Author(s):
Santhoshkumar Rajendran, Shankar Krishnan, Mayuri Iyer, Ravi Kannan, Litika Vermani, Rajeev Kumar
Email(s):
rajeev.kvhfc@cacharcancerhospital.org
DOI:
10.52711/0974-360X.2026.00631 
Address:
Santhoshkumar Rajendran1,2, Shankar Krishnan1, Mayuri Iyer2, Ravi Kannan3,Litika Vermani2, Rajeev Kumar2*
1Electronics and Instrumentation Engineering, National Institute of Technology Silchar, Assam - 788010, India.
2Research Division, Cachar Cancer Hospital and Research Centre, Silchar, Assam - 788015, India.
3Surgical Oncology, Cachar Cancer Hospital and Research Centre, Silchar - 788015, India.
*Corresponding Author
Published In:
Volume - 19,
Issue - 10,
Year - 2026
ABSTRACT:
Tobacco related cancers are major threat to public health in north east region of India, particularly the oral cancer. To reveal mechanism behind the molecular changes, studying their gene expression profile is essential. However, the accurate quantification of gene expression remains challenging due to variability in reference gene stability in quantitative polymerase chain reaction (qPCR). Hence, this study aimed to identify an optimal reference gene for the assessment of gene expression in oral squamous cell carcinoma (OSCC) and to evaluate molecular markers of tumour progression in relation to overall survival. Twenty histopathologically confirmed OSCC tissues from chronic tobacco users were analysed. Using qPCR, six candidate reference genes (B2M, GAPDH, GUSB, PUM1, PMM1, and RPN1) were evaluated. Stability was assessed using multiple algorithms including the ?Ct method and RefFinder. The most stably expressed gene was used for normalisation to evaluate the expression pattern of biomarkers including ALDH1, E-cadherin, Vimentin, and TWIST1. Expression profiles were compared between patients with overall survival of <18 and =18 months. In this preliminary study, PUM1 was identified as the most stable reference gene by consensus ranking across algorithms, followed by RPN1 gene. When PUM1 applied for normalisation, elevated mRNA expression of vimentin was observed in patients with poor survival (p<0.05). No significant changes were found in the expression of TWIST1, ALDH1, and E-cadherin. The pertinency of PUM1 as a suitable reference gene is supported by this preliminary data. Further validation across larger and more diverse OSCC cohorts is recommended to substantiate these findings.
Cite this article:
Santhoshkumar Rajendran, Shankar Krishnan, Mayuri Iyer, Ravi Kannan,Litika Vermani, Rajeev Kumar. PUM1 as Reference Gene for EMT and CSC Marker Expression in Tobacco-Associated Oral Squamous Cell Carcinoma. Research Journal Pharmacy and Technology. 2026;19(10):4535-2. doi: 10.52711/0974-360X.2026.00631
Cite(Electronic):
Santhoshkumar Rajendran, Shankar Krishnan, Mayuri Iyer, Ravi Kannan,Litika Vermani, Rajeev Kumar. PUM1 as Reference Gene for EMT and CSC Marker Expression in Tobacco-Associated Oral Squamous Cell Carcinoma. Research Journal Pharmacy and Technology. 2026;19(10):4535-2. doi: 10.52711/0974-360X.2026.00631 Available on: https://rjptonline.org/AbstractView.aspx?PID=2026-19-10-7
REFERENCES:
1. Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians. 2024 Aug; 74(3): 229–263. doi: 10.3322/caac.21834
2. Chaturvedi P, Singh A, Chien CY, Warnakulasuriya S. Tobacco related oral cancer. The BMJ. 2019; 365: l2142. doi: 10.1136/bmj.l2142
3. Shobiya D, Prince V. A study to assess the oral health status and knowledge on oral health hazards among tobacco consuming adults in Nanchiyampalayam, Dharapuram in view of preparing a self-instructional module. International Journal of Nursing Education and Research. 2019; 7(1): 15–20. doi: 10.5958/2454-2660.2019.00004.8
4. Shiv Kumar Patel, Moumita Sinha, Mitashree Mitra. Glutathione-S-transferase M1 and T1 gene Polymorphism as Risk Factors of Oral Squamous Cell Carcinoma: A Preliminary Investigation. Research J. Pharm. and Tech. 2012; 5(7): 918-920.
5. ICMR-NCDIR. Report on monitoring survey of cancer risk factors and health system response in North East Region (NER) of India. 2022. Available from: https://www.ncdirindia.org
6. Sangeetha. R, Deepa Rajendiran, Soniya. R, Savithri. S. Therapeutic Strategies in Oral Cancer - A Review. Research Journal of Pharmacy and Technology. 2024; 17(3): 1398-2. doi: 10.52711/0974-360X.2024.00221
7. Shiv Kumar Patel, Moumita Sinha, Mitashree Mitra. Epidemiological and Socio-demographic Profile of Oral Cancer Patients of Chhattisgarh: A Retrospective Study. Research J. Science and Tech. 2012; 4(4): 145-147.
8. Roshan Kumar Jha, Ranjit S. Ambad, Priya Koundal, Akansha Singh. Effect of Tobacco on Lipoprotein Profile: A Comparative study among Smokers and Chewers. Research Journal of Pharmacy and Technology. 2021; 14(9): 4973-6. doi: 10.52711/0974-360X.2021.00865
9. Ananya Madiyal, Subhas G. Babu, Suchetha Kumari N, Sharmila K. P, Prajna U, Deesha Kumari, Mohammed Faizal Asan. Status of salivary human beta defensin-2 in oral potentially malignant disorders and oral cancer: Quest for a novel non-invasive biomarker. Research Journal of Pharmacy and Technology.2024; 17(4):1799-4. doi: 10.52711/0974-360X.2024.00286
10. Syed Saif Imam. Sublingual Tablets Amalgamated with Nano-particles and natural products to treat Oral Cancer. Research Journal of Pharmacy and Technology. 2024; 17(5): 2056-2. doi: 10.52711/0974-360X.2024.00326
11. Smith A, Teknos TN, Pan Q. Epithelial to mesenchymal transition in head and neck squamous cell carcinoma. Oral Oncology. 2013 Aug; 49(4): 287–292. doi: 10.1016/j.oral oncology.2012.10.009
12. Vermani L, Kumar R, Kannan RR, Deka MK, Talukdar A, Kumar NS. Expression pattern of ALDH1, E-cadherin, vimentin and Twist in early and late onset sporadic colorectal cancer. Biomarkers in Medicine. 2020 Aug; 14(14): 1371–1382. doi: 10.2217/bmm-2020-0206
13. Wagner EM. Monitoring gene expression: quantitative real-time RT-PCR. Methods in Molecular Biology. 2013; 1027: 19–45. doi: 10.1007/978-1-60327-369-5_2
14. Bustin SA, Ruijter JM, van den Hoff MJB, Kubista M, Pfaffl MW, Shipley GL, et al. MIQE 2.0: revision of the minimum information for publication of quantitative real-time PCR experiments guidelines. Clinical Chemistry. 2025 Aug; 71(6): 634–651. doi: 10.1093/clinchem/hvaf043
15. V. Padmaharish. Salivary Biomarkers of Oral Cancer – A Review. Research J. Pharm. and Tech. 2016; 9(7): 1007-1012. doi: 10.5958/0974-360X.2016.00190.6
16. Nauma Hafeez. Creatine phosphokinase levels in Oral cancer Patients. Research J. Pharm. and Tech 2016; 9(10): 1577-1580. doi: 10.5958/0974-360X.2016.00310.3
17. James B, Zaidi S, N BS, V R, Dokhe Y, Shetty V, et al. Reference gene evaluation for normalization of gene expression studies with lymph tissue and node derived stromal cells of patients with oral squamous cell carcinoma. Oncology Letters. 2024 Aug;28(5). doi: 10.3892/ol.2024.14673
18. Jo J, Choi S, Oh J, Lee SG, Choi SY, Kim KK, et al. Conventionally used reference genes are not outstanding for normalization of gene expression in human cancer research. BMC Bioinformatics. 2019 Aug; 20: 245. doi: 10.1186/s12859-019-2809-2
19. Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics. 2012; 13: 134.
20. Vermani L, Kumar R, Senthil Kumar N. GAPDH and PUM1: optimal housekeeping genes for quantitative polymerase chain reaction-based analysis of cancer stem cells and epithelial-mesenchymal transition gene expression in rectal tumors. Cureus. 2020 Dec;12(12):e12020. doi: 10.7759/cureus.12020
21. Gu Y, Tang S, Wang Z, et al. A pan-cancer analysis of the prognostic and immunological role of β-actin (ACTB) in human cancers. Bioengineered. 2021; 12(1): 6166-6185. doi:10.1080/21655979.2021.1973220
22. Yang H, Chen W, Tan Z, Lin J, Liu Z, Ji F, et al. Let-7b miRNA-loaded nanostructures inhibited human tongue squamous cell carcinoma metastasis by the MOR-SRC-EGFR axis. Current Research in Biotechnology. 2024 Jan; 7: 100213. doi: 10.1016/j.crbiot.2024.100213
23. Chen KC, Schmuck K, Tietze LF, Roffler SR. Selective cancer therapy by extracellular activation of a highly potent glycosidic duocarmycin analogue. Molecular Pharmaceutics. 2013 Aug; 10(5): 1773–1782. doi: 10.1021/mp300581u
24. Ghasemi O, Karami F, Salahshourifar I, Noori Daloii MR. Expression analysis of genes in oral squamous cell carcinoma patients. Journal of Sciences, Islamic Republic of Iran. 2021 May; 32(2): 115–120. doi: 10.22059/jsci.2020.310939.1007576
25. Mukherjee S, Chakraborty S, Basak U, Pati S, Dutta A, Dutta S, et al. Breast cancer stem cells generate immune-suppressive T regulatory cells by secreting TGFβ to evade immune-elimination. Discover Oncology. 2023 Dec; 14(1): 87. doi: 10.1007/s12672-023-00787-z
26. Haznedaroglu Benlioglu E, Dogan AL. Inhibition of serum and glucocorticoid inducible kinase 1 (SGK1) in triple negative breast cancer. UHOD - Uluslararasi Hematoloji-Onkoloji Dergisi. 2023 Jan; 33(1): 37–43. doi: 10.4999/uhod.236832
27. Xie F, Wang J, Zhang B. RefFinder: a web-based tool for comprehensively analyzing and identifying reference genes. Functional & Integrative Genomics. 2023; 23(2): 125.
28. Ng HF, Ngeow YF. A simple spreadsheet-based method for relative quantification using quantitative real-time PCR. Biochemistry and Molecular Biology Education. 2022 Aug; 50(1): 99–103. doi: 10.1002/bmb.21596
29. Orlov YL, Pindyurin AV, Kiselev V, Yang S, Krasnov GS, Kudryavtseva AV, et al. Pan-cancer analysis of TCGA data revealed promising reference genes for qPCR normalization. Frontiers in Genetics. 2019; 10: 97. doi: 10.3389/fgene.2019.00097
30. Satutya Wicaksono, Jola Rahmahani, Diah Savitri Ernawati, Fedik Abdul Rantam, Theresia Indah Budhy, et al. Mesenchymal Stem Cell Secretome as Novel Regenerative Approach in Oral Ulcerative Lesions Management: A Review. Research Journal of Pharmacy and Technology. 2024; 17(3): 1408-3. doi: 10.52711/0974-360X.2024.00223
31. Reethu Ravichandiran, J. Venkatesh, A. John William Felix, K. Ramya. Prevalence of Oral mucosal lesion in patients with Tobacco related habits among Chidambaram population – A Cross-sectional study. Research Journal of Pharmacy and Technology. 2022; 15(8): 3377-1. doi: 10.52711/0974-360X.2022.00565
32. Nowak E, Bednarek I, Diomede F, Trubiani O, Pizzicanella J, Marconi GD. Aspects of the epigenetic regulation of EMT related to cancer metastasis. Cells. 2021 Aug; 10(12): 3435. doi: 10.3390/cells10123435
33. da Silva SD, Marchi FA, Su J, Yang L, Valverde L, Hier J, et al. Co-overexpression of TWIST1-CSF1 is a common event in metastatic oral cancer and drives biologically aggressive phenotype. Cancers (Basel). 2021 Aug; 13(1): 153. doi: 10.3390/cancers13010153
34. Zhou J, Tao D, Xu Q, Gao Z, Tang D. Expression of E-cadherin and vimentin in oral squamous cell carcinoma. International Journal of Clinical and Experimental Pathology. 2015; 8(3): 3150–3154.
35. Chaw SY, Majeed AA, Dalley AJ, Chan A, Stein S, Farah CS. Epithelial to mesenchymal transition (EMT) biomarkers – E-cadherin, beta-catenin, APC and vimentin – in oral squamous cell carcinogenesis and transformation. Oral Oncology. 2012 Aug; 48(10): 997–1006. doi: 10.1016/j.oraloncology.2012.05.011
36. Puneeta N, Santosh T, Mishra I, Gaikwad P, Sahu A. Evaluation of E-cadherin and vimentin expression for different grades of oral epithelial dysplasia and oral squamous cell carcinoma: an immunohistochemical study. Journal of Oral and Maxillofacial Pathology. 2022 Aug; 26(2): 285. doi: 10.4103/jomfp.jomfp_166_20