长链非编码RNA-CCDC144NL反义RNA1通过海绵吸附miR-940诱导WDR5表达促进肝细胞癌发展
LncRNA-CCDC144NL-AS1 Promotes the Development of Hepatocellular Carcinoma by Inducing WDR5 Expression via Sponging miR-940.
作者信息
Zhang Yingying, Zhang Hongyu, Wu Shuhuan
机构信息
Department of Infectious Diseases, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, 450052, People's Republic of China.
出版信息
J Hepatocell Carcinoma. 2021 May 3;8:333-348. doi: 10.2147/JHC.S306484. eCollection 2021.
PURPOSE
This work was initiated to offer solid evidence regarding the expression and roles of long noncoding RNA (lncRNA) CCDC144NL-AS1 in hepatocellular carcinoma (HCC).
PATIENTS AND METHODS
Cell Counting Kit-8 assay, flow cytometric analysis, and invasion assays were used to explore the malignant biological characteristics of cells. Immunohistochemistry (IHC), Western blotting analysis, and real-time quantitative PCR (RT-qPCR) were used to analyze the expression level of related proteins and nucleic acids. Bl6/Rag2/GammaC double knockout mice were used for HCC modeling to address the therapeutic value of CCDC144NL-AS1.
RESULTS
CCDC144NL-AS1 was significantly upregulated in HCC tissue and had a marked relationship with the 5-year prognosis. In vitro study revealed that CCDC144NL-AS1 was highly expressed in HCC cell line MHCC97H but lowly expressed in normal hepatic cell line L02. Overexpression of CCDC144NL-AS1 in L02 enhanced the invasion and proliferation abilities of cells but inhibited the apoptosis rate. Knockdown of CCDC144NL-AS1 in MHCC97H weakened the invasion and proliferation abilities of cells but increased the apoptosis rate. CCDC144NL-AS1 was found to sponge miR-940 to induce the expression of WD repeat domain 5 (WDR5). ChIP-seq analysis identified that matrix metalloproteinase (MMP) 2, MMP9, and cyclin-dependent kinase (CDK) 1, CDK2, and CDK4 were all targets of WDR5. The recruitment of WDR5 to the promoter of these target genes upregulated the histone H3 lysine 4 trimethylation (H3K4me3) level in these regions and further induced the transcription of MMP2, MMP9, CDK1, CDK2, and CDK4. In vivo study revealed that compared to the normal liver tissue, CCDC144NL-AS1, WDR5, MMP2, MMP9, CDK1, CDK2, and CDK4 were all significantly upregulated in HCC tissue from the same mouse, while miR-940 was decreased. Besides, knockdown of CCDC144NL-AS1 or WDR5 or overexpression of miR-940 could all inhibit tumor growth.
CONCLUSION
CCDC144NL-AS1 drives HCC development by inducing MMP2/MMP9 and CDK1/CDK2/CDK4 expressions through miR-940/WDR5-regulated epigenetic pathway.
目的
开展本研究以提供关于长链非编码RNA（lncRNA）CCDC144NL-AS1在肝细胞癌（HCC）中的表达及作用的确凿证据。
患者与方法
采用细胞计数试剂盒-8检测、流式细胞术分析及侵袭实验来探究细胞的恶性生物学特性。运用免疫组织化学（IHC）、蛋白质免疫印迹分析及实时定量聚合酶链反应（RT-qPCR）来分析相关蛋白质和核酸的表达水平。利用Bl6/Rag2/γC双敲除小鼠建立HCC模型，以探讨CCDC144NL-AS1的治疗价值。
结果
CCDC144NL-AS1在HCC组织中显著上调，且与5年预后显著相关。体外研究显示，CCDC144NL-AS1在HCC细胞系MHCC97H中高表达，而在正常肝细胞系L-02中低表达。在L-02中过表达CCDC144NL-AS1可增强细胞的侵袭和增殖能力，但抑制细胞凋亡率。在MHCC97H中敲低CCDC144NL-AS1可削弱细胞的侵袭和增殖能力，但增加细胞凋亡率。发现CCDC144NL-AS1可吸附miR-940以诱导WD重复结构域5（WDR5）表达。染色质免疫沉淀测序（ChIP-seq）分析确定基质金属蛋白酶（MMP）2、MMP9以及细胞周期蛋白依赖性激酶（CDK）1、CDK2和CDK4均为WDR5的靶标。WDR5募集至这些靶基因的启动子可上调这些区域的组蛋白H3赖氨酸4三甲基化（H3K4me3）水平，并进一步诱导MMP2、MMP9、CDK1、CDK2和CDK4的转录。体内研究显示，与正常肝组织相比，同一小鼠的HCC组织中CCDC144NL-AS1、WDR5、MMP2、MMP9、CDK1、CDK2和CDK4均显著上调，而miR-940则降低。此外，敲低CCDC144NL-AS1或WDR5或过表达miR-940均可抑制肿瘤生长。
结论
CCDC144NL-AS1通过miR-940/WDR5调控的表观遗传途径诱导MMP2/MMP9和CDK1/CDK2/CDK4表达，从而推动HCC发展。
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