长非编码LINC00622调控UM增殖和侵袭转移的机制研究

Uveal melanoma (UM) is the most common primary intraocular malignant tumor in adults with high death rate because of the high relapse and metastasis rate. Preliminary study found: 1.Various of long non-coding RNAs (lncRNAs) are over-expressed in UM and LINC00622 is the most obvious one. Knockdown of LINC00622 expression could significantly inhibit the growth and metastasis in UM cells. 2.Gene and protein expression level, biological experiments and correlation analysis confirmed that IGSF11 is the downstream target gene of LINC00622, which is involved in the proliferation and metastasis of UM. 3.LINC00622 located in the nucleus of UM cells. Above all, we suggest that abnormally up-regulated LINC00622 promoted the expression level of IGSF11 through the key factor, and then lead the increased cell proliferation and metastasis. This study will use the UM cell lines to study the mechanism of LINC00622 regulating IGSF11 through key factor by the technology of oligonucleotide precipitation (CHOP), RNA oligonucleotides chromosome conformation capture (R3C) and so on, so as to reveal the molecular biological mechanism of LINC00622 in UM proliferation and metastasis, and to provide theoretical basis for the pathogenesis of UM and potential target for clinical treatment of UM patients.

葡萄膜黑色素瘤(Uveal Melanoma，UM)是成人最常见的眼内恶性肿瘤，易复发转移，致死率高。前期研究发现：1、UM中多条长非编码RNA（lncRNA）异常高表达，其中LINC00622上调最明显。敲除LINC00622表达显著抑制UM细胞增殖和侵袭转移；2、基因蛋白表达水平和生物学实验以及相关性分析证实IGSF11是LINC00622的下游靶基因，参与UM增殖转移；3、LINC00622表达位于UM细胞核内。据此推测：UM细胞核内异常高表达的LINC00622通过关键作用因子调控IGSF11表达，进而促进UM细胞增殖转移。本研究将借助UM细胞系，采用寡核苷酸沉淀（CHOP）和RNA染色体构象捕获（R3C）等技术筛选并检测关键因子在LINC00622调控IGSF11中的作用机制，以期揭示LINC00622调控UM增殖转移的作用机制，为UM患者临床治疗提供理论依据和潜在治疗靶点。

葡萄膜黑色素瘤(Uveal Melanoma，UM)是成人最常见的眼内恶性肿瘤，易复发转移，致死率高。前期研究发现：UM中多条非编码RNA（lncRNA、microRNA）异常高表达，本项目将系统性研究非编码RNA对UM生长转移的影响及调控作用。1、LINC00622是UM中新发现上调最明显的长非编码RNA，敲除LINC00622表达显著抑制UM细胞增殖和侵袭转移；本研究发现LINC00622通过调控下游靶基因IGSF11促进UM的侵袭转移。研究包括：a、基因蛋白表达水平和体内、体外生物学实验以及相关性分析证实IGSF11是LINC00622的下游靶基因，敲除IGSF11表达显著抑制UM细胞增殖和侵袭转移；b、UM细胞株中证实LINC00622表达位于UM细胞核内，cDNA末端的快速扩增实验明确新发现LINC00622在UM细胞中的全长。c、UM细胞核内异常高表达的LINC00622通过调控IGSF11甲基化水平，进而影响IGSF11的表达，进而促进UM细胞增殖转移。本研究明确了LINC00622调控UM增殖转移的作用机制，为UM患者临床治疗提供理论依据和潜在治疗靶点。2、microRNA是短非编码RNA，mir-181s家族成员包括mir-181a、-181b、-181c和-181d，目前UM中关于mir-181s家族作用机制亟待解决。本课题组首次在UM中研究mir-181s对UM生长转移的影响。a：RT-PCR检测发现mir-181s家族在UM组织和UM细胞中明显上调，抑制miR-181s表达，明显抑制UM细胞周期进展，其中mir-181b作用最明显。b：miR-181s家族成员在不同物种间具有高度同源性，miR-181s表达上调促进UM细胞周期进展。在家族成员中，mir-181b在UM组织和大多数UM细胞中明显高表达。c：生物信息学和双荧光素酶报告分析证实CTDSPL是mir-181s的作用靶点。mir-181b过度表达抑制了CTDSPL的表达，进而导致RB基因磷酸化和下游周期因子E2F1积累，进而促进UM细胞周期进展。研究表明：mir-181s家族成员是CTDSPL介导的细胞周期进展的主要负调控因子。mir-181家族成员，特别是mir-181b，可作为UM诊断和治疗的新靶点。