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目的:探讨lncRNA-AK058003在缺氧诱导胃癌侵袭和转移中的作用。方法:SGC7901、MKN45、MKN28三个胃癌细胞系,分别经常氧/缺氧孵育24h后,Trizol法提取3例配对的胃癌细胞总RNA,利用高通量lncRNA芯片比较它们之间的表达谱差异。通过差异倍数、邻近编码基因信息分析、长度特征分析等策略初步筛选与缺氧诱导胃癌侵袭转移相关的关键分子。RT-PCR法检测lncRNA-AK058003在缺氧诱导的胃癌细胞中相对于常氧条件下的表达水平,进一步检测其在20例胃癌临床标本中相对于癌旁组织的表达水平。构建小干扰RNA慢病毒,稳定下调AK058003在SGC7901及MKN45中的表达。Transwell、划痕实验、裸鼠尾静脉注入不同胃癌细胞等体外和体内实验检测抑制AK058003后对常氧和缺氧条件下胃癌侵袭转移的影响。结果:高通量lncRNA芯片比较发现:缺氧诱导的胃癌细胞SGC7901、MKN45和MKN28与常氧条件下的细胞相比,有84个分子的表达在缺氧诱导的胃癌细胞中显著上调(P<0.05),其中差异倍数在3倍以上的共有5个。多重筛选策略提示AK058003可能是缺氧诱导胃癌侵袭转移的关键lncRNA分子之一。RT-PCR结果显示:AK058003在缺氧诱导的胃癌细胞中显著上调,其分别在SGC7901缺氧16h,MKN45缺氧24h,MKN28缺氧48h时达到峰值。进一步RT-PCR结果发现,AK058003在胃癌组织中的表达水平显著高于癌旁组织。缺氧条件下的Transwell实验结果显示缺氧能够显著增加SGC7901和MKN45的侵袭转移力,而抑制AK058003的表达后,胃癌细胞侵袭转移力明显下降。结论:通过高通量lncRNA芯片比较,首次发现了一系列胃癌细胞中缺氧相关的lncRNAs分子。通过临床标本验证以及功能缺失试验证实lnc-AK058003是系列分子中影响缺氧诱导胃癌侵袭转移的关键lncRNAs分子之一。
Objective: To investigate the role of lncRNA-AK058003 in invasion and metastasis of gastric cancer induced by hypoxia. METHODS: Three gastric cancer cell lines SGC7901, MKN45 and MKN28 were respectively treated with oxygen and oxygen for 24 hours. Trizol method was used to extract the total RNA of gastric cancer cells from three matched pairs. The expression profiles of the three gastric cancer cell lines were compared by high-throughput lncRNA. Differentiation fold, adjacent coding gene information analysis, length characteristics analysis and other strategies for the initial screening of key molecules associated with hypoxia-induced invasion and metastasis of gastric cancer. The expression level of lncRNA-AK058003 in hypoxia-inducible gastric cancer cells under normoxia was detected by RT-PCR, and the expression level of lncRNA-AK058003 was further detected in 20 gastric cancer specimens. Construction of small interfering RNA lentivirus down-regulated the expression of AK058003 in SGC7901 and MKN45 cells. Transwell, scratch test, different gastric cancer cells injected into tail vein of nude mice and other in vitro and in vivo experiments were performed to detect the effect of inhibiting AK058003 on the invasion and metastasis of gastric cancer under normoxia and hypoxia. Results: The results of high-throughput lncRNA microarray showed that the expression of 84 molecules in hypoxia-inducible gastric cancer cells SGC7901, MKN45 and MKN28 were significantly up-regulated in hypoxia-induced gastric cancer cells compared with those in normoxia (P < 0.05), in which the difference in multiples of 3 times more than a total of five. Multiple screening strategies suggest that AK058003 may be one of the key lncRNA molecules in hypoxia-inducible gastric cancer invasion and metastasis. RT-PCR results showed that AK058003 up-regulated in hypoxia-inducible gastric cancer cells, which peaked at 16h after SGC7901 hypoxia, 24h after MKN45 hypoxia and 48h after MKN28 hypoxia respectively. Further RT-PCR results showed that the expression level of AK058003 in gastric cancer tissues was significantly higher than that in paracancerous tissues. Hypoxia under Transwell experimental results showed that hypoxia can significantly increase the invasion and metastasis SGC7901 and MKN45, while inhibiting the expression of AK058003 gastric cancer cell invasion and metastasis decreased significantly. Conclusion: A series of hypoxia-associated lncRNAs were found in gastric cancer cells by high-throughput lncRNA chip comparison. Through clinical specimen verification and loss of function test, it was confirmed that lnc-AK058003 is one of the key molecules of lncRNAs that affect the invasion and metastasis of hypoxia-inducible gastric cancer.