Department of Cell Biology and Cancer Institute of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China; Department of Molecular Biology, Princeton University, Princeton, New Jersey; Institute of Gastroenterology, Zhejiang University, Hangzhou, China.
Department of Cell Biology and Cancer Institute of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Gastroenterology. 2019 Feb;156(3):676-691.e11. doi: 10.1053/j.gastro.2018.10.054. Epub 2018 Nov 13.
BACKGROUND & AIMS: We aimed to identify long noncoding RNAs (lncRNAs) that are up-regulated in gastric cancer tissues from patients and study their function in gastric tumor metastasis.
We collected gastric tumor and nontumor tissues from patients in China and analyzed levels of lncRNAs by microarray analysis, proteins by immunohistochemistry, and RNAs by quantitative reverse-transcription polymerase chain reaction; we compared these with survival times of patients and tumor progression. RNA levels were knocked down or knocked out in BGC-823, SGC-7901, and MKN45 cell lines using small interfering or short hairpin RNAs or clustered regularly interspaced short palindromic repeats (ie, CRISPR)/CRISPR associated protein 9 (ie, Cas9) vectors. Genes were overexpressed from transfected plasmids in HGC-27 cells. Cells were analyzed by Northern blot and immunoblot, polysome profiling assay, and cell invasion assay. Cells were injected into the tail veins or spleens of nude mice or SCID mice; lung and liver tissues were collected, and metastases were counted. lncRNAs were cloned by using rapid amplification of complementary DNA ends. Their interactions with other genes were determined by RNA pulldown and mapping assays.
In microarray analyses, we identified 151 lncRNAs expressed at significantly higher levels in gastric tumor vs nontumor tissues. Levels of an lncRNA that we called gastric cancer metastasis associated long noncoding RNA (GMAN) were increased in gastric tumor tissues, compared with nontumor tissues; its up-regulation was associated with tumor metastasis and shorter survival times of patients. The GMAN gene overlaps with the ephrin A1 gene (EFNA1) and was highly expressed in BGC-823 and MKN45 cells. Knockdown of GMAN in these cells did not affect proliferation, colony formation, or adhesion but did reduce their invasive activity in Transwell assays. Ectopic expression of GMAN increased the invasive activity of HGC-27 cells. BGC-823 and MKN45 cells with knockdown of GMAN formed fewer metastases after injection into tail veins of nude mice. Knockdown or knockout of GMAN also reduced levels of ephrin A1 protein in cells. We found that GMAN promoted translation of ephrin A1 messenger RNA into protein by binding to the antisense GMAN RNA (GMAN-AS)-this antisense sequence is also complementary to that of ephrin A1 mRNA. Levels of ephrin A1 protein were also increased in gastric tumors from patients with metastases than in those without metastases. Knockout of ephrin A1 in BGC-823 cells reduced their invasive activity in Transwell assays and ability to form metastases after injection into SCID mice. Ectopic expression of ephrin A1 in BGC-823 cells with knockdown or knockout of GMAN restored their invasive activities and ability form metastases in nude or SCID mice. A CRISPR/Cas9-based strategy to disrupt the GMAN gene significantly reduced the numbers of metastases formed from SGC-7901 cells in mice.
We identified an lncRNA, which we call GMAN, that is increased in gastric tumors from patients and associated with survival and formation of metastases. It regulates translation of ephrin A1 mRNA by binding competitively to GMAN-AS. Knockdown or knockout of GMAN or ephrin A1 in gastric cancer cell lines reduces their invasive activity and ability to form metastases after injection into mice. These genes might be targeted to prevent or reduce gastric cancer metastasis.
我们旨在鉴定在胃癌组织中上调的长非编码 RNA（lncRNA），并研究其在胃肿瘤转移中的功能。
我们收集了来自中国患者的胃癌组织和非肿瘤组织，通过微阵列分析、免疫组织化学、定量逆转录聚合酶链反应分析 lncRNA、蛋白质和 RNA 的水平；我们将这些与患者的生存时间和肿瘤进展进行了比较。使用小干扰 RNA 或短发夹 RNA 或成簇规律间隔的短回文重复（即 CRISPR）/CRISPR 相关蛋白 9（即 Cas9）载体在 BGC-823、SGC-7901 和 MKN45 细胞系中敲低或敲除 RNA 水平。使用转染质粒在 HGC-27 细胞中转基因表达。通过 Northern blot 和免疫印迹、多核糖体谱分析和细胞侵袭试验分析细胞。将细胞注射到裸鼠或 SCID 小鼠的尾静脉或脾脏中；收集肺和肝组织，计数转移灶。通过快速扩增互补 DNA 末端克隆 lncRNA。通过 RNA 下拉和作图试验确定它们与其他基因的相互作用。
在微阵列分析中，我们鉴定了 151 个在胃癌组织中表达水平明显高于非肿瘤组织的 lncRNA。与非肿瘤组织相比，我们称之为胃癌转移相关长非编码 RNA（GMAN）的 lncRNA 在胃癌组织中表达上调；其上调与肿瘤转移和患者生存时间缩短有关。GMAN 基因与 Ephrin A1 基因（EFNA1）重叠，在 BGC-823 和 MKN45 细胞中高表达。在这些细胞中敲低 GMAN 不会影响增殖、集落形成或粘附，但会降低它们在 Transwell 测定中的侵袭活性。GMAN 的异位表达增加了 HGC-27 细胞的侵袭活性。在裸鼠尾静脉注射后，GMAN 敲低的 BGC-823 和 MKN45 细胞形成的转移灶较少。GMAN 的敲低或敲除也降低了 Ephrin A1 蛋白在细胞中的水平。我们发现 GMAN 通过与反义 GMAN RNA（GMAN-AS）结合促进 Ephrin A1 mRNA 翻译成蛋白质-该反义序列也与 Ephrin A1 mRNA 互补。来自有转移的患者的胃癌肿瘤中的 Ephrin A1 蛋白水平也高于没有转移的患者。在 BGC-823 细胞中敲除 Ephrin A1 降低了它们在 Transwell 测定中的侵袭活性和在 SCID 小鼠中形成转移的能力。在 BGC-823 细胞中敲低或敲除 GMAN 后，异位表达 Ephrin A1 恢复了它们在裸鼠或 SCID 小鼠中的侵袭活性和形成转移的能力。基于 CRISPR/Cas9 的策略破坏 GMAN 基因显著降低了 SGC-7901 细胞在小鼠中形成的转移灶数量。
我们鉴定了一种在胃癌组织中上调的 lncRNA，我们称之为 GMAN，它与患者的生存和转移形成有关。它通过与 GMAN-AS 竞争结合来调节 Ephrin A1 mRNA 的翻译。在胃癌细胞系中敲低或敲除 GMAN 或 Ephrin A1 可降低其侵袭活性和在小鼠中形成转移的能力。这些基因可能成为预防或减少胃癌转移的靶点。
