Abstract:

Abstract: Objective To study the effect of interfering long-chain non-coding RNA nuclear paraspeckle assembly transcript 1 ( NEAT1 ) up-regulation of miR-126 in the inhibition of gastric cancer cells growth, epithelialmesenchymal transition and tumor formation in nude mice. Method The expression level of NEAT1 in gastric cancer MGC-803 cell, SGC-7901 cell and normal gastric epithelial GES-1 cell were analyzed by RT-PCR, the silencing efficiency of sh-NEAT1 was detected as well. After NEAT1 silencing, EDU staining was used to analyze the proliferation of tumor cells. Flow cytometry was used to analyze the apoptosis. Transwell chamber and scratch assay were used to analyze the invasion and migration of tumor cells. Western blot was used to analyze the expressions of Ki67, Caspase-3, N-cadherin and E-cadherin in tumor cells. Luciferase reporter assay was used to validate the targeting relationship between NEAT1 and miR-126, and the effects of NEAT1 / miR-126 on regulation of growth and movement of gastric cancer SGC-7901 cell. By constructing xenograft model in nude mice of SGC- 7901 cells transfected with sh-NEAT1, tumor volume and weight within 30 days were detected. The expression levels of Ki67 and caspase-3 were detected by immunohistochemistry. The expression levels of NEAT1 and miR-126 were detected by RT-PCR. The expression levels of N-cadherin and E-cadherin were detected by Western blot.Result The expression level of NEAT1 in tumor cells was significantly higher than that in normal cells ( P <0. 05) . After sh-NEAT1 silencing, the expression level of NEAT1 in tumor cells was significantly decreased, and the proliferation was significantly inhibited, and the proportion of apoptotic cells was significantly increased, and the invasion and migration were significantly weakened, and the expression levels of Ki67 and N-cadherin in tumor cells were significantly decreased while the expression levels of Caspase-3 and E-cadherin were significantly increased ( P<0. 05) . Luciferase reporter assay showed that NEAT1 had a targeting relationship with miR-126, and sh-NEAT1 could significantly promote the expression of miR-126 ( P < 0. 05 ) , and miR-126 inhibitor could

significantly promote proliferation, invasion and migration of SGC-7901 cell by sh-NEAT1 and inhibit apoptosis. Xenograft model in nude mice showed that sh-NEAT1 could inhibit increase of tumor volume, down-regulate the expression levels of NEAT1, Ki67 and N-cadherin in tumor tissues, and up-regulate the expression levels of miR-126, caspase-3 and E-cadherin, but tumor quality was significantly increased compared with control group ( P <

0. 05) . Conclusion Interfering long-chain non-coding RNA NEAT1 can up-regulate the expression of miR-126 and inhibit the proliferation of gastric cancer cells. And it can reduce the invasion and metastasis of tumor cells and inhibit the tumor formation in nude mice by blocking EMT mechanism.

摘要：

摘要:目的 研究干扰长链非编码 RNA 核富集的转录物 1( NEAT1) 上调 miR-126 抑制胃癌细胞的生长,间质转换及裸鼠肿瘤形成的影响。 方法 通过 RT-PCR 分析 NEAT1 在胃癌 MGC-803、SGC-7901 细胞和正常胃上皮 GES-1细胞中的表达,并检测 sh-NEAT1 的沉默效率。 NEAT1 沉默后,EDU 染色分析肿瘤细胞的增殖能力;流式细胞术分析细胞凋亡;Transwell 小室和划痕实验分析肿瘤细胞的侵袭、迁移能力;Western blot 分析肿瘤细胞 Ki67、Caspase-3、N-cadherin 和 E-cadherin 的表达。 荧光素酶报告实验验证 NEAT1 与 miR-126 的靶向关系,分析 NEAT1 / miR-126 对胃癌 SGC-7901 细胞生长和运动的调节影响;通过构建转染 sh-NEAT1 的 SGC-7901 细胞的移植瘤模型裸鼠,检测30 d 内肿瘤体积及质量变化;免疫组化检测 Ki67 和 Caspase-3 的表达,RT-PCR 检测 NEAT1 和 miR-126 的表达;Western blot 检测 N-钙黏蛋白( N-cadherin) 及 E-钙黏蛋白( E-cadherin) 的表达。 结果 NEAT1 在肿瘤细胞中的表达水平显著高于正常细胞( P< 0. 05) ,sh-NEAT1 沉默后,肿瘤细胞 NEAT1 的表达量显著降低,增殖能力明显受到抑制,凋亡细胞比例显著增高,肿瘤细胞的侵袭、迁移能力明显减弱,肿瘤细胞中 Ki67 和 N-cadherin 表达量明显降低,Caspase-3 和 E-cadherin 的表达水平显著升高,差异均具有统计学意义( P< 0. 05) 。 荧光素酶报告实验表明NEAT1 与 miR-126 存在靶向关系,sh-NEAT1 能显著促进 miR-126 的表达( P< 0. 05) ,miR-126 inhibitor 能显著促进 sh-NEAT1 对 SGC-7901 细胞增殖、侵袭、迁移,并抑制细胞凋亡。 移植瘤模型裸鼠表明 sh-NEAT1 能够抑制肿瘤体积增大,下调肿瘤组织中 NEAT1、Ki67 及 N-cadherin 的表达水平,上调 miR-126、Caspase-3 和 E-cadherin 的表达量,肿瘤的 质 量 与 对 照 组 相 比 显 著 增 加,差 异 均 有 统 计 学 意 义 ( P < 0. 05) 。 结 论 干 扰 长 链 非 编 码 RNANEAT1 能够上调 miR-126 表达,从而抑制胃癌细胞的增殖,并通过阻断 EMT 机制降低肿瘤细胞的侵袭、转移能力,抑制裸鼠肿瘤的形成。