Objective:
Concerning the association between smoking and cancer, the relative risk of death in Japanese oral and pharyngeal cancer due to smoking is more than doubled. On the other hand, heat-not-burn cigarettes have begun to be sold in Japan, and it has recently been reported to release carcinogens at higher concentrations than conventional heating cigarettes.
In this research, it was clarified whether the heat-not-burn cigarettes, as well as conventional heating cigarettes, are involved in carcinogenesis by causing multi-step gene mutation. The purpose of this study was to obtain new evidence based on comprehensive gene expression analysis focusing on microRNA.

Methods:
The Keratinocyte‐derived cell lines HaCaT was routinely cultured in DMEM and 10% (v/v) FBS. All cells were grown in antibiotic‐free media at 37°C and 5% (v/v) CO2. Keratinocyte cells were serum‐starved for 16-18 h before experimentation. For the viability assays and preparation of conditioned media, Keratinocyte cells were treated with 0–100 μg/ml CSC for 48 h at 37°C and 5% (v/v) CO2. Cells were collected and stored at −20°C. MTS reagent (Promega) was added and OD measured at 490 nm using a spectrophotometer following incubation at 37°C, 5% (v/v) CO2 for 48 h. The RNeasy Mini Kit (Quiagen, Hilden, Germany) was used to extract total RNA from keratinocyte according to the manufacturer's instructions, and RNA was quantified using a bioanalyzer 2100 (Agilent, California, US). Subsequently, using miRNA PCR array platform (Human Cancer Pathway Finder miScript miRNA PCR array, MIHS-102Z, Qiagen), we analyzed the miRNA profiles in these cell lines. To identify the possible targets of this miRNA, we performed bioinformatics analysis by TargetScan algorithm and integrated analysis across the data of human cancer cell lines and mRNA microarray data to identify miRNAs whose expression correlated with the inverse expression of mRNA targets predicted in silico.

Results and Conclusions:
Heat map generated from miRNA microarray data and scatterplot of miRNA gene expression level in keratinocyte cells exposed to heat-not-burn cigarette smoke extract against cigarette smoke extract are differences in miRNA expression were observed.