长链非编码RNA Linc00467在肺腺癌中的表达及功能

Expression and roles of long non-coding RNA Linc00467 in lung adenocarcinoma

目的长链非编码RNA Linc00467在肺腺癌发生发展中的作用尚不明确。文中旨在研究Linc00467在人肺腺癌中的表达情况及临床意义,并阐明Linc00467在体外对肺腺癌细胞及内皮细胞功能的影响。方法选取2012年1月至2013年12月南京军区南京总医院心胸外科60例行肺叶切除并经术后病理证实为肺腺癌I-IIIa期患者的肺腺癌组织标本,及癌周正常组织标本。HUVEC细胞系分为HUVEC实验组(转染pccl-Linc00467过表达质粒)、HUVEC对照组(转染pccl空质粒);A549细胞系分为A549实验组(转染Linc00467-siRNA)、A549对照组(转染阴性siRNA);H1299细胞系分为H1299实验组(转染Linc00467-siRNA)、H1299对照组(转染阴性siRNA)。使用qRT-PCR验证Linc00467在人肺腺癌中的表达情况,分析其与患者临床病理参数的相关性;CCK-8法研究Linc00467在体外对肺腺癌细胞A549、H1299及内皮细胞HUVEC增殖能力的影响;探究Linc00467对HUVEC内皮细胞形成新生血管能力的影响。结果 Linc00467在肺腺癌组织中的表达水平是正常肺组织的(2.72±1.31)倍;Linc00467在A549、H1299中的相对表达量分别为HBE的(3.45±0.25)、(3.22±0.33)倍,差异具有统计学意义(P<0.01)。患者肿瘤大小、血管侵犯与Linc00467的相对表达水平显著相关(P<0.05)。转染Linc00467-siRNA后,A549实验组、H1299实验组Linc00467的表达水平分别较A549对照组、H1299对照组下调72%和68%(P<0.01)。培养48、72、96 h后,A549实验组活细胞数量较A549对照组显著减少[(1.29±0.07)vs(1.51±0.09)个、(1.53±0.15)vs(2.13±0.11)个、(1.98±0.18)vs(3.02±0.12)个];H1299实验组较H1299对照组亦显著减少;HUVEC实验组较HUVEC对照组显著增加(P<0.01)。实验第5天,HUVEC实验组生成血管的分支数量、相对血管长度较HUVEC对照组均明显增加(7.36个/微珠vs 4.25个/微珠、3.12 vs 1,P<0.01)。结论 Linc00467能在体外促进肺腺癌细胞增殖和血管生成,人肺腺癌组织中高表达Linc00467与肿瘤大小和血管侵犯等因素密切相关,提示其为潜在的生物标记物和治疗靶标。

Objective The role of long non-coding RNA Linc00467 in human lung adenocarcinoma is not yet clear. This study was to investigate the expression of long non-coding RNA Linc00467 in human lung adenocarcinoma,its clinical significance,and the effects of Linc00467 on the functions of the tumor and endothelial cells in vitro. Methods Lung adenocarcinoma tissue and normal tissue surrounding the malignance were obtained from 60 patients with pathologically proved stage I-Ⅲa lung adenocarcinoma.Human umbilical vein endothelial cells（HUVECs） were transfected with the over-expressed plasmid pccl-Linc00467（HUVEC experimental group） or the empty vector pccl（HUVEC control group）,A549 cells with Linc00467-siRNA（A549 experimental group） or negative siRNA（A549 control group）,and H1299 cells,too,with Linc00467-siRNA（H1299 experimental group） or negative siRNA（H1299 control group）. The expression level of Linc00467 in the lung adenocarcinoma tissue was detected by qRT-PCR with an analysis of its correlation with the clinicopathological characteristics of the patients; the influence of Linc00467 on the proliferation of the A549,H1299 and HUVEC cells was assayed with CCK-8; and the role of Linc00467 in the angiogenesis of the HUVECs was assessed by fibrin bead sprouting assay. Results The expression of Linc00467 in the lung adenocarcinoma tissue was 2.72±1.31 times as high as that in the normal lung tissue（P〈0.01）,and those in the A549 and H1299 cells were 3.45±0.25 and 3.22±0.33 times as high as those in the human bronchial epithelial（HBE） cells（P〈0.01）. The expression level of Linc00467 was significantly correlated with the tumor size and vascular invasion（P 0. 05）. After transfection of Linc00467-siRNA,the expressions of Linc00467 in the A549 and H1299 experimental groups were down-regulated by 72% and 68%as compared with those in the A549 and H1299 control groups（P〈0.01）. The number of living cells was remarkably decreased in the A549 experimental group in comparison with the A549 control at 48 h（1.29±0.07 vs 1.51±0.09）,72 h（1.53±0.15 vs 2.13±0.11）,and 96 h after culturing（1.98±0.18 vs 3. 02 ± 0. 12）,and so was it in the H1299 experimental versus the H1299 control group,but markedly increased in the HUVEC experimental versus the HUVEC control group（P〈0.05）. At 5 days,HUVEC experimental group,as compared with the HUVEC control,showed a significantly increased number of newly formed vascular branches（7.36 vs 4.25/superbead,P〈0.01） and relative length of the blood vessels（3.12 vs 1,P〈0.01）. Conclusion Linc00467 promotes tumor cell proliferation and angiogenesis and is highly expressed in the lung adenocarcinoma tissue,which is correlated with the tumor size and vascular invasion and suggests that Linc00467 could be a potential biomarker and therapeutic target.