Microarray and bioinformatic analysis of conventional ameloblastoma: an observational analysis

Abstract

Ameloblastoma is a highly aggressive odontogenic tumor, and its pathogenesis is associated with many participating genes. Objective We aimed to identify and validate new critical genes of conventional ameloblastoma using microarray and bioinformatics analysis. Methodology Gene expression microarray and bioinformatic analysis were performed using CHIP H10KA and DAVID software for enrichment. Protein-protein interactions (PPI) were visualized using STRING-Cytoscape with MCODE plugin, followed by Kaplan-Meier and GEPIA analyses that were used for the candidate’s postulation. RT-qPCR and IHC assays were performed to validate the bioinformatic approach. Results 376 upregulated genes were identified. PPI analysis revealed 14 genes that were validated by Kaplan-Meier and GEPIA resulting in PDGFA and IL2RA as candidate genes. The RT-qPCR analysis confirmed their intense expression. Immunohistochemistry analysis showed that PDGFA expression is parenchyma located. Conclusion With bioinformatics methods, we can identify upregulated genes in conventional ameloblastoma, and with RT-qPCR and immunoexpression analysis validate that PDGFA could be a more specific and localized therapeutic target.

LRP5, SLC6A3, and SOX10 Expression in Conventional Ameloblastoma

“…Briefly, 50 μm slides of each sample were obtained, de-paraffinized, and total RNA was obtained following the manufacturer’s protocol and diluted in 30 μL of RNase-free water. To determine the RNA concentration and purity, a NanoDrop ND-2000 spectrophotometer (Thermo Fisher, Rochester, NY, USA) was employed, considering only samples with a 260/280 ratio of ≥1.8 [ 14 ]. RT-qPCR was performed using a QuantiNova SYBR Green RT-PCR Kit (Qiagen, Cat.…”

“…Antigenic retrieval was performed in 10 mM citrate buffer in a microwave histoSTATION at 100 °C for 5 min in GPR/20S histomodule (KOS Millestone, Sorisole, BG, Italy). Endogenous peroxidase nonspecific background blocking was performed, as reported previously [ 14 ]. The slides were incubated overnight at 4 °C with primary antibodies for SOX10 (sc-365692, Santa Cruz Biotechnology, Paso Robles, CA, USA), SLC6A3 (sc-32259, Santa Cruz Biotechnology, Paso Robles, CA, USA), and LRP5 (GTX64412, GeneTex, Irvine, CA, USA), all adjusted to concentrations of 1:200.…”