Supplementary MaterialsSupplementary information 41598_2018_25498_MOESM1_ESM. significantly dysregulated miRNAs (4 fold; p??0.05) in chewing tobacco treated cells and 6 in cigarette smoke exposed cells. We integrated this data with global proteomic data and recognized 36 protein targets that showed inverse expression pattern in chewing tobacco treated cells and 16 protein targets that showed inverse expression in Celastrol pontent inhibitor smoke uncovered cells. In addition, we recognized 6 novel miRNAs in chewing tobacco treated cells and 18 novel miRNAs in smoke uncovered cells. Integrative analysis of dysregulated miRNAs and their targets indicates that signaling mechanisms leading to oncogenic transformation are unique between both forms of tobacco. Our study demonstrates alterations in miRNA expression in oral cells in response to two frequently used forms of tobacco. Introduction Oral squamous cell carcinoma (OSCC) is one of the most common cancers worldwide and remains the most common malignancy of the head and neck cancers. Tobacco use, alcohol consumption and human papilloma computer virus (HPV) 16/18 have been identified as the main risk factors for the initiation and progression of OSCC1. Tobacco is mainly consumed worldwide in the form of manufactured smokes. Tobacco is also consumed in the form of smokeless tobacco, especially chewing tobacco in South-East Asian countries2. Despite being one of the most Celastrol pontent inhibitor common cancers in India, molecular alterations in oral malignancy development in tobacco chewers and smokers is not well understood. MicroRNAs have been established as important regulators of oncogenic potential in cells. Alterations at the genetic and epigenetic levels in the complex enzymatic machinery involved in miRNA biogenesis can result in aberrant miRNA expression3. Post-transcriptional regulation of gene expression by miRNAs has an influence on multiple pathways, including those involved in cellular transformation and proliferation4. miRNAs function as either oncogenes or tumor suppressors, playing crucial functions in tumorigenesis, tumor invasion and Celastrol pontent inhibitor metastasis5. In recent years multiple studies have revealed the altered expression of miRNAs which play a Rabbit Polyclonal to ARMX1 role in the development and progression of diverse cancers including oral squamous cell carcinoma6C8. Increased expression of microRNAs including miR-155 and miR-23a, have been observed in oral cancer patients that are tobacco chewers compared to non-chewers9,10. Fanconi anemia complementation group G protein (FANCG) is usually a miR-23a target with a role in DNA double strand break repair pathway. Decreased expression of FANCG in normal oral Celastrol pontent inhibitor fibroblasts contributes to the development of carcinogenesis on treatment with areca nut10. In contrast, microRNAs such as miR-145 are found Celastrol pontent inhibitor to be significantly downregulated upon cigarette smoke condensate treatment in oral fibroblasts while its target protein MMP-2 is usually overexpressed which plays a key role in perturbation of stromal-epithelial communication and promotes pro-tumorogenic interactions11. Similarly, a decrease in miR-101-3p and a corresponding increase in expression levels of its target protein COX2 was observed in an esophageal non-tumorigenic cell collection upon treatment with cigarette smoke condensate thereby facilitating cell transformation and cancer development12. Downregulation of miR-200c levels in human bronchial epithelial cells with increased expression of IL-6 and activation of nuclear factor-B (NF-B) pathway by cigarette smoke extract is also shown to regulate epithelial-mesenchymal transition and carcinogenesis13. Taken together, these studies show that molecular mechanisms for cellular transformation may vary depending upon the form of tobacco used. Till date no study has systematically investigated the differences in molecular alterations induced in oral cells upon exposure to different forms of tobacco. To achieve this, we developed two cellular models where immortalized, oral keratinocytes (OKF6/TERT1) were chronically treated with either chewing tobacco or exposed to cigarette smoke for a period of six months. To understand specific molecular alterations brought about by each form of tobacco, we performed miRNA sequencing of oral keratinocytes chronically treated with chewing tobacco/cigarette smoke. miRNA dysregulation is usually in turn known to impact expression of their target proteins leading to diverse functional effects. Hence, in addition to studying miRNA dysregulation, we have investigated proteomic alterations associated with exposure to these two forms of tobacco. We observed that chronic treatment of, immortalized oral keratinocytes with either chewing tobacco extract or cigarette smoke condensate affected expression of distinct set of miRNAs and their corresponding protein targets. Results Chronic exposure to chewing tobacco and cigarette smoke results in phenotypic changes in oral cells In this study, we chronically treated immortalized oral keratinocytes (OKF6/TERT1) with chewing tobacco extract and cigarette smoke condensate to model the effects of tobacco chewing and smoking in oral cancer. We observed an increase in proliferative capability of both OKF6/TERT1-Tobacco and OKF6/TERT1-Smoke.