Conclusions
WRAP53 β showed significantly higher expression level in HSNC, and was significantly associated with p53 pathway genes. ATM, CDK1 and MDM4 inhibitors may be potential WRAP53 β co-inhibitory agents. RNA interference of WRAP53 β expression may cause inhibition of DNA damage, thereby indicating therapeutic potential for HNSC. 目的: 探讨端粒酶新核心亚单位WD40-encoding RNA antisense to p53（WRAP53β）与头颈鳞癌（squamous cell carcinoma of the head and neck, HNSC）的临床、基因组和免疫浸润特征的关系，寻求HNSC的潜在靶向联合治疗方法。 方法: 采用TIMER在线模块预测WRAP53β表达与TCGA队列中头颈肿瘤的临床特征、癌基因或免疫浸润之间的关系，TISCH网站分析其在单细胞水平的表达，通过CARE软件分析靶向WRAP53β的小分子抑制剂。体外实验验证中，合成shWRAP53β序列的重组慢病毒颗粒，构建稳定表达shWRAP53β的口腔鳞癌Cal27细胞（shWRAP53β组），并设置两个对照组（shNC组：Cal27细胞加入含非特异性对照序列的慢病毒颗粒，Con组：未处理的Cal27细胞）；MTT法检测3组细胞的增殖能力，细胞免疫荧光检测进一步定性检测shWRAP53β组和shNC组细胞中P53蛋白的表达；Western blot检测第18、23、28代shWRAP53β组和shNC组细胞的WRAP53β和DNA损伤蛋白γ-H2AX的表达。最后，收集口腔鳞癌病例样本13例，口腔黏膜炎性病例样本7例，通过免疫组化验证口腔鳞癌临床标本中WRAP53β和γ-H2AX的表达情况。 结果: TIMER分析发现WRAP53β表达在HNSC中较正常组织显著高表达。WRAP53β基因水平与p53通路基因如CCNB1、CCNB2及CDK1呈显著正相关，与HPV阳性的头颈肿瘤炎症因子IFN-γ、IL-23A呈正相关，而与IL-1A、IL-6呈负相关，但与浸润免疫细胞无显著相关。TISCH单细胞测序数据集同样显示该基因在恶性细胞中呈高表达，在免疫细胞中表达水平极低或无表达。CARE评分预测对WRAP53β共抑制效果最强的药物为靶向ATM、CDK1和MDM4的抑制剂。体外细胞实验证实，与对照组相比，shWRAP53β组Cal27细胞的第5天到第7天的增殖能力下降（P<0.05），P53表达降低。Western blot检测示，与对照组相比，shWRAP53β组WRAP53β的表达水平在第18、23、28代均降低（P<0.05），γ-H2AX表达仅在第18、28代降低（P<0.05）。临床标本显示口腔鳞癌组织中γ-H2AX的阳性表达检出率高（12/13），口腔黏膜炎样本未检出WRAP53β阳性表达（0/7）。 结论: WRAP53β在HSNC中呈高表达水平，且与p53通路基因显著相关。ATM、CDK1和MDM4抑制剂可能是WRAP53β的潜在共抑制药物。干扰WRAP53β可能导致DNA损伤降低，从而具有对HNSC的治疗潜力。
Methods
Tumor IMmune Estimation Resource (TIMER) online modules were adopted to predict the association between WRAP53 β expression and the clinical features, oncogene, and immune infiltration of HNSC in the Cancer Genome Atlas (TCGA) cohort. Tumor Immune Single-cell Hub (TISCH) was used to analyze WRAP53 β expression at the single cell level. Analysis of the small molecule inhibitors potentially targeting WRAP53 β was carried out by Computational Analysis of REsistance (CARE). In the in vitro verification experiment, recombinant lentiviral particles with the sh WRAP53 β sequence were synthesized. Then, the oral squamous cell carcinoma cell line Cal27 (the sh WRAP53 βgroup) stably expressing sh WRAP53 β were constructed, and two control groups were set up (the shNC group consisting of Cal27 cells added with lentiviral particles containing non-specific control sequences and the Con group consisting of untreated Cal27 cells). MTT assay was done to examine the proliferation of cells in the three groups. Cellular immunofluorescence assay was done for further qualitative examination of the expression of P53 protein in the cells of the sh WRAP53 β group and the shNC group. Western blot was done to measure the expression of WRAP53β and γ-H2AX, a DNA damage protein, in the 18 th, 23 rd and 28 th passages of the sh WRAP53 β group and the shNC group. Finally, specimens of 13 cases of oral squamous cell carcinoma and 7 cases of oral mucosal inflammation were collected, and the expression of WRAP53β and γ-H2AX in the clinical specimens of oral squamous cell carcinoma was verified with immunohistochemistry. Resluts: TIMER analysis revealed that the expression level of WRAP53 β in HNSC tissues was significantly higher than that in normal tissues. There was a significant positive correlation between WRAP53 β expression and multiple genes in the p53 pathway, including CCNB1, CCNB2 and CDK1. Although no significant correlation between WRAP53 β expression and infiltrating immune cells was found, WRAP53 β was significantly positively correlated with the inflammatory factors IFN-γ and IL23A, and negatively correlated with IL-1A and IL-6 in HPV-positive carcinoma of the head and neck. TISCH single cell sequencing datasets also showed higher expression of WRAP53 β in malignant cells, and very low or zero expression in immune cells. According to the CARE scores, the most potent WRAP53 β co-inhibitory drugs were ATM, CDK1 and MDM4 targeted inhibitors. In vitro cell experiments showed that the proliferation ability of Cal27 cells decreased significantly in the sh WRAP53 β group as compared with that of the control group between Day 5 and Day 7 ( P<0.05). Furthermore, the expression of P53 decreased significantly in the sh WRAP53 β group. As compared with the control group, the expression of WRAP53β in sh WRAP53 β group significantly decreased in the 18 th, 23 rd and 28 th passages ( P<0.05), while γ-H2AX expression only decreased in the 18 th and 28 th passages ( P<0.05) according to the
Objective
To investigate the association between WD40-encoding RNA antisense to p53 ( WRAP53 β), a telomerase new core subunit, and the clinical, genomic and immune infiltration characteristics of squamous cell carcinoma of the head and neck (HNSC), and to explore for potential joint targeted therapy of HNSC.