Babu Niraj, Advani Jayshree, Solanki Hitendra S, Patel Krishna, Jain Ankit, Khan Aafaque Ahmad, Radhakrishnan Aneesha, Sahasrabuddhe Nandini A, Mathur Premendu Prakash, Nair Bipin, Keshava Prasad Thottethodi Subrahmanya, Chang Xiaofei, Sidransky David, Gowda Harsha, Chatterjee Aditi
Institute of Bioinformatics, International Technology Park, Bangalore 560066, India.
Manipal Academy of Higher Education, Manipal 576104, India.
Microrna. 2018;7(1):38-53. doi: 10.2174/2211536607666180103165343.
Dysregulation of miRNAs is associated with the development of non-small cell lung cancer (NSCLC). It is imperative to study the dysregulation of miRNAs by cigarette smoke which will affect their targets, either leading to the overexpression of oncoproteins or downregulation of tumor suppressor proteins.
In this study, we carried out miRNA sequencing and SILAC-based proteomic analysis of H358 cells chronically exposed to cigarette smoke condensate. Using bioinformatics analysis, we mapped the dysregulated miRNAs to differentially expressed target proteins identified in our data. Gene ontology-based enrichment and pathway analysis was performed using the deregulated targets to study the role of cigarette smoke-mediated miRNA dysregulation in NSCLC cell line.
miRNA sequencing resulted in the identification of 208 miRNAs, of which 6 miRNAs were found to be significantly dysregulated (2 fold, Log Base 2; p-value ≤ 0.05) in H358-Smoke cells. Proteomic analysis of the smoke exposed cells compared to the untreated parental cells resulted in the quantification of 2,610 proteins, of which 690 proteins were found to be differentially expressed (fold change ≥ 2). Gene ontology based analysis of target proteins revealed enrichment of proteins driving metabolism and a decrease in expression of proteins associated with immune response in the cells exposed to cigarette smoke. Pathway study using Ingenuity Pathway Analysis (IPA) revealed activation of NRF2-mediated oxidative stress response and actin-cytoskeleton signaling, and repression of protein kinase A signaling in H358-Smoke cells. We also identified 5 novel miRNAs in H358-Smoke cells using unassigned reads of small RNA-Seq dataset.
In summary, this study indicates that chronic exposure to cigarette smoke leads to widespread dysregulation of miRNAs and their targets, resulting in signaling aberrations in NSCLC cell line. The miRNAs and their targets identified in the study need to be further investigated to explore their role as potential therapeutic targets and/or molecular markers in NSCLC especially in smokers.
微小RNA（miRNA）失调与非小细胞肺癌（NSCLC）的发生发展相关。研究香烟烟雾对miRNA的失调作用及其如何影响其靶标至关重要，这可能导致癌蛋白的过表达或肿瘤抑制蛋白的下调。
在本研究中，我们对长期暴露于香烟烟雾冷凝物的H358细胞进行了miRNA测序和基于稳定同位素标记氨基酸法（SILAC）的蛋白质组学分析。通过生物信息学分析，我们将失调的miRNA映射到数据中鉴定出的差异表达靶蛋白。使用失调的靶标进行基于基因本体论的富集和通路分析，以研究香烟烟雾介导的miRNA失调在NSCLC细胞系中的作用。
miRNA测序鉴定出208种miRNA，其中6种miRNA在H358-烟雾细胞中显著失调（2倍，以2为底的对数；p值≤0.05）。与未处理的亲代细胞相比，对暴露于烟雾的细胞进行蛋白质组学分析，定量了2610种蛋白质，其中690种蛋白质差异表达（倍数变化≥2）。基于基因本体论对靶蛋白的分析显示，暴露于香烟烟雾的细胞中驱动代谢的蛋白质富集，而与免疫反应相关的蛋白质表达下降。使用 Ingenuity Pathway Analysis（IPA）进行的通路研究显示，H358-烟雾细胞中NRF2介导的氧化应激反应和肌动蛋白细胞骨架信号激活，蛋白激酶A信号受到抑制。我们还使用小RNA测序数据集的未分配读数在H358-烟雾细胞中鉴定出5种新的miRNA。
总之，本研究表明长期暴露于香烟烟雾会导致miRNA及其靶标广泛失调，从而导致NSCLC细胞系中的信号异常。本研究中鉴定出的miRNA及其靶标需要进一步研究，以探索它们作为NSCLC潜在治疗靶点和/或分子标志物的作用，尤其是在吸烟者中。
