh, Chandrasekar Lakshmi Krithika, Asokan Kannan, Ganesan Anuradha, Yesoda K Aniyan
Department of Oral Medicine and Radiology, SRM Dental College, Ramapuram, Chennai, India
|Date of Submission||08-Jan-2022|
|Date of Decision||20-Apr-2022|
|Date of Acceptance||22-Apr-2022|
|Date of Web Publication||28-Jun-2022|
Dr. Nandhini Ramesh
Department of Oral Medicine and Radiology, SRM Dental College, Ramapuram, Chennai 600089
Source of Support: None, Conflict of Interest: None
Aim: To evaluate Sirtuin 3, a protein-coding gene regulator that is contemporary in research and has gained importance rapidly in the recent years, and to correlate its expression in normal tissues, smokers without lesion (SWL), oral leukoplakia (OL), and oral cancer. Materials and Methods: The prospective study of sample size of 52 tissues was divided into 4 groups such as normal (n = 13), SWL (n = 13), OL (n = 13), and oral cancer (n = 13) based on simple randomization. The tissue samples of OL and oral squamous cell carcinoma (OSCC) were subjected to histologic examination. Real-time polymerase chain reaction was carried out in 52 samples to evaluate the expression of Sirtuin 3. The Shapiro–Wilk test was used for normality testing. The Kruskal–Wallis test was used for intergroup comparison of cycle threshold, followed by Dunn’s post hoc analysis. Results: The post hoc analysis showed that there was a statistically significant difference between normal group and other study groups (smokers, leukoplakia, and OSCC) (P < 0.001). A statistically significant difference was also observed between leukoplakia and oral cancer for the concentration of Sirtuin (P = 0.0004). Intergroup comparison for cycle threshold revealed no statistically significant difference between the study groups. Conclusion: Sirtuin 3 was well expressed in oral cancer and OL tissues, suggesting that it could be added to the oncogenic profile. The expression of Sirtuin 3 increases with an increase in grades of dysplasia and could aid in understanding its role in the pathogenesis of potentially malignant disorders and oral cancer and can also be an ideal evaluator of prognosis.
Keywords: Molecular Pathogenesis, Oral Leukoplakia, Oral Squamous Cell Carcinoma, Sirtuin 3
|How to cite this article:|
Ramesh N, Krithika CL, Kannan A, Anuradha G, Aniyan YK. Expression of Sirtuin 3 in oral cancer and oral leukoplakia: A cross-sectional observational study. J Int Oral Health 2022;14:290-7
|How to cite this URL:|
Ramesh N, Krithika CL, Kannan A, Anuradha G, Aniyan YK. Expression of Sirtuin 3 in oral cancer and oral leukoplakia: A cross-sectional observational study. J Int Oral Health [serial online] 2022 [cited 2022 Aug 17];14:290-7. Available from: https://www.jioh.org/text.asp?2022/14/3/290/348425
| Introduction|| |
Oral squamous cell carcinoma (OSCC) is the 8th most common human cancer with higher mortality rate and ranks 10th in fatality. The malignant transformation occurs in a multistep and multifactorial pathway, in which there are changes in both cellular and genetic levels. Premalignant lesions are the morphologically altered tissues that are more prone to develop into cancer than the normal tissues. Premalignant lesions like oral leukoplakia (OL) have malignant transformation rate of 0.3–2.5%. Smoking form of tobacco, being the most common etiology, has over six times higher risks of developing into OL. The incidence rate is higher in males. OL occurs due to changes at the cellular level, and these changes depend on the cellular environment and genetic susceptibility; therefore, the genetic expressions should also be studied in smokers without lesions (SWL). The epithelial changes are due to the alteration in the cellular level in which the normal cells transform into dysplastic cells and these occur due to gene regulators. The process of transformation from normal to tumor cells is not induced by a single gene. Among the various genes that are involved in the molecular and genetic pathogenesis of oral cancer, Sirtuins also plays a role in tumor progression.
Sirtuins are NAD+-dependent histone deacetylases commonly seen in cellular substructure. The cellular proteins are complex tissues, and they bind with the cofactor NAD+ to exert normal biological functions. Sirtuins are also known as silent information regulator (SIR). They are a class of proteins, which is possessed by mammals in seven different types. Among the seven, each protein was found to be evident on a different cellular substructure, i.e., in nucleus (1, 2, 6, 7), cytoplasm (1 and 2), and mitochondria (4 and 5). The NAD+-dependent histone subunits have a role in influencing cellular processes such as inflammation, oxidative stress resistance, aging, longevity, apoptosis, cell survival, and DNA repair, in which its expression causes the modulations that bring about the changes. Sirtuin 1 and 3 have predominant roles in cancers. They show varied regulations pertaining to the site. As lots of research have been done in Sirtuin 1 in literature that has proved Sirtuin to be a tumor suppressor, the study focusses on Sirtuin 3. In oral carcinoma, Sirtuin 3 is shown to have altered regulations. The role of Sirtuin 3 in cancer is debatable, as it has diverse behavior in cancer as both tumor suppressor and promoter. Studies have proved that Sirtuins act as both oncogenic and tumor suppressors. This action is brought about by the reactive oxygen signaling (ROS) pathway, which is responsible for the arrest of tumor progression. The gene has direct control over the ROS pathway, so in few cancers it prevents the ROS actions and acts as oncogenic and in other few cancers it initiates ROS action leading to tumor suppression.
Theories of Sirtuin expression on cancerous cells have been proved. Sirtuins have altered regulation patterns with paucity of research in determining the expression of Sirtuin 3 in tissue samples of OL and smokers. The rationale of this study is to determine the expression of Sirtuin 3 in oral cancer, leukoplakia, smoker and normal tissues, so that it could be used as a diagnostic or prognostic marker in the future.
| Materials and Methods|| |
The presented cross-sectional study was started with the approval of Institutional Review Board (IRB) and Ethical Committee (SRMDC/IRB/2019/MDS/No.903). Based on the Declaration of Helsinki, informed consent was obtained from the study participants. The details were fully explained to them. The study was carried out from January 2020 to October 2021 for almost 2 years.
Sample size estimation
Sample size was calculated using G*Power software with a total sample size of 52. They were divided into four groups: Group 1: normal (n = 13), Group 2: SWL (n = 13), Group 3: OL (n = 13), and Group 4: OSCC (n = 13).
The study groups of 52 patients were considered with 13 study volunteers from the Outpatient Department of Oral Medicine and Radiology in each group (normal, smokers, OL, OSCC, respectively). Patients satisfy the criteria of age group 40–70 years (Group 1–4). Patients under Groups 3 and 4 were clinically diagnosed with OL and OSCC based on the habit of tobacco, either smoking or chewing. Group 3 OL patients were selected based on the clinical diagnostic criteria by World Health Organization in 1980—“homogeneous leukoplakia” with uniform white and non-scrapable lesion. Due to the paucity of cases, only homogeneous leukoplakia was considered in the study. OL cases were not evaluated based on the degree of dysplasia. Group 4 OSCC study was selected based on the American Joint Committee on Cancer (AJCC), 2010, with TNM stages I–III included. There were no cases considered with both leukoplakia and OSCC or leukoplakia turning into carcinoma in this study. Patients with other oral lesions (premalignant condition and lesion), autoimmune, systemic conditions, pregnancy, history of treated or untreated malignancies of any other system, and patients contraindicated for biopsy were excluded. The participants not willing to participate in the study were excluded from all four groups. The participants willing to participate were explained about the study, and written consent was signed before the start of the procedure.
Tissue samples from smokers and healthy volunteers were collected in an extraction socket, and flap surgeries were done. Tissue biopsies were performed in OL and OSCC in clinically diagnosed patients with differing histopathological features from the outpatient department. Biopsy tissues were separated into two parts: one half was saved in 10% formaldehyde for histological examination and other half was saved in “RNA later” aliquots (RNA stabilization reagent) [Figure 1]. Aliquots were reserved at 4°C for 48 h and was preserved at ‒20°C. Tissue from healthy individuals, both SWL and normal tissues, were collected from the extraction sites and stored in the aliquots containing RNA later reagent and the storage was done for 48 h at 4°C.
Extraction of RNA
Real-time polymerase chain reaction (RT-PCR) was used to produce complementary DNA from RNA. The RNA-specific primer was used to multiply the corresponding RNA molecules, followed by the complementary DNA synthesis. The image [Figure 2] represents the amplification curves in Sirtuin expression.
Establishment of standards for quantitative RT-PCR:
In order to quantitatively determine the copy numbers of SIRT3 in each sample (relative to each other and among the samples), a standard curve was established with serial dilutions of 177 bp PCR product amplified from control SIRT3 cDNA with primers mentioned earlier. They were gel-purified (cat#NA1111, Sigma-Aldrich, USA) and fractionated in 40 μL of buffer. The concentration of the eluate was determined by analyzing 1 μL of the eluate in a Qubit fluorometer (Invitrogen, Austria) using QuantiFluor ONE dsDNA system (cat#E4871, Promega, USA).
To determine the copy numbers of each of the Sirtuin 3 molecules, the amplification curves were normalized to establish a cutoff threshold value, in reference to a linear standard graph. To confirm the specificity of amplification of SIRT3 signals, the samples were subjected to melt curve analysis at the termination of each run. Relative quantification of Sirtuin in each group showed significant expression in OSCC [Figure 3].
The statistical analysis was performed using Stata/SE 17.0 statistical software. The Shapiro–Wilk test was performed to test for normality pertaining to the study variables. Intergroup comparison for the variable cyclic threshold and calculated concentration (copies per microliter) was performed using the Kruskal–Wallis test followed by Dunn’s post hoc analysis. P < 0.05 was considered statistically significant. Spearman’s rank correlation coefficient was used to determine the correlation between concentration of Sirtuin and histopathological staging. Receiver operating characteristic curve was generated to determine the association between sensitivity and specificity values for the calculated concentration of Sirtuin for detecting each group.
| Results|| |
The descriptive statistics [mean ± SD, median, interquartile range (IQR), range, 95% confidence interval (CI)] for groups (normal, smokers, OL and OSCC) are also presented in [Table 1]. Median, IQR, minimum and maximum values for the groups (normal, smokers, OL, and OSCC) were also represented using box and whisker plot [Figure 4]. As the normality assumption was rejected, non-parametric tests were performed for intergroup comparison [Table 2].
|Table 1: Descriptive statistics among normal, smokers, leukoplakia, and OSCC|
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Between the groups, a statistically significant difference was observed (P < 0.001) for the concentration of SIRT3 with higher values observed with the OSCC group (130,519.7 ± 109,120.8 copies/μL) [Table 3]. The post hoc analysis showed that there was statistically significant difference between normal group and other study groups (smokers, OL, and OSCC) (P < 0.001). Although Sirtuin 3 expression was elevated in SWL when compared with normal tissues and statistical difference between them was obtained, optimal cutoff point with degree of sensitivity and specificity could not be determined for smokers. A statistically significant difference was also observed between OL and OSCC for the concentration of Sirtuin (P = 0.0004) [Table 3]. Intergroup comparison revealed no statistically significant difference between the study groups for cycle threshold (P = 0.6848) [Table 3].
|Table 3: Intergroup comparison for variables cycle threshold and calculated concentration (copies/μL) using Kruskal–Wallis test followed by Dunn’s post hoc test|
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Spearman’s rank correlation between concentration of Sirtuin and histopathological staging was determined to be 0.7813 (P < 0.001), indicating strong correlation.
The empirical cut point (concentration of Sirtuin) for OSCC was estimated to be 49,478.5 copies/μL with sensitivity of 1.0, specificity of 0.92, and area under the curve (AUC) of 0.96 (95% CI=39,090.94, 59,866.06; P < 0.001) [Table 4]. The estimated empirical cut point for OSCC had high sensitivity and specificity values. Hence, the estimated empirical cut point was accurate in correctly identifying as well as differentiating OSCC from the other groups. The empirical cut point (concentration of Sirtuin) for OL was estimated to be 15,016 copies/μL with sensitivity of 0.46, specificity of 1.0, and AUC of 0.73 (95% CI=4288.144, 25,743.86; P = 0.006) [Table 4]. The estimated empirical cut point for OL had low sensitivity and high specificity values. Hence, the estimated empirical cut point was not accurate in correctly identifying OL (true positive). But it was accurate in ruling out true negative cases. The empirical cut point (concentration of Sirtuin) for smokers was estimated to be 2407 copies/μL with sensitivity of 0.38, specificity of 0.65, and AUC of 0.52 (95% CI=1208.066, 3605.934; P < 0.001) [Table 4]. The estimated empirical cut point for smokers had low sensitivity and specificity values.
Cluster analysis for concentration of Sirtuin shows that the accuracy of clustering was 53.8% for OSCC and only 23% for OL. The dendrogram showed that 100% of poorly differentiated OSCC lesions, 60% of well-differentiated OSCC lesions, and only 75% of moderately differentiated OSCC lesions could be accurately clustered using the concentration of Sirtuin [Figure 5].
| Discussion|| |
Among various cancers worldwide, oral cancer ranks 8th with an incidence of 354,964 cases and 177,384 deaths annually (GLOBOCAN 2018). The incidence rate is higher in India, Sri Lanka, and Papua New Guinea. The 5-year relative survival rate for OSCC is around 55–60% for patients diagnosed at early stage of cancer, whereas at the advanced stage it is decreased to 30–40%. The transformation of normal to dysplastic cells may occur due to chronic insult or irritation to the mucosa, from previously existing oral potentially malignant disorder and metastasis from other sites in the body. The malignant transformation from normal cells to cancer cells involves various steps and pathways at cellular, genetic, or at both levels. In premalignant lesions, the cells are more susceptible to develop cancer. Few premalignant lesions of oral cavity include OL, erythroplakia, and lesions involving the palate due to reverse smoking.
OL has a global prevalence rate of 1.49–2.6% in males, whereas the prevalence rate varies according to population. The annual incidence rate in India is approximately 1.1–2.4/1000/year and 0.2–1.3/1000/year in men and women, respectively. It occurs more commonly in males when compared with females with 3.2:1. The causative factors of OL are smoking tobacco, pouching of smokeless tobacco, consumption of alcohol, nutritional deficiencies, and rarely by ultraviolet radiation and microorganisms. Presence of dysplasia favors the malignant transformation of OL. The dysplastic features are categorized as mild, moderate, and severe and 0.13–17.9% of OL develop into malignancies.
Smoking and smokeless tobacco chewers are more susceptible to develop oral cancer. In India culture, men predominantly use tobacco in the form of smoking or chewing, compared with women. Tobacco smokers are 27 times at higher risk to develop oral cancer than non-smokers. Tumor grading reveals the prognosis rate and aids in the formulation of treatment protocol. The histopathological evaluation reveals the changes at the cellular level. The molecular changes include the epigenetic and genetic alterations in the mucosal cells due to both intrinsic and extrinsic factors. To enhance the treatment strategies and survival rate for the patients, it is essential to understand the cancer biology. Molecular biomarkers provide an in-depth insight on cancer biology. Sirtuins, a mammalian homolog, was initially found in yeast Saccharomyces cerevisiae. They were first discovered by Dr. Amr Klar in the year 1970. They are proteins with NAD+-dependent histone deacetylase, which are required to emphasize normal functions. They are complex and widely expressed in normal tissues. There are about seven types of Sirtuin (1–7) each with diverse cellular location and function. Sirtuins are type of NAD-dependent proteins. The profuse presence of Sirtuin and its pluripotent actions facilitate the identification and its role in carcinogenesis. They are contributors to oncogenesis as they act as tumor suppressors and oncogenes.
The expression of Sirtuin differs in each type of tissues as it maintains a unique tissue expression profile. It is distinguished in expression from the normal and tumor tissues. Variations in the expression of Sirtuin among the tissues have also been confirmed through various researches. Among the Sirtuin members, SIRT3 has received much attention for its role in cancer genetics, aging, neurodegenerative disease, and stress resistance. SIRT3 is well known to avert the growth of OSCC by eliminating ROS. Sirtuins are located on 8q24 chromosome. The main function of Sirtuin 3 is to control and regulate energy demand during cellular stress situations and metabolism through deacetylation.
Cancer-specific protein SIRT3 may be a biomarker that helps identify cancer patients in a particular stage of the disease.In-vitro study showed that silencing of the SIRT3 gene inhibited the proliferation, invasion, and cells migration but increased the apoptosis in the cultured colon cell lines. As SIRT3 has a tissue-specific pattern of expression, evaluating their levels in tissue samples will be more reliable than other samples. Alhazzazi et al. have demonstrated the upregulation of SIRT3 in OSCC cell lines when compared with normal cell lines. The results are in accordance with the study, in which they assessed the expression of SIRT3 in tissue samples that are more specific. Literature review reveals no research on evaluation of SIRT3 in tissue samples of OL. Hence, this study evaluated SIRT3 expression. Zhao et al. and Liu et al. have also shown the overexpression of SIRT3 in esophageal cancer tissues and in colon cancer, respectively. In the present study, tissue samples of oral cancer demonstrated overexpression of SIRT3 in accordance with the literature. They act as tumor promotors in esophageal cancer, renal cancer, oral squamous cancer, colon cancer, and melanoma and act as suppressors in breast, pancreatic, hepatocellular, basal cell cancer.
According to Xiong et al. and Kamarajan et al., Fas, a tumor necrosis factor, binds to receptor-interacting protein and initiates cell death. Sirtuin acts on receptor-inhibiting protein and prevents cell death and thus acts as tumor oncogenes. Our study in accordance with this theory shows very minimum expression in normal cells to over expressed in OSCC, explaining that increasing expression of Sirtuins is in accordance with severity in our present study. Saito et al. in their study explain that OL has a higher chance of malignant transformation. Literature study reveals that there is paucity of research in evaluation of expression of SIRT3. Though Sirtuin 3 has been evaluated in OSCC, their pathogenesis can be studied on evaluation of their expression at each stage. There are numerous studies executed in various demographic regions; literature does not show any studies pertaining to this demographic area in tissue samples. Hence, expression of OSCC, OL, SWL, and normal tissues of healthy individuals was carried out in and around Chennai. Napier and Speight stated that OL commonly occurs between the age of 40 and 70 years of life and predominantly in male population with variation according to geographic locations. So, the study recruited individuals within 4th–7th decade and the study groups were predominantly males in India. Liu et al. stated that expression of genes occurs in various forms of cancer. The group IV individuals (OSCC) were recruited with no past history of treated/untreated malignancies of same or different sites. Alhazzazi et al. have claimed that SIRT3 expression patterns in tissues samples are more reliable and definitive when compared with those of saliva and serum. Hence, in this study, tissue samples were chosen over serum and saliva. Sathish et al. have suggested that “RNA later” is one of the best non-toxic reagents that stabilize and protect the cellular RNA intact in unfrozen tissue and saliva samples. The traditional methods to detect Sirtuin are western blotting, quantitative RT-PCR, immunohistochemical staining, cell line culture, cell proliferation assay, cell migration assay, cell invasion assay, and microarray-based hybridization with tissues, cell lines, or blood. Considering the sensitivity, specificity, reliability, ease of use, precision, and accuracy, the RT-qPCR was chosen to estimate SIRT3 in collected tissue samples.
In literature, there is no reported evidence of research, evaluating the expression of OL in comparison with OSCC (based on histopathological staging), smokers without oral lesions, and normal tissues. The results showed significant difference between OL and OSCC (P = 0.0004). Intergroup comparison revealed no statistically significant difference between the study groups for cycle threshold (P = 0.6848). Histopathological examination of OSCC samples revealed five mild differentiation, four moderate differentiation, and four poorly differentiation. Spearman’s correlation coefficient of histopathological grading and expression was at 0.7813 (P < 0.001), respectively, indicating a strong positive correlation.
The study has various strengths and limitation. The strong positive aspect is that it was carried out on tissue samples that are more specific compared to serum, saliva, and cell lines. Quantitative RT-PCR was used which is the most reliable technique for evaluation of Sirtuin 3. There is inadequate research reported in literature evaluating SIRT3 expression in OSCC with grades of differentiation, OL, SWL, and normal. The study revealed that SIRT3 is expressed significantly in oral cancer followed by precancerous condition. The study stands novel as there is no literature evidence that have evaluated the Sirtuin 3 expression in normal, smokers, OL, and OSCC. This study reveals an alteration in Sirtuin 3 level in OL and OSCC when compared with that of normal tissues, thereby rejecting the null hypothesis (N0) and emphasizing that Sirtuin 3 can be used as a biomarker in OSCC, OL, SWL, and normal tissues. Hence, it can be added to the “oncogenic profile.” Hence, there is a need for further research with larger sample size for it to be used as a biomarker and that it could aid as a diagnostic or prognostic marker in near future. The limitations include restricted sample size, evaluation of single gene expression as it was a completely self-funded pilot research. Moreover, the target gene coding for Sirtuin 3 also could not be evaluated, which could have provided more insight on its role in molecular pathogenesis of oral cancer. The target gene in vitro proved to downregulate SIRT3, which was not evaluated in the study. Earlier studies performed in vitro revealed that downregulating Sirtuin arrests progression by preventing the cell colony formation. However, in-vivo study evidence is required to prove the pathogenesis. Evaluation of Sirtuin 3 along with its target gene is required to validate and generalize our results. This could have provided an insight over its role in cancer regulation. Further studies with more samples at multicentric studies evaluating target gene should be performed to validate the results of the study. This can pave way for establishing Sirtuin3 as a molecular marker either in cancer diagnostics or as prognostic indicator.
| Conclusion|| |
This is a novel study to evaluate the expression of Sirtuin 3 in OL in comparison with OSCC, SWL, and normal tissues by quantitative RT-PCR, which will add up to the current literature. As there was a significant elevation in the expression of Sirtuin 3 in OL when compared with SWL and normal tissues, it can be listed in “oncogenic profile” in forthcoming years. Future research with larger samples at multicentric studies evaluating the target gene is required for validation of the study.
I would like to thank my guides Dr C. L. Krithika, Dr G. Anuradha, Dr A. Kannan, Dr Arvind Ramanathan, Dr K. Velavan, and Dr K. S. Sethna Muthlakshmi for providing support throughout the research.
Financial support and sponsorship
It is a self-funded research.
Conflicts of interest
There are no conflicts of interest.
NR, CLK, and AK formulated the study. NR, CLK, and YA carried out the study. NR, CLK, and GA prepared, checked, and formulated the final manuscript.
Ethical policy and Institutional Review Board statement
This is to certify that the study entitled “Expression of Sirtuin 3 in oral cancer and oral leukoplakia: A cross-sectional observational study” has been completed as per the research protocol approved by the Institutional Review Board and Institutional Ethical Committee.
Patient declaration consent
Informed signed consent was obtained from all the patients willing to participate in the study. Patients not willing to give concern were not included in the study.
Data availability statement
All the data were collected from the patients attending the Outpatient Department in SRM Dental College, Ramapuram, Chennai and can be accessed for further references.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4]