糖酵解参与的黑色素瘤细胞放射敏感性lncRNAs的初步筛选及相关性分析

Preliminary screening and correlation analysis for lncRNAs related to radiosensitivity in melanoma cells by inhibiting glycolysis

目的:通过微阵列芯片技术筛选糖酵解参与的黑色素瘤细胞放射治疗(以下简称放疗)敏感性相关的lncRNA和mRNA表达谱,初步探索lncRNA调控糖酵解途径影响黑色素瘤细胞放疗敏感性的分子机制。方法:以不同浓度(1.25、2.50、5.00、10.00 mmol/L)的2-脱氧-D-葡萄糖(2-deoxy-D-glucose,2-DG)处理、不同剂量(0、2、4、6、8 Gy)的X射线照射黑色素瘤细胞WM35。采用MTT法检测NC-0 Gy组(阴性对照组)、NC-4 Gy组(仅4 Gy X射线照射)、2-DG组(仅2.50 mmol/L 2-DG处理)、2-DG-4 Gy组(2.50 mmol/L 2-DG处理,4 Gy X射线照射)增殖能力;lncRNA和mRNA表达谱微阵列芯片检测NC-4 Gy组和2-DG-4 Gy组的lncRNA和mRNA表达谱的改变。采用CNC(coding-noncoding)分析预测表达最显著的10个lncRNA潜在靶基因,构建差异lncRNA和mRNA共表达网络。运用基因本体(gene ontology,GO)功能富集分析和KEGG(Kyoto EncyclopediaofGenesandGenomes)通路富集分析预测差异表达的lncRNA的功能分布;real-time RT-PCR定量检测差异表达上调最显著的5个和下调最显著的5个lncRNA的表达。结果:2-DG处理WM35细胞48和96 h,细胞增殖均受到明显抑制(均P<0.05),呈剂量依赖关系;大剂量X射线照射抑制WM35细胞的增殖,造成细胞大量死亡,4 Gy X射线照射后细胞增殖活性较阴性对照组下降61%;对NC-4 Gy组和2-DG-4 Gy组行lncRNA芯片分析,发现有1206个lncRNA和543个mRNA差异表达,real-time RT-PCR示lncRNA的变化与mRNA芯片基本一致;进一步CNC分析示这10个表达最显著的lncRNA与333个靶基因之间存在正/负相关,GO功能富集分析结果显示lncRNA主要富集于DNA结合、DNA损伤和修复、细胞周期阻滞、氧化应激等功能,KEGG通路富集分析结果显示这10个lnRNA主要与放疗敏感性相关。结论:采用微阵列芯片技术筛选出糖酵解参与的黑色素瘤细胞放疗敏感性相关的差异表达lncRNA,lncRNA RPL34-AS1可能是黑色素瘤放疗潜在的生物靶点。

Objective:To screen the expression profiles of lncRNA and mRNA related to the radiosensitivity of melanoma cells by inhibiting glycolysis through microarray technology.Methods:WM35 melanoma cells were treated with different concentrations(1.25,2.50,5.00,10.00 mmol/L)of 2-deoxy-D-glucose(2-DG)and different doses(0,2,4,6,8 Gy)of X-ray irradiation.MTT assay was used to detect the proliferation ability of NC-0 Gy group(negative control group),NC-4 Gy group(only 4 Gy X-ray irradiation),2-DG group(only 2.50 mmol/L DG treatment),and 2-DG-4 Gy group(2.50 mmol/L 2-DG treatment,4 Gy Xray irradiation).Microarray chip was used to detect the changes in the expression profiles of lncRNA and mRNA in the NC-4 Gy group and the 2-DG-4 Gy group.Real-time RTPCR was used to quantitatively detect the top 5 upregulated and the top 5 downregulated expression lncRNA.CNC analysis was used to predict potential target genes for the 10 most significantly expressed lncRNAs,after which the co-expression network of lncRNA and co-regulated mRNA were constructed.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses were used to predict the functional distribution of differentially expressed lncRNA.Real-time RT-PCR was used to quantitatively detect the top 5 upregulated and the top 5 downregulated expression lncRNAs.Results:After 48 and 96 h,the cell proliferation of WM35 treated with 2-DG was significantly inhibited in a dose-dependent manner(all P<0.05).The cell proliferation of WM35 was inhibited by a high dose of X-ray irradiation,resulting in the death of mass cells.The cell proliferation activity of WM35 after 4 Gy X-ray irradiation descended 61%compared to the negative control group.Microarray analysis showed that there were 1206 lncRNAs and 543 differentially expressed mRNAs between the NC-4 Gy group and the 2-DG-4 Gy group,while real-time RT-PCR showed basically consistent changes in lncRNA and mRNA microarray.Further CNC analysis showed that these 10 lncRNAs had a positive or negative correlation with 333 target genes.GO analysis was mainly concentrated in DNA binding,DNA damage repair,cell cycle arrest,and oxidative stress,while KEGG pathway analysis showed the 10 lncRNAs were related to radiosensitivity.Conclusion:Microarray chip screens the expression profiles of differentially expressed lncRNA related to the radiosensitivity of melanoma cells via inhibiting glycolysis,and lncRNA RPL34-AS1 might be a potential biological target for melanoma radiotherapy.