Transcriptomic Profiling of Select Genes Implicated in Cervical Intraepithelial Neoplasia among Individuals of Reproductive Age in Ibadan, Nigeria
Description
Cervical cancer remains a leading cause of cancer-related mortality among women in Nigeria, primarily due to late detection. Genetic alterations in host genes contribute significantly to its pathogenesis. This study aims to validate the differential expression, mutational status, and clinical significance of SPP1, ANXA1, ANXA3, TMEM45A, IL-18, IGHG2, KRT10, CD177, ERO1α, ANKRD31, GSTA4, and MREG genes in patients presenting with symptoms of cervical cancer. A total of twenty-four (24) consenting female patients were recruited for this study; twenty-two (22) of them were those presenting with symptoms of cervical cancer, and the remaining two (2) were those who were clinically and histologically confirmed not to have any symptoms of cervical cancer. Tissue samples were obtained surgically and examined histopathologically. Total RNA was isolated, and gene expression levels were quantified using RT-qPCR. Gene expression across normal, tumour, and metastatic tissues, and their association with cancer hallmarks, were analysed using TNMPlot, muTarget, and Cancer Hallmark Enrichment Plot. Gene ontology and functional enrichment analyses were also performed. Histopathological analysis identified eight morphological categories: normal control (NC), mild infiltration/inflammation (MI), low-grade (LSIL) and high-grade squamous intraepithelial lesions (HSIL), chronic cervicitis (CC), well-differentiated squamous cell carcinoma (WISCC), moderately-differentiated squamous cell carcinoma (MSCC), and poorly-differentiated squamous cell carcinoma (PISCC). Most of the genes were upregulated in MI, LSIL, MSCC, HSIL, WISCC, and CC, indicating roles in proliferation, immune evasion, and metastasis. Notably, ERO1α, IGHG2, IL-18, TMEM45A, GSTA4, ANXA1, and ANXA3 were downregulated in early-stage lesions, supporting their potential as early diagnostic biomarkers. Collectively, the present study findings highlight the studied genes as key contributors to cervical cancer progression and underscore their potential utility as biomarkers for early detection and targeted therapy in Nigerian women.
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Ethical approval was obtained from the UI/UCH Ethics Committee, Institute for Advanced Medical Research and Training (IAMRAT) (UI/EC/23/0381). All procedures complied with the Declaration of Helsinki, and informed consent was obtained from participants. Twenty-four women were recruited: 22 with cervical cancer symptoms and 2 histologically confirmed as negative controls. Eligible participants were females aged 25–50 years with cervical dysplasia symptoms, while those with tumour recurrence or other cancers were excluded. Samples and gynaecological assessments were conducted at the University College Hospital (UCH), Ibadan (Jan–Nov 2023). Tissues were preserved in DNA/RNA Shield™ at –80 °C or fixed in 10% buffered formalin for histopathology. Molecular analyses were performed at Mols and Sims, Ado-Ekiti. Biopsies were processed at the Department of Pathology, UCH. After fixation, tissues underwent dehydration, clearing, paraffin embedding, and sectioning (3–5 μm). Sections were mounted, stained with H&E, and examined microscopically for epithelial, stromal, and transformation zone features by a certified pathologist. RNA was extracted using a modified Omotuyi et al (2018) protocol. Tissues were thawed (90–100 °C, 10 min), homogenised, centrifuged, and the supernatant were treated with sodium acetate and ethanol before incubation on ice (2 h). Pellets obtained by centrifugation were washed in 70% ethanol, air-dried, dissolved in RNase-free buffer, and quantified spectrophotometrically. Microarray datasets (GSE120691 and GSE223804) were retrieved from GEO. Twelve candidate genes (CD177, IGHG2, IL18, SPP1, ERO1α, ANKRD31, TMEM45A, KRT10, GSTA4, MREG, ANXA1, and ANXA3) were identified with differential fold-change values. Candidate genes were validated using qRT-PCR. One microgram of RNA was reverse-transcribed with iScript™ cDNA kit. Expression analysis was performed with SYBR Green Master Mix (KAPA SYBR® FAST) on an Applied Biosystems system in 20 μl reactions for 40 cycles (95 °C 10 min; 95 °C 15 s; 60 °C 30–60 s). GAPDH was the internal control, and expression levels were normalised using the 2−ΔΔCt method. TNMplot was used to compare gene expression across normal (15,648), tumour (40,442), and metastatic (848) cervical tissue samples. muTarget identified mutations influencing gene expression in cervical cancer using default thresholds (p ≤ 0.01; 0.714 > FC > 1.4), focusing on somatic mutations. Cancer Hallmark Enrichment Plot was applied to link the studied genes to established hallmarks of cancer. Functional analysis was conducted with WebGestalt using Over-Representation Analysis (ORA), covering GO categories (biological process, cellular component, molecular function) and KEGG pathways. qRT-PCR data were analysed in GraphPad Prism v9.5.1. Results are presented as mean ± SD. One-way ANOVA with Tukey’s post hoc test determined significance at p < 0.05.
Institutions
- University of IbadanOyo, Ibadan