Multiomic analysis of oral keratinocytes chronically exposed to shisha

Abstract

Background Tobacco is smoked in different form including cigarettes and water pipes. One popular form of water pipe smoking especially in Middle Eastern countries is shisha smoking. Shisha has been associated with various diseases including oral cancer. However, genomic alterations and gene expression changes associated with chronic shisha exposure have not been previously investigated. Objectives Whole‐exome sequencing and gene expression profiling of immortalized human oral keratinocytes (OKF6/TERT1) cells chronically treated with 0.5% shisha extract for a period of 8 months was undertaken to characterize molecular alterations associated with shisha exposure. Methods Genomic DNA and RNA were extracted and preprocessed as per manufacturer's instruction and subjected to whole‐exome and transcriptome sequencing using Illumina HiSeq2500 platform. Exome was analyzed using GATK pipeline whereas RNA‐Seq data was analyzed using HiSat2 and HTSeq along with DESeq to elucidate differentially expressed genes. Results Whole‐exome sequence analysis led to identification of 521 somatic missense variants corresponding to 389 genes RNA‐Seq data revealed 247 differentially expressed genes (≥2‐fold, P‐value<0.01) in shisha treated cells compared to parental cells. Pathway analysis of differentially expressed genes revealed that interferon‐signaling pathway was significantly affected. We predict activation of MAPK1 pathway which is known to play a key role in oral cancer. We also observed allele specific expression of mutant LIMA1 based on RNA‐Seq dataset. Conclusion Our findings provide insights into genomic alterations and gene expression pattern associated with oral keratinocytes chronically exposed to shisha.

Whole-Exome Sequencing Analysis of Oral Squamous Cell Carcinoma Delineated by Tobacco Usage Habits

“…A recent study by Maitra et al reported a similar observation of higher C>G transversion in gingiva-buccal OSCC patients with a tobacco-chewing history (12). Another study by our group employed whole-exome and RNA-Seq analyses of oral keratinocytes exposed to Shisha tobacco and demonstrated enrichment of Interferon signaling pathway and activation of MAPK1 pathway (13).…”

Interventions to prevent periodontal disease in tobacco‐, alcohol‐, and drug‐dependent individuals

“…47 The effects of waterpipe smoking on periodontal and oral health have been examined through cross-sectional observations [51][52][53][54][55][56][57][58][59][60][61][62][63][64][65][66][67][68][69] and limited in vitro assays. 70,71 However, the demographics of waterpipe usage limits the majority of human studies to males of Middle Eastern origin. These methodologically limited studies demonstrate lower mean age-adjusted periodontal bone levels, deeper vertical bony defects, deeper periodontal probe depths, lower gingival bleeding, and higher plaque levels in waterpipe users.…”

“…Waterpipe smoking also increased the risk of developing peri-implantitis. 51,52,54,63,64,69 Microbiologic, immunologic, and histochemical studies have demonstrated a dose-dependent cytotoxic effect 68,70,71,[73][74][75] and mutagenic effect 75 on buccal mucosa, as well as enrichment of specific pathogenic bacteria 76 and Candida albicans 61,67 in waterpipe users.…”

“…The effects of waterpipe smoking on periodontal and oral health have been examined through cross‐sectional observations and limited in vitro assays . However, the demographics of waterpipe usage limits the majority of human studies to males of Middle Eastern origin.…”

Signaling alterations in oral keratinocytes in response to shisha and crude tobacco extract

“…Recently, a series of multiomic studies carried out by our group provided substantial evidence to indicate that chronic exposure to WS leads to oncogenic transformation in normal human oral keratinocytes. [5][6][7][8] The main component used in these studies is 'shisha'-a processed tobacco variant blended with flavors and additives.…”