Parvapan Bhattarakosol

Virome capture sequencing for comprehensive HPV genotyping in cervical samples

Objective This study aims to explore HPV genotyping in the cervical specimen using VirCapSeq by comparing the results with the reverse blot hybridization assay (REBA). Methods A secondary cross-sectional data of HPV genotypes in 35 cervical specimens was obtained from VirCapSeq and REBA methods. The .FASTQ files were downloaded from the NCBI Sequence Read Archive (SRA) (accession number PRJNA766412) and HPV genotyping was bioinformatically analyzed by mapping the sequences to the PaVE database. HPV genotypes detected by REBA and NGS were compared. All specimens were stratified by histology into cervical intraepithelial neoplasia grades 1 (CIN1) and 2/3 (CIN2/3). Results NGS via VirCapSeq detected HPV DNA in 100% of the samples, whereas the REBA (hybridization-based) assay diagnosed HPV DNA in 85.71%. While the limitation of the conventional methods for HPV genotyping is the use of primers or probes, NGS detected a broader range. The results showed that mixed infections were detected in all samples by NGS, with HPV16 and HPV52 being the most abundant genotypes. Conclusions HPV genome abundance, coverage, and diversity were associated with detection discrepancies between the methods, highlighting the enhanced sensitivity and diagnostic capabilities of NGS. These findings underscore the potential of NGS technologies for comprehensive HPV genotyping, advancing cervical cancer screening, and epidemiological studies. Future research should address cost barriers and expand cohort sizes to validate these findings.

Effect of NQO1 Downregulation on the Migration and Invasion of HPV16-Positive Cervical Cancer Cells

This study aimed to identify upregulated genes in HPV16-positive cervical cancer cells and investigate the impact of downregulating NAD(P) H:quinone oxidoreductase 1 (NQO1) on the survival of these cells. Transcriptomic sequencing (RNA-seq) was utilized to pinpoint upregulated genes and associated cancer-related pathways in HPV16-positive cervical cancer cells, comparing them to HPV-negative cervical cancer cells. NQO1 gene knockdown was performed in HPV16-positive cervical cancer cell lines to assess its effect on cell survival, including parameters such as cell proliferation, migration, invasion, cell cycle progression, apoptosis, and the expression of key proteins in the PI3K/AKT pathway, p53, and RECK. Genes with a fold change ≥4.0 in HPV16-positive cervical cancer cell lines were predominantly localized to the extracellular region and plasma membrane. These genes were involved in protein binding and cell adhesion, influencing cellular responses to stimuli and tissue development. KEGG pathway analysis identified the most significant pathways, including metabolic pathways, cancer pathways, MAPK signaling, and PI3K-AKT signaling. Knockdown of NQO1 significantly decreased cell proliferation, migration, and invasion, while increasing apoptosis in HPV16-positive cervical cancer cells (p ≤ 0.01). Additionally, proteins associated with the PI3K-AKT pathway were downregulated, while p53 and RECK protein levels were elevated. Our findings suggest that NQO1 plays a crucial role in promoting migration and invasion in HPV16-positive cervical cancer cells, highlighting its potential as a therapeutic target.

HPV16 E1 dysregulated cellular genes involved in cell proliferation and host DNA damage: A possible role in cervical carcinogenesis

HPV16 is the most prominent cause of cervical cancer. HPV16 E1, a helicase required for HPV replication exhibits increased expression in association with cervical cancer progression, suggesting that E1 has a similar effect on the host as the HPV16 E6 and E7 oncoproteins. This study aimed to determine whether expression of HPV16 E1 correlated with carcinogenesis by modulating cellular pathways involved in cervical cancer. HEK293T cells were transfected with pEGFP, pEGFPE1 or truncated forms of HPV16 E1. Cell proliferation, cell death, and the impact of HPV16 E1 on host gene expression was then evaluated. HPV16 E1 overexpression resulted in a significant reduction of cell viability and cellular proliferation (p-value<0.0001). Moreover, prolonged expression of HPV16 E1 significantly induced both apoptotic and necrotic cell death, which was partially inhibited by QVD-OPH, a broad-spectrum caspase inhibitor. Microarray, real time RT-PCR and kinetic host gene expression analyses revealed that HPV16 E1 overexpression resulted in the downregulation of genes involved in protein synthesis (RPL36A), metabolism (ALDOC), cellular proliferation (CREB5, HIF1A, JMJDIC, FOXO3, NFKB1, PIK3CA, TSC22D3), DNA damage (ATR, BRCA1 and CHEK1) and immune response (ISG20) pathways. How these genetic changes contribute to HPV16 E1-mediated cervical carcinogenesis warrants further studies.

Detection of Global DNA Methylation in Cervical Intraepithelial Neoplasia and Cancerous Lesions by Pyrosequencing and Enzyme-Linked Immunosorbent Assays

Cervical cancer is one of the most significant cancer found in women worldwide especially in developing countries. Previous reports showed that global DNA hypomethylation was correlated with various types of cancer including cervical cancer. Long interspersed nuclear element-1 (LINE1) pyrosequencing and Enzyme linked-immunosorbent assay (ELISA) assays were used for detection of global DNA methylation. The ELISA results were compared to bisulfite LINE1 pyrosequencing assay. Different cervical cancer cell lines (CaSki, SiHa, HeLa, ME180, MS751, C33A) showed low global methylation percentage when compared to normal white blood cells by ELISA assay (1.47%-5.09% vs 8.20%, respectively) and by LINE1 pyrosequencing (20%-45% vs 62%, respectively). Global DNA methylation levels in cervical cancer samples were lower than precancerous lesions (Normal-CIN3) by LINE1 pyrosequencing (mean, 48.8% vs 56.9%, respectively, p<0.05) and ELISA assay (mean, 3.03% vs 3.85%, respectively, p<0.05). Global DNA hypomethylation was predominantly found in cervical cancer samples detected by ELISA and LINE1 pyrosequencing assays and could be used as triage tests in cervical cancer screening. ELISA assay is a suitable method for detection of global  DNA methylation in large population; however, it should be further evaluated in a large clinical samples in order to be used as screening method.