Dynamic changes in the gene expression profile during rat oral carcinogenesis induced by 4-nitroquinoline 1-oxide

Abstract

The typical progression of oral cancer is from hyperplastic epithelial lesions through dysplasia to invasive carcinoma. It is important to investigate malignant oral cancer progression and development in order to determine useful approaches of prevention of dysplastic lesions. The present study aimed to gain insights into the underlying molecular mechanism of oral carcinogenesis by establishing a rat model of oral carcinogenesis using 4‑nitroquinoline 1‑oxide. Subsequently, transcription profile analysis using an integrating microarray was performed. The dynamic gene expression changes of the six stages of rat oral carcinogenesis (normal, mild epithelial dysplasia, moderate dysplasia, severe dysplasia, carcinoma in situ and oral squamous cell carcinomas) were analyzed using component plane presentations (CPP)‑self‑organizing map (SOM). Six genes were verified by quantitative polymerase chain reaction, immunohistochemistry and succinate dehydrogenase (SDH) activity assay kit. Numerous differentially expressed genes (DEGs) were identified during rat oral carcinogenesis. CPP‑SOM determined that these DEGs were primarily enriched during cell cycle, apoptosis, inflammatory response and tricarboxylic acid cycle, indicating the coordinated regulation of molecular networks. In addition, the expression of specific DEGs, such as janus kinase 3, cyclin‑dependent kinase A‑1, B‑cell chronic lymphocytic leukaemia/lymphoma 2‑like 2, nuclear factor‑κB, tumor necrosis factor receptor superfamily member 1A, cyclin D1 and SDH were identified to have high concordance with the results from microarray data. The current study demonstrated that oral carcinogenesis is a multi‑step and multi‑gene process, with a distinct pattern alteration along a continuum of malignant transformation. In addition, this comprehensive investigation provided a theoretical basis for the understanding of the molecular alterations associated with oral carcinogenesis.

Figure 1

Pathological evidence of carcinogenesis in rat tongues. (A) Normal squamous epithelium of the tongue. Magnification, ×400. (B) Mild epithelial dysplasia of the tongue exhibited loss of polarity of the deeper cell layers of the epithelium and mild nuclear pleomorphism of cells. Magnification, ×200. (C) Moderate epithelial dysplasia of the tongue indicated a basaloid appearance, loss of polarity of cells and intercellular cohesion. Magnification, ×400. (D) Severe epithelial dysplasia of the tongue exhibited a proliferation of basal cells, grossly disturbed stratification, the loss of polarity of cells, individual cell keratinization and nuclear pleomorphism of cells. Magnification, ×400. (E) Carcinoma in situ of the tongue exhibited the proliferation of primitive basal epithelial cells from the basement membrane to the surface, marked nuclear atypia and the full thickness of the epithelium. Magnification, ×200. (F) Squamous cell carcinoma of the tongue showed gross disruption of normal epithelial architecture, prominent cellular pleomorphism, the formation of dyskeratosis with keratin pearl and invasion into underlying connective tissues. Magnification, ×100.

Figure 2

Differentially expressed genes and SOM output data at each stage during rat oral carcinogenesis. (A) The numbers of up/downregulated genes in tongue tissues at different stages during carcinogenesis. (B) Illustration of SOM output microarray data by component plane presentations. Each of these presentations appears as genome-wide transcriptional display, in which all upregulated units (red), downregulated units (blue) and moderately transcribed units (green and yellow) are well-delineated. Color-coding index represents log₂-transformed ratios, and the brighter color denotes the higher value. The following 7 major regulatory categories are recognizable: i) 1/7, Down/upregulated genes at the early stage (MiD and MoD); ii) 2/6, down/upregulated genes at the late stage (SCC); iii) 3/5, down/upregulated genes at the intermediate stage (SD and CIS); iv) 4, upregulated genes at the intermediate stage (SD and CIS). SOM, self-organizing map; MiD, mild epithelial dysplasia; MoD, moderate epithelial dysplasia; SCC, squamous cell carcinoma; SD, severe epithelial dysplasia; CIS, carcinoma in situ.

Figure 3

Functional enrichment of DEGs during rat oral carcinogenesis. (A) Gene ontology enrichment analysis of DEGs at different pathological stage. Illustration of DEGs underlying (B) TCA cycle and and (C) cell cycle during oral carcinogenesis. DEGs at the early stage are highlighted by background pink (upregulation) and light-blue (downregulation), whereas DEGs at intermediate and late stage are indicated by red (upregulation) and blue (downregulation). Synergistically/additively impacted genes in the cotreatment series are designated with asterisks. Unchanged/unmentioned genes in the experimental data are marked and framed by black with light background. DEG, differentially expressed gene; TCA, tricarboxylic acid cycle.

Figure 4

Verification of mRNA levels of Jak3, Cdc2a, Bcl2I2, NF-κB and Tnfrsf1a by reverse transcription-quantitative polymerase chain reaction. ^*P<0.05 vs. MiD and MoD, ^#P<0.05 vs. MiD, MoD and SD. Jak3, janus kinase 3; Cdc2a, cyclin dependant kinase; Bcl2l2, Bcl-2-like 2; NF-κB; nuclear factor-κB; Tnfrsf1a, tumor necrosis factor receptor 1; MiD, mild epithelial dysplasia; MoD, moderate epithelial dysplasia; SD, severe epithelial dysplasia; CIS, carcinoma in situ; SCC, squamous cell carcinoma.

Figure 5

Immunohistochemical staining of NF-κBp65 and cyclin D1 in tongue tissues during rat oral carcinogenesis. (A) Immunostaining for NF-κBp65 in mild epithelial dysplasia tissue. (B) Immunostaining for NF-κBp65 in squamous cell carcinoma tissue. (C) Immunostaining for cyclin D1 in mild epithelial dysplasia tissue. (D) Immunostaining for cyclin D1 in squamous cell carcinoma tissue. Original magnification, ×400. NF-κBp65, nuclear factor-κBp65.

Figure 6

(A) mRNA expression and activity of (B) SDH during rat oral carcinogenesis. ^*P<0.05 and ^**P<0.05 vs. normal group. SDH, succinate dehydrogenase; Mid, mild epithelial dysplasia; Mod, moderate epithelial dysplasia; SD, severe epithelial dysplasia; SCC, squamous cell carcinoma.