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首页> 外文期刊>Tobacco Induced Diseases >Effects of cigarette smoke extract and heat-not-burn cigarette smoke extract on microRNA expression in keratinocyte cells
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Effects of cigarette smoke extract and heat-not-burn cigarette smoke extract on microRNA expression in keratinocyte cells

机译：香烟烟雾提取物和热不燃烧香烟烟雾提取物对角质形成细胞微RNA表达的影响

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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.
机译：目的：关于吸烟与癌症的关系，日本人因吸烟导致的口腔和咽喉癌的相对死亡风险增加了一倍以上。另一方面，不燃烧的热卷烟已开始在日本销售，并且最近据报道，它释放的致癌物质的浓度高于传统的加热卷烟。在这项研究中，明确了不燃烧的香烟和传统的加热香烟是否通过引起多步基因突变而参与了致癌作用。本研究的目的是基于针对微小RNA的全面基因表达分析获得新的证据。方法：将角化细胞衍生的HaCaT细胞系常规培养于DMEM和10％（v / v）FBS中。所有细胞均在37°C和5％（v / v）CO2的无抗生素培养基中生长。实验前将角质形成细胞血清饥饿16-18小时。为了进行活力分析和条件培养基的制备，将角质形成细胞用0–100μg/ ml CSC在37°C和5％（v / v）的CO2下处理48小时。收集细胞并保存在约20℃。加入MTS试剂（Promega），并在37°C，5％（v / v）CO2孵育48小时后，使用分光光度计在490 nm下测量OD。根据制造商的说明，使用RNeasy Mini Kit（Quiagen，Hilden，德国）从角质形成细胞中提取总RNA，并使用生物分析仪2100（Agilent，美国加利福尼亚州）对RNA进行定量。随后，使用miRNA PCR阵列平台（Human Cancer Pathway Finder miScript miRNA PCR array，MIHS-102Z，Qiagen），我们分析了这些细胞系中的miRNA图谱。为了确定该miRNA的可能靶标，我们通过TargetScan算法进行了生物信息学分析，并对人类癌细胞系和mRNA微阵列数据进行了综合分析，以鉴定其表达与计算机模拟预测的mRNA靶标的反向表达相关的miRNA。结论：从miRNA芯片数据生成的热图和暴露于未燃烧的香烟烟雾提取物对香烟烟雾提取物的角质形成细胞中miRNA基因表达水平的散点图观察到miRNA表达的差异。

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