非小细胞肺癌并发肺栓塞患者差异基因lncRNA MALAT1的筛选与验证

Screening of differential gene lncRNA MALAT1 in patients with non-small cell lung cancer concurrent pulmonary embolism and validation analysis

目的 通过高通量测序(RNA-Seq)技术筛选非小细胞肺癌(NSCLC)并发肺血栓栓塞症(PTE)患者中差异基因表达谱,探讨长链非编码RNA(lncRNA)肺腺癌转移相关转录本1(MALAT1)与NSCLC并发PTE疾病的关联性。方法 收集2020-01-01-2020-12-30就诊于新疆医科大学附属肿瘤医院确诊的NSCLC并发PTE患者、单纯NSCLC患者及同期门诊健康体检者的临床资料及其外周血样本各4例。(1)差异基因lncRNA MALAT1的筛选:NSCLC并发PTE患者、单纯NSCLC患者和健康体检者的外周血各4例,应用RNA-Seq行差异基因lncRNA表达谱测序,行基因本体论(GO)和京都基因与基因组百科全书(KEGG)数据库分析差异基因相关的富集及信号通路。(2)对筛选出的lncRNA MALAT1差异表达的验证:上述3组各30例,应用实时荧光定量聚合酶链反应法检测患者外周血MALAT1、缺氧反应诱导因子1α(HIF-1α)、尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4(NOX4)及活性氧(ROS)的表达水平,通过单因素方差分析、LSD-t检验比较组间差异,采用简单线性回归分析MALAT1表达水平与HIF-α、NOX4及ROS表达水平之间的相关性。结果 (1)NSCLC并发PTE患者对比单纯NSCLC患者,共筛选出727个差异表达长链非编码RNA(lncRNA),其中327个表达上调,400个表达下调;单纯NSCLC患者与健康对照组对比,共筛选出794个差异表达lncRNA,其中414个表达上调,380个表达下调。利用Excel 2019得到NSCLC并发PTE组对比单纯NSCLC组,以及单纯NSCLC组对比健康对照组患者共同的差异基因,分别为MERGE.7330.1、MALAT1、MERGE.9869.8、MERGE.11060.20、MERGE.32224.4、MERGE.27691.1、MERGE.8536.1、MERGE.3176.3等8个基因。进一步发现,共同参与的KEGG通路主要涉及HIF-1α信号通路、B细胞受体信号通路、系统性红斑狼疮等。(2)3组中MALAT1、HIF-1α、NOX4及ROS的表达水平有共同的规律,即由高到低依次为NSCLC并发PTE组、单纯NSCLC组及健康对照组,MALAT1测值分别为1.78±0.64、1.33±0.33和0.96±0.08,HIF-1α测值分别为2.29±0.48、1.60±0.34和1.01±0.06,NOX4测值分别为2.33±0.63、1.59±0.39和0.98±0.08,ROS测值分别为1.82±0.53、1.60±0.39和0.97±0.09,差异均有统计学意义,F值分别为28.936、106.716、74.699及30.648,均P<0.001。上述指标在2组中的Person相关分析显示,MALAT1与HIF-1α表达呈中度正相关(r=0.696,P<0.001),与NOX4表达呈高度正相关(r=0.785,P<0.001),与ROS表达呈中度正相关,r=0.680,P<0.001。结论 MALAT1与HIF-1α/NOX4/ROS轴相关,从而参与NSCLC并发PTE患者疾病的发生发展。

Objective To screen for differential gene expression profiles in patients with non-small cell lung cancer(NSCLC) with pulmonary embolism(PTE) by high-throughput sequencing(RNA-Seq) and investigate the correlation between long non-coding RNA(lncRNA) metastasis-associated lung adenocarcinoma transcript 1(MALAT1) and NSCLC complicated with PTE.Methods Clinical data and peripheral blood samples of 4 patients with NSCLC complicated with PTE,4 patients with NSCLC,and 4 healthy outpatients were collected from January 1,2020 to December 30,2020 at the Cancer Hospital Affiliated to Xinjiang Medical University.(1)Screening of differential gene lncRNA MALAT1:There were 4 cases of peripheral blood in patients with NSCLC complicated with PTE,patients with NSCLC and health examiners,respectively.RNA-Seq was used to sequence the differential gene lncRNA expression profile.The enrichment and signaling pathways of differential gene were analyzed by using genetic ontology(GO)and Kyoto encyclopedia of genes and genomics(KEGG)database.(2)To verify the differential expression of lncRNA:There were 30 cases in the above three groups.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the expression levels of peripheral blood MALAT1,hypoxia-inducible factor-1α(HIF-1α),NOX4,and reactive oxygen species(ROS)in patients.One-way ANOVA and LSD-t test were used to compare the difference among groups.Simple linear regression was used to analyze the correlation between MALAT1 expression level and HIF-1α,NOX4 and ROS expression levels.Results(1)Compared to patients with NSCLC,727 different expression lncRNAs were screened in patients with NSCLC complicated with PTE,of which 327 were upregulated and 400 were downregulated.Compared with normal health group,794 different expression lncRNAs were screened in patients with NSCLC,414 expressions were upregulated,380 expressions were downregulated,Excel 2019 was used to obtain common differential genes in patients with NSCLC and PTE compared with patients with NSCLC alone,patients with NSCLC alone,and healthy controls:namely,MERGE.7330.1,MALAT1,MERGE.9869.8,MERGE.11060.20,MERGE.32224.4,MERGE.27691.1,MERGE.8536.1,MERGE.3176.3.It was further found that the KEGG pathway involved HIF-1αsignaling pathway,B cell receptor signaling pathway and systemic lupus erythematosus.(2)The expressions of MALAT1,HIF-1α,NOX4 and ROS in 3 group have a common pattern,namely,NSCLC concurrent PTE,NSCLC and normal health.MALAT1measurements were 1.78±0.64,1.33±0.33,0.96±0.08,the values HIF-1α were 2.29±0.48,1.60±0.34,1.01±0.06,respectively.NOX4 measurements were 2.33±0.63,1.59±0.39,0.98±0.08,the ROS measurements were 1.82±0.53,1.60±0.39and 0.97±0.09respectively,and the differences were statistically significant,Fvalues were 28.936,106.716,74.699and 30.648,all P<0.001.The correlation analysis of the above indexes in 2 case groups showed that MALAT1 expression level was moderately positively correlated with HIF-1α expression(r=0.696,P<0.001)and highly positively correlated with NOX4 expression(r=0.785,P<0.001);moderately positive correlated with ROS expression(r=0.680,P<0.001).Conclusion MALAT1 may associate with the HIF-1α/NOX4/ROS axis and involve in the occurrence and development of patients with NSCLC complicated by PTE.