LncRNA-AK058003在缺氧诱导胃癌侵袭转移中的作用

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。

The role of lncRNA - AK058003 in the metastasis and invasion of gastric cancer under hypoxia

Objective:To investigate the role of long non - coding RNA AK058003 in the hypoxia - induced inva-sion and metastasis in GC. Methods:High throughput lncRNA microarrays(12 × 135k lncRNA Expression Microar-ray:lncRNA from NCBI RefSeq,UCSC,RNAdb,lncRNAs from literatures and UCRs and references)were used to compare the lncRNA expression profile difference between normoxia - induced and hypoxia - induced GC cell lines (SGC7901,MKN45 and MKN28). Screen hypoxia - induced invasion and metastasis related lncRNAs through strate-gies such as change fold,neighboring genes analysis and characteristics(length)analysis. The different expression of the key molecular between hypoxia - induced GC and normoxia - induced GC were valitaed using RT - PCR. The ex-pression levels of the key molecular in 20 GC tissues and corresponding non - tumor mucosa were detected by RT -PCR. SiRNA - mediated antisense lncRNA - AK058003 gene transfer technique was employed to downregulate AK058003 expression in human GC cell lines SGC7901 and MKN45. Transwell migration and invasion assays,wound- healing assays between normoxia and hypoxia and tail vein injection metastasis assays were employed for investiga-ting the hypoxia - induced metastatic properties of each cell subline. Results:Compare with the gastric cancer cell lines under normoxia 254 lncRNAs which had more than 1. 5 times variation and significant diference(P < 0. 05)by statistical analysis,while 84 increased more than 1. 5 times and 5 increased more than 3 times. After screening hypoxia- induced invasion and metastasis related lncRNAs through some strategies,we found that lncRNA - AK058003 may play the key role in the hypoxia - induced GC metastasis and invasion. RT - PCR showed that lncRNA - AK058003 was frequently up - regulated under hypoxic GC cell lines relative to expression under normoxia. The AK058003 was also detected in 20 pairs of GC and corresponding noncancerous gastric samples using RT - PCR. The results showed that the expression of AK058003 was significantly up - regulated in GC when compared to the noncancerous gastric samples. Functional studies of AK058003 revealed that down - expression of AK058003 resulted in an reduction in migration and invasion. This suggests that AK058003 involved in GC metastasis and invasion. To further explore whether AK058003 mediated the hypoxia - induced GC metastasis and invasion. Transwell migration and invasion as-sys revealed that hypoxia can promote the GC cells metastasis and invasion. To further investigate the promotion of in vivo tumor metastasis by AK058003,we implanted SGC7901 - LV - siAK cells that were stably knocked down AK058003 or control cells into nude mice through the tail vein injection. The average liver metastasis nodule numbers were apparent in mice that injected with SGC7901 - LV - control cells. In contrast,fewer metastasis nodules were de-tected in mice injected with SGC7901 - LV - siAK cells. All these results demonstrated that AK058003 invovled in the hypoxia - induced metastasis and invasion. Conclusion:Based on high - throughput screening of lncRNAs mi-croarrays,we firstly reported a group of hypoxic gastric cancer related lncRNAs. Further study revealed that lncRNA -AK058003 is one of the most key moleculars invovled in hypoxia induced GC metastasis and invasion.