Ferroptosis-Related LncRNAs Signature Predicts the Prognosis of Stomach Adenocarcinoma
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摘要:
目的 通过研究胃癌细胞铁死亡相关LncRNA,建立预测胃腺癌患者的生存预后情况的预后模型,从而为其生物标志物与治疗靶点的开发提供理论依据。 方法 对 TCGA数据库中的胃腺癌患者的转录本测序数据进行分析,并与铁死亡相关基因取交集,通过共表达和差异分析方法,从而筛选出与铁死亡相关的 LncRNA。采用单因素和多因素 Cox回归分析,筛选出与胃腺癌患者预后相关的 LncRNA,从而建立预后评分模型。在此基础上,对每一样本进行风险值计算,并对模型的可靠性进行充分验证;根据模型结果对高低风险组之间进行免疫浸润与免疫反应等差异分析。 结果 肿瘤组织中相较正常组织筛选到与铁死亡相关的503个 LncRNA (上调431个,下调72个);单因素Cox回归分析得出33个可作为独立风险因子的 LncRNA,而多因素Cox回归分析构建出一个由17个 LncRNA组成的预测模型。生存曲线表明高风险的患者比低高风险的患者的存活率显著降低(P < 0.001);单因素和多因素独立预后分析表明,年龄、分期与风险值是患者的独立危险因素;时间依赖的ROC曲线提示,模型的1,2,3 a生存率预测AUC值为0.751,0.799,0.779,证明模型具备可靠与稳定性。高低风险组间多个免疫激活反应、免疫细胞的浸润程度与免疫检查点的表达水平存在显著差异。 结论 以铁死亡相关lncRNA为基础,建立胃腺癌患者预后预测模型,可较好地评估患者预后情况,纳入模型的LncRNA具备开发为生物标志物与治疗靶点的可行性。 Abstract:Objective To establish a prognostic model that predicts the survival and prognosis of gastric adenocarcinoma patients by studying the LncRNAs related to iron death in gastric cancer cells, thereby providing a theoretical basis for the development of their biomarkers and therapeutic targets. Methods The transcript sequencing data of gastric adenocarcinoma patients in the TCGA database were analyzed and intersected with iron death-related genes, which were screened for iron death-related LncRNAs by co-expression and differential analysis methods. One-way and multifactorial Cox regression analyses were used to screen out the prognostic-related LncRNAs in gastric adenocarcinoma patients, so as to establish the prognostic scoring models. On this basis, risk values were calculated for each sample, and the reliability of the model was fully verified. According to the model results, differences in the immune infiltration and immune response between the high- and low-risk groups were analyzed. Results Tumor tissues were screened for 503 LncRNAs (431 up-regulated and 72 down-regulated) associated with iron death compared to the normal tissues; univariate Cox regression analysis yielded 33 LncRNAs that could be used as the independent risk factors, whereas multivariate Cox regression analysis constructed a predictive model consisting of 17 LncRNAs. Survival curves indicated that patients with the high risk had the significantly lower survival rates than those with the low risk (P < 0.001). Unifactorial and multifactorial independent prognostic analyses showed that age, stage, and risk value were independent risk factors for patients; Time-dependent ROC curves suggested that the predicted AUC values of the model's 1-, 2-, and 3-year survival rates were 0.751, 0.799, and 0.779 respectively, proving that the model was reliable and stable. There were significant differences in multiple immune activation responses, the degree of immune cell infiltration, and the expression levels of immune check points between the high- and low-risk groups. Conclusion The established prognostic prediction model based on iron death-related lncRNAs for gastric adenocarcinoma patients can better assess the prognosis of patients, and the lncRNAs included in the model have the feasibility of being developed into biomarkers and therapeutic targets. -
Key words:
- Stomach adenocarcinoma /
- Ferroptosis /
- LncRNA /
- Prognostic model
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头颈部鳞癌中最常见的是喉癌,占所有癌症死亡的1%[1],据文献报道[2]预计2022年中国(不包括台湾地区)将有大约3万人新诊断出喉癌,1.7万人死于喉癌;其中,新发病例男性约2.7万人,女性约0.3万人;死亡病例男性约1.4万,女性约0.3万。喉乳头状瘤,儿童发病多于成人,成人有恶变倾向,因此,喉癌的癌前病变是喉乳头状瘤[3-4]。喉息肉多由慢性炎症、刺激等因素引起的,发病率占喉部良性肿瘤的20%以上[5]。
目前,关于喉鳞状上皮病变的不同阶段(喉息肉、喉乳头状瘤、喉鳞状细胞癌)关系比较的报道少见。而喉癌目前的治疗方法仍以手术为主,但传统手术方法创伤较大,且术后患者生存质量较低。随着微创手术概念的兴起,需要对喉癌作出早期诊断,并正确估计患者治疗后的预后,以便选择合理的治疗方法,避免造成不必要的创面并尽最大可能地保留喉的功能。当前,肿瘤患者手术方法的确定和预后的评价主要基于肿瘤的临床分期,但同一分期的喉癌患者常常显示出不同的临床过程,这就表明单一的TNM分期对于喉癌患者预后的推测存在不足。为此,本课题主要通过免疫组化检测增殖分化相关基因COX2、CDX2在喉息肉、喉乳头状瘤、喉鳞癌中的表达,同时探讨检测指标与喉癌患者预后之间关系以找出判断喉鳞癌预后的最佳免疫组化检测指标,为喉鳞癌患者预后判断和治疗方案的选择提供一定的理论依据。
1. 资料与方法
1.1 研究材料
1.1.1 样本资料
样本选自昆明医科大学第二附属医院手术切除的临床资料完整的存档蜡块,共51例。全部病例术前均未经放疗和化疗。其中,喉息肉15例、喉乳头状瘤11例、喉鳞癌25例;25例喉鳞癌样本,高分化8例,中分化11例,低分化6例。淋巴结有转移8例,淋巴结无转移17例。男性23例,女性2例,年龄39~79岁,平均62.12岁。按WHO喉癌TNM分类标准[6]:T1~T2期16例,T3~T4期9例。4%中性甲醛固定,5 µm连续切片。
所有患者的随访以电话方式进行,随访日期从病理报告确诊日期算起,至2008年12月,严格记录随访情况。共随访到25例喉鳞癌患者,失访0例;随访结局定义为死亡。
1.1.2 主要试剂
COX2即用型兔抗人单克隆抗体、CDX2即用型鼠抗人单克隆抗体、DAB显色试剂盒、即用型非生物素免疫组化EliVisionTM plus检测试剂盒等均为美国Maxin公司产品,购自福州迈新生物技术有限公司。
1.2 研究方法
1.2.1 免疫组化染色步骤
严格按试剂盒说明进行。每次实验均设阳性对照和阴性对照。
细胞质和/或细胞膜为COX2主要着色部位,淡黄色~棕黄色。细胞核为CDX2主要着色部位,呈淡黄色。
采用HPIAS-1000高清晰度病理图文分析系统对免疫组化结果进行定量分析。每个样本均在400倍下,按无偏采样原则取5个视野,每个视野测定5个细胞。最终结果由阳性单位(positive unit,PU)来判断[7]。
1.3 统计学处理
应用SPSS21. 0统计软件包,均值±标准差(
$ \bar x \pm s $ )表示PU值[6],采用单因素方差分析(单因素ANOVA检验)和t检验;生存分析使用Kaplan-Meier曲线进行单因素分析;P < 0.05为差异有统计学意义。2. 结果
2.1 COX2蛋白免疫组化检测结果
COX2在喉鳞癌组织中的表达低于喉息肉、喉乳头状瘤中的表达(图1~3),差异有统计学意义(P = 0.004,P = 0.001),在喉息肉和喉乳头状瘤中阳性着色集中于棘细胞层和颗粒细胞层;T3-T4期喉鳞癌COX2表达比T1-T2期高,且差异有统计学意义(P = 0.001);有淋巴结转移喉鳞癌组高于无淋巴结转移喉鳞癌组,低、中、高分化喉鳞癌中的表达逐渐升高,但差异均无统计学意义(P > 0.05),见 表1~2。
表 1 COX2在喉息肉、喉乳头状瘤和喉鳞癌中的表达($\bar x \pm s $ )Table 1. The expression of COX2 in laryngeal polyps,laryngeal papilloma and laryngeal squamous cell carcinoma ($ \bar x \pm s $ )组别 n COX2表达 P 阳性单位(PU) 喉息肉 15 20.5987 ± 6.1766 1.000,0.004▲ 喉乳头状瘤 11 23.0000 ± 2.4537 0.001▲ 喉鳞癌 25 11.3664 ± 10.4782 与喉鳞癌比较,▲P < 0.05。 表 2 COX2在喉鳞癌中的表达与临床病理参数之间的关系($\bar x \pm s $ )Table 2. The relationship between the expression of COX2 and clinicopathologic parameters in laryngeal squamous cell carcinoma ($\bar x \pm s $ )临床病理参数 n COX2表达 P 阳性单位(PU) 临床分期 T1~T2 16 6.6875 ± 8.9408 0.001 T3~T4 9 19.5778 ± 7.6142 颈淋巴结转移 (+) 8 13.1500 ± 10.9172 0.571 (−) 17 10.5294±10.5008 分化程度 高分化 8 15.2275 ± 9.6195 1.000,0.169 中分化 11 12.3945±9.9813 0.381 低分化 6 4.3333±10.6145 2.2 CDX2蛋白免疫组化检测结果
CDX2在喉息肉、喉乳头状瘤(图4~5)中多为弱阳性~阳性表达,阳性细胞散在分布,喉鳞癌(图6)中未表达CDX2,但两两比较差异无统计学意义(P > 0.05),见 表3。
表 3 CDX2在喉息肉、喉乳头状瘤和喉鳞癌中的表达($\bar x \pm s $ )Table 3. The expression of CDX2 in laryngeal polyps,laryngeal papilloma and laryngeal squamous cell carcinoma ($\bar x \pm s $ )组别 n CDX2表达 P 阳性单位(PU) 喉息肉 15 8.73 ± 1.20 0.455,0.052 喉乳头状瘤 11 5.01 ± 0.05 0.132 喉鳞癌 25 0 2.3 COX2的表达与喉鳞癌患者预后的关系
单因素生存分析中,COX2阴性表达的喉鳞癌患者1 a、3 a、5 a生存率高于COX2阳性表达的喉鳞癌患者,COX2阴性表达的喉鳞癌中位生存期比COX2阳性表达的长,但是,两者比较差异无统计学意义(P > 0.05),见 表4和图7。
表 4 COX2的表达与喉鳞癌患者预后的关系Table 4. Relationship between expression of COX2 and prognosis of laryngeal squamous cell carcinoma patientCOX2表达 年生存率(%) 中位生存期 1 a 3 a 5 a 95%可信区间 P (+) 85.71 34.29 34.29 1.70~4.30 0.078 (−) 90.91 90.91 40.40 3.00~7.00 3. 讨论
3.1 COX2的表达特点及分析
喉鳞癌是一种高度致残的疾病,影响患者的语言、吞咽和呼吸技能[8],临床上早期诊断极为重要。环氧合酶2(cyclooxygenase 2,COX2)作为体内前列腺素生物合成的限速酶,其蛋白由604个氨基酸组成,表皮生长因子结构域、膜结合结构域及酶活性结构域,构成其基本结构。人COX2基因位于1号染色体1q25.2~25.3上,全长约8.3 kb,含10个外显子和9个内含子[9]。COX2被认为是一种“应答基因”,在健康组织中呈低表达或不表达[10-11],而在多种癌前病变和恶性病变时呈高表达[12]。其在体内呈诱导性表达,在被细胞内外各种刺激因素如脂多糖、白介素-1、血小板激活因子等因素所诱导下表达COX2。病理情况下,COX2参与炎症及肿瘤的形成[13-14]。研究显示,COX2与喉鳞癌[15-16]、食管鳞癌[17]和头颈部鳞癌[18]的淋巴结转移及预后有关,而有报道[19-20]却发现口腔鳞癌的复发或生存率与COX2无关,因此,在头颈部鳞癌中,COX2的表达无预后意义。本实验发现COX2在喉鳞癌中的表达低于喉息肉和喉乳头状瘤中的表达,表明COX2参与了炎症反应及喉鳞癌的发生;COX2在喉鳞癌低、中、高分化的表达逐渐升高,但其表达差异无统计学意义;随着喉鳞癌临床分期的增高,COX2的表达也增加,且差异有统计学意义。笔者认为COX2在不同病理阶段含量不同,对肿瘤细胞的促增殖作用也不同。
本实验还发现喉鳞癌中有淋巴结转移组比无淋巴结转移组的COX2阳性表达高,但差异无统计学意义。考虑与标本量太少有关。COX2表达阴性的喉鳞癌患者比阳性的患者中位生存期长,差异无统计学意义,故笔者认为COX2不能预测喉鳞癌患者的预后。
3.2 CDX2的表达特点及分析
CDX2基因,包括3个外显子和2个内含子,全长22~23 kb,蛋白含311个单氨基酸,与DNA的相应区域结合,方式为螺旋-环-螺旋,DNA表达的调节靠转录因子的形式。这种核同源异构体转录因子,调控着组织的分化和增殖[21-22]。
据报道,CDX2是胆管癌[23]和胰腺浸润性导管癌[24]患者预后的指标之一;CDX2对Barrett食管进展的早期检测具有潜在的预后效用,其表达可显著预测初次活检后40 ~ 45个月杯状细胞肠化生发生的风险[22]。有学者推测CDX2对肠上皮的鉴别、发生发展起着重要作用[25],是结直肠腺癌[26]的标志物,具有高度特异性和敏感性,CDX2的表达可能有助于乳腺Paget病的分型[27];但也有学者认为CDX2多态性不能作为预测中国人结直肠癌易感性的有用标记[28]。60例食管鳞癌中CDX2的阳性率为5%,而CDX2在43例肺鳞癌中仅2例为散在阳性,42例膀胱鳞癌中也发现有CDX2表达,但表达较少为1例,提示CDX2在鳞癌中表达极低[29]。
本实验发现CDX2在喉息肉、喉乳头状瘤中多为弱阳性~阳性表达,CDX2在喉鳞癌中不表达,且差异无统计学意义,初步推测CDX2可能不是喉鳞癌的危险因素。
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表 1 患者临床基线资料表[n(%)]
Table 1. The clinical characteristics of patients in the TCGA database [n(%)]
临床特征 分类 人数 死亡人数 n 375 150 性别 女性 134 (35.7) 51(34) 男性 241 (64.3) 99(66) 年龄 ≤65 173 (46.1) 60(40) >65 202 (53.9) 90(60) 组织学分级 G1 10 (2.7) 3(2) G2 131 (34.9) 49(32.7) G3 234 (62.4) 98(65.3) 病理分期 Stage I 46 (12.3) 11(7.3) Stage II 123 (32.8) 34(22.7) Stage III 165 (44) 79(52.7) Stage IV 41 (10.9) 26(17.3) T 分期 T1 15 (4) 1(0.7) T2 80 (21.3) 27(18) T3 179 (47.8) 79(52.7) T4 101 (26.9) 43(28.6) N 分期 N0 114 (30.4) 29(19.3) N1 102 (27.2) 43(28.7) N2 75 (20) 31(20.7) N3 84 (22.4) 47(31.3) M 分期 M0 339 (90.4) 133(88.7) M1 36 (9.6) 17(11.3) -
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