LINC01614和细胞外基质-受体相互作用通路基因表达及遗传变异与头颈部鳞癌发生发展关系及机制研究

Long non-coding RNAs (lncRNAs) play an important role in regulating gene expression and maintaining cell homeostasis. The abnormal expression or function of lncRNAs are closely related to tumorigenesis and development. Through the analysis of head and neck squamous cell carcinoma (HNSCC) RNA sequencing data in TCGA in the earlier time, our team found that LINC01614 in cancer tissues and adjacent non-cancerous tissues were significantly different expression. Through the analysis of WGCNA, KEGG pathway and GO, we found that the genes in ECM- receptor interaction pathway (COL1A1, COL1A2, COL6A3, ITGA11, FN1) are highly correlated with LINC01614. This project aims to study the differential expression and co-expression of LINC01614 and the above genes in tumor tissues and adjacent non-cancerous tissues by sequencing and real-time quantitative PCR in Chinese population and analyze the association between these genes expression and the occurrence and development of HNSCC. We will explore the association of genetic variation with susceptibility of HNSCC and prognosis of HNSCC through case-control study and follow-up study. Furthermore, we will verify the function of genetic variation using dual luciferase reporter assay. The relationship between LINC01614 and cell apoptosis, proliferation and invasion in HNSCC will be also studied by LNCRNA functional assay. RIP will be used to study the mechanism. The results will provide a new theoretical basis for the screening of HNSCC genetic biomarkers and tumor biomarkers for the detection of high-risk populations, and for the study of new targets of treatment and prognosis monitoring in HNSCC.

长链非编码RNA表达或功能异常与肿瘤发生发展密切相关。本项目前期通过对TCGA头颈部鳞癌(HNSCC)RNA测序数据分析，发现LINC01614在癌与癌旁组织间显著性差异表达，WGCNA与KEGG通路及GO分析发现ECM-受体相互作用通路基因(COL1A1、COL1A2、COL6A3、ITGA11，FN1)表达与LINC01614高度相关。因此，本项目拟通过测序和实时定量PCR方法研究中国人LINC01614和上述基因表达与HNSCC发生发展的关系；开展病例对照和随访研究探讨基因遗传变异与HNSCC发生发展的关系；通过双荧光素酶报告基因实验对遗传变异进行功能学验证；细胞表型实验研究LINC01614与HNSCC细胞凋亡、增殖、侵袭等的关系；以及RIP等技术研究机制。研究结果将为HNSCC遗传标记和肿瘤标志物的筛选及高危人群的发现提供新理论依据，为HNSCC的治疗新靶点及预后监测提供基础。

长链非编码RNA表达或功能异常与肿瘤发生发展密切相关。本项目通过测序以及基于TCGA数据库的生信分析发现，LINC01614在HNSCC组织中的表达量显著高于癌旁组织，且LINC01614的高表达与HNSCC患者较差的生存预后显著相关（P<0.05）。KEGG通路富集和GO功能注释分析显示LINC01614共表达基因显著富集于“ECM-receptor interaction”、 “Focal adhesion”和“PI3K/AKT signaling pathway”等和癌症相关的信号通路。通过两阶段配对病例-对照研究策略研究了LINC01614功能性SNPs与HNSCC易感性的关系，以及通过非配对病例-对照研究策略研究与LINC01614高度相关的细胞外基质（ECM）-受体相互作用通路基因(COL1A1、COL1A2、COL6A3、ITGA11，FN1)功能性SNPs与HNSCC易感性的关联。结果发现LINC01614的遗传变异位点rs16854802 A>G和rs3113503 G>C增加了HNSCC的患病风险，在COL6A3 rs2291794的共显性模型中，发现携带AG基因型的个体比携带AA基因型的个体具有更高患HNSCC发病风险的可能。通过数据库预测及双荧光素酶报告基因实验验证，发现rs3113503 G>C破坏了miR-616-3p与LINC01614的结合位点。进一步功能实验发现rs3113503 G>C干扰了miR-616-3p对LINC01614[G]表达的抑制，从而影响了LINC01614的表达水平。 敲低Fadu和Cal-27细胞中LINC01614的表达，减弱了癌细胞的增殖、迁移和侵袭能力，抑制了PI3K/AKT信号通路的激活。进一步在Fadu和Cal-27细胞中过表达不同基因型LINC01614的表达，结果发现与过表达含rs3113503[C]突变体的LINC01614相比， 过表达rs3113503[G]LINC01614时，促进了PI3K/AKT信号通路的激活。本研究通过组织测序以及基于TCGA数据库分析寻找新的HNSCC发病相关lncRNAs，并通过两阶段1∶1匹配的病例-对照研究探讨lncRNA遗传变异和HNSCC易感性的关联，最后通过分子生物学实验验证lncRNA遗传变异及其表达在HNSCC发生发展中所起的作用和有关机制，从而为