Low abundance of TFPI-2 by both promoter methylation and miR-27a-3p regulation is linked with poor clinical outcome in gastric cancer
Low abundance of TFPI-2 by both promoter methylation and miR-27a-3p regulation is linked with poor clinical outcome in gastric cancer
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DOI：
10.1002/jgm.3166
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发表时间：
2020-05-01
影响因子：
3.5
通讯作者：
Yuan, Xiao
中科院分区：
文献类型：
--
作者：
Geng, Guangyong;Liu, Xin;Yuan, Xiao
背景组织因子途径抑制物2（TFPI-2）在多种肿瘤中的抑癌作用已被报道。本研究旨在提高对TFPI-2在胃癌中致癌特性的认识。方法分别采用实时荧光定量PCR和免疫印迹法检测胃癌组织中TFPI-2的相对表达。通过细胞计数试剂盒-8测定法测量细胞活力，并通过集落形成测定法评价增殖。caspase-3活性检测试剂盒检测细胞凋亡，transwell小室法检测细胞侵袭能力。甲基化特异性PCR检测TFPI-2基因启动子甲基化水平。用荧光素酶报告基因测定分析miR-27 a-3p对TFPI-2的调节作用。结果TFPI-2在胃癌组织中表达下调，与临床预后不良相关。TFPI-2的异位导入大大降低了细胞的存活率、集落形成和侵袭能力，同时也诱导了细胞凋亡。与正常组织相比，胃癌组织中TFPI-2启动子区广泛甲基化，提示TFPI-2表达受到表观遗传抑制。我们进一步鉴定了TFPI-2作为海绵RNA针对miR-27 a-3p发挥作用。最重要的是，miR-27 a-3p特异性抑制剂发挥了类似于TFPI-2本身的肿瘤抑制功能，TFPI-2的抗肿瘤活性被完全消除。结论我们发现，表观遗传学抑制的TFPI-2削弱了胃癌中miR-27 a-3p的海绵效应，从而导致胃癌的肿瘤生物学。
Background The tumor suppressor role of tissue factor pathway inhibitor 2 (TFPI-2) has been reported in various tumors. The present study aimed to improve the understanding of the oncogenic properties of TFPI-2 in gastric cancer.Methods Relative expression of TFPI-2 was determined by a real-time polymerase chain reaction (PCR) and western blotting, respectively. Cell viability was measured via a cell counting kit-8 assay and proliferation was evaluated by a colony formation assay. Cell apoptosis was assessed with a caspase-3 activity kit and invasion was evaluated by a transwell chamber assay. The methylation level of TFPI-2 promoter was assayed by methylation-specific PCR. The regulatory effect of miR-27a-3p on TFPI-2 was analyzed with a luciferase reporter assay. The direct association between miR-27a-3p and TFPI-2 was shown by biotin-labelling pulldown.Results TFPI-2 was down-regulated in gastric cancer, which associated with an unfavorable prognosis clinically. Ectopic introduction of TFPI-2 greatly compromised cell viability, colony formation and invasive capacity, and also induced cell apoptosis simultaneously. The promoter region of TFPI-2 was extensively methylated in gastric cancer tissues compared to normal tissues, suggesting the epigenetic inhibition of TFPI-2 expression. We further identified that TFPI-2 functioned as sponge RNA against miR-27a-3p. Most importantly, miR-27a-3p-specific inhibitor significantly exerted a tumor suppressor function akin to TFPI-2 itself, and the anti-tumoral activities were completely abolished by TFPI-2 knockdown.Conclusions We found that the epigenetically suppressed TFPI-2 compromised sponging effects with respect to miR-27a-3p in gastric cancer, which consequently and mechanistically contributed to the tumor biology of gastric cancer.