背景:晚期诊断是口腔鳞状细胞癌(OSCC)的主要混杂因素之一。尽管分子诊断最近取得了进展,临床上没有疾病特异性生物标志物可用于OSCC的早期风险预测.因此,重要的是要确定使用非侵入性液体活检技术可检测的稳健生物标志物,以促进口腔癌的早期诊断.这项研究确定了潜在的唾液外泌体来源的miRNA生物标志物和负责OSCC进展的关键miRNA-mRNA网络/潜在机制。
方法:进行小RNASeq(n=23)以鉴定源自OSCC患者的组织和唾液外泌体中的潜在miRNA生物标志物。Further,癌症基因组图谱(TCGA)数据集的综合分析(n=114),对更大的患者队列(n=70)进行qPCR验证,并利用各种临床病理参数进行统计分析,以评估所鉴定的miRNA特征的有效性。通过整合转录组测序和TCGA数据进行miRNA-mRNA网络和通路分析。用鉴定的miRNA标记转染OECM-1细胞系以观察其对各种功能机制如细胞增殖的影响。细胞周期,凋亡,侵袭和迁移潜力以及这些miRNA-mRNA网络调节的下游信号通路。
结果:小RNASeq和TCGA数据鉴定了与对照组相比OSCC患者中12种差异表达的miRNA。在更大的患者队列中验证这些发现,miR-140-5p,miR-143-5p,和miR-145-5p被发现显著下调。该3-miRNA特征在预测疾病进展方面表现出更高的功效,并且临床上与不良预后相关(p<0.05)。转录组,TCGA,和miRNA-mRNA网络分析鉴定出HIF1a,CDH1,CD44,EGFR,和CCND1作为受miRNA标签调控的hub基因。Further,转染介导的3-miRNA标记的上调显著降低了细胞增殖,诱导细胞凋亡,通过逆转OECM-1细胞系中的EMT过程,导致G2/M期细胞周期停滞,并降低了侵袭和迁移潜力。
结论:因此,这项研究确定了一个3-miRNA特征,该特征可用作预测OSCC疾病进展的潜在生物标志物,并揭示了将正常上皮细胞转化为恶性表型的潜在机制.
BACKGROUND: Late diagnosis is one of the major confounders in oral squamous cell carcinoma (OSCC). Despite recent advances in molecular diagnostics, no disease-specific biomarkers are clinically available for early risk prediction of OSCC. Therefore, it is important to identify robust biomarkers that are detectable using non-invasive liquid biopsy techniques to facilitate the early diagnosis of oral cancer. This study identified potential salivary exosome-derived miRNA biomarkers and crucial miRNA-mRNA networks/underlying mechanisms responsible for OSCC progression.
METHODS: Small RNASeq (n = 23) was performed in order to identify potential miRNA biomarkers in both tissue and salivary exosomes derived from OSCC patients. Further, integrated analysis of The Cancer Genome Atlas (TCGA) datasets (n = 114), qPCR validation on larger patient cohorts (n = 70) and statistical analysis with various clinicopathological parameters was conducted to assess the effectiveness of the identified miRNA signature. miRNA-mRNA networks and pathway analysis was conducted by integrating the transcriptome sequencing and TCGA data. The OECM-1 cell line was transfected with the identified miRNA signature in order to observe its effect on various functional mechanisms such as cell proliferation, cell cycle, apoptosis, invasive as well as migratory potential and the downstream signaling pathways regulated by these miRNA-mRNA networks.
RESULTS: Small RNASeq and TCGA data identified 12 differentially expressed miRNAs in OSCC patients compared to controls. On validating these findings in a larger cohort of patients, miR-140-5p, miR-143-5p, and miR-145-5p were found to be significantly downregulated. This 3-miRNA signature demonstrated higher efficacy in predicting disease progression and clinically correlated with poor prognosis (p < 0.05). Transcriptome, TCGA, and miRNA-mRNA network analysis identified HIF1a, CDH1, CD44, EGFR, and CCND1 as hub genes regulated by the miRNA signature. Further, transfection-mediated upregulation of the 3-miRNA signature significantly decreased cell proliferation, induced apoptosis, resulted in G2/M phase cell cycle arrest and reduced the invasive and migratory potential by reversing the EMT process in the OECM-1 cell line.
CONCLUSIONS: Thus, this study identifies a 3-miRNA signature that can be utilized as a potential biomarker for predicting disease progression of OSCC and uncovers the underlying mechanisms responsible for converting a normal epithelial cell into a malignant phenotype.