Gene expression in cell lines from human ameloblastoma immortalized using hTERT and HPV16‐E6/E7

Abstract

Objective The aim of this study was to evaluate and compare alterations in gene expression using two distinct immortalization methods (hTERT and HPV16‐E6/E7) in ameloblastoma cell lines. Materials and Methods A primary cell culture derived from human ameloblastoma (AME‐1) was established and immortalized by two different methods using a transfection processes to hTERT and HPV‐E6/E7. The RNA‐seq was used to verify which immortalization method had less influence on gene expression. It was performed in four steps: extraction and collection of mRNA, PCR amplification, comparison with the human reference genome, and analysis of differential expression. The genes with differentiated expression were identified and mapped. Results RNA‐seq revealed genetic alterations in ameloblastoma cell lines after the immortalization process, including increased expression of tumor genes like MYC, E2F1, BRAF, HRAS, and HTERT, and a decrease in tumor suppressor genes like P53, P21, and Rb. Conclusions It is possible to affirm that cell immortalization is not an inert method regarding gene regulation mechanisms and the hTERT method (AME‐TERT) presented fewer changes in gene expression levels.

Interrogation of TERT promoter hotspot mutations in ameloblastoma and ameloblastic carcinoma

“…12 In most human cancers telomerase is reactivated during carcinogenesis by expression of the catalytic subunit telomerase reverse transcriptase (TERT), resulting in replicative immortality. 5 Immortalization of ameloblastoma cells through TERT overexpression is a successful model for in vitro research, 13,14 and previous studies showed elevated levels of TERT expression and telomerase activity in ameloblastoma. 10,11,15 Several studies demonstrated that activation of telomerase activity is highly correlated with TERT gene transcription.…”

Immunoexpression of stem cell markers SOX-2, NANOG AND OCT4 in ameloblastoma

“…The clinical data of the AME samples were acquired through medical records with reports present in the files, collected manually, and histologically classified by two oral pathologists. For the in vitro study, the cell line derived from human AME, called AME-hTERT, established at the Cell Culture Laboratory of the Faculty of Dentistry, Federal University of Pará (UFPA) ( Cruz et al, 2021 ), was used. This study was registered and approved by the Human Research Ethics Committee of the Health Sciences Institute of the Federal University of Pará—CEP/ICS/UFPA (CAAE: 30647720.6.0000.0018).…”

Expression of stem cell markers SALL4, LIN28A, and KLF4 in ameloblastoma

“…For the in vitro study, the cell line derived from human AME, called AME-hTERT, established at the Cell Culture Laboratory of the Faculty of Dentistry, Universidade Federal do Pará (UFPA), was used [ 32 ]. For the in situ study, 21 cases of conventional AME, ten cases of dentigerous cysts (DC), and ten cases of dental follicles (DF) were collected at the Laboratory of Pathological Anatomy and Immunohistochemistry of the Graduate Program in Dentistry, Universidade Federal do Pará, and Centro Universitário do Pará (CESUPA).…”