The effect of Maras powder and smoking on the microRNA deregulation of oral mucosa

Abstract Objective This study aimed to investigate the effects of Maras powder (a type of smokeless tobacco obtained from Nicotiana rustica Linn and mixed with the ashes of wood, especially from oak, walnut or grapevine) on the microRNA (miRNA) deregulation of oral mucosa, and it compares these effects with those of smoking. Methodology Oral mucosal samples were collected from 74 patients, consisting of 16 nonusers, 26 smokers, and 32 Maras powder users. Genes associated with oral cancer were selected and 90 microRNAs targeting these genes were identified. MicroRNA were isolated and purified using the microRNA isolation kit. MicroRNA were expressed using Fluidigm RT-PCR. Results A positive correlation between the duration of Maras powder use with miR-31 expression levels, and a negative correlation between the Maras powder chewing time and miR-372 expression levels was found. In addition, there is a negative correlation between the amount of Maras powder consumed and expression levels of miR-375, miR-378a, miR-145, and miR-10b; moreover, another negative correlation is observed between the number of cigarettes consumed and the expression levels of miR-23a, miR-23b, miR-203a, miR-200b, and miR-375. However, miR-200b and miR-92a levels were downregulated significantly more in Maras powder users when compared with smokers and nonusers (p<0.05). Conclusion The results show both chewing Maras powder and smoking have an effect on deregulation of miR-200b and miR-92a expressions. This leads to the belief that assessing the expression of these two miRNAs is a promising noninvasive method of analysis, especially in mutagen exposures. Finally, large-scale and high-throughput studies may help to identify an extensive miRNA expression profile associated with tobacco use and improve the understanding of oral malignancies.

摘要
研究目的：本研究旨在探究马拉斯烟粉（Maras powder，一种由黄花烟草（Nicotiana rustica Linn）制成并混合木材灰（尤其是橡木、胡桃木或葡萄藤灰）的无烟烟草制品）对口腔黏膜微核糖核酸（microRNA，简称miRNA）表达失调的影响，并将其与吸烟的影响进行对比。
研究方法：本研究共收集74例患者的口腔黏膜样本，其中非使用者16例、吸烟者26例、马拉斯烟粉使用者32例。筛选与口腔癌相关的基因，并鉴定出靶向这些基因的90种miRNA。采用微核糖核酸提取试剂盒对miRNA进行分离纯化，通过Fluidigm RT-PCR进行miRNA表达检测。
研究结果：本研究发现，马拉斯烟粉使用时长与miR-31的表达水平呈正相关，而马拉斯烟粉咀嚼时长与miR-372的表达水平呈负相关。此外，马拉斯烟粉的每日消耗量与miR-375、miR-378a、miR-145及miR-10b的表达水平呈负相关；同时，每日吸烟量与miR-23a、miR-23b、miR-203a、miR-200b及miR-375的表达水平亦呈负相关。相较于吸烟者与非使用者，马拉斯烟粉使用者的miR-200b与miR-92a表达水平显著下调（P<0.05）。
研究结论：结果表明，咀嚼马拉斯烟粉与吸烟均可导致miR-200b及miR-92a的表达失调。据此认为，检测这两种miRNA的表达水平是一种极具应用前景的无创分析方法，尤其适用于致突变物暴露的相关研究。最后，大规模高通量研究有助于鉴定与烟草使用相关的全面miRNA表达谱，进而加深对口腔恶性肿瘤的认知。