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  Objective  To investigate the association between WD40-encoding RNA antisense to p53 (WRAP53β), 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.

  Methods  Tumor IMmune Estimation Resource (TIMER) online modules were adopted to predict the association between WRAP53β 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 WRAP53β expression at the single cell level. Analysis of the small molecule inhibitors potentially targeting WRAP53β was carried out by Computational Analysis of REsistance (CARE). In the in vitro verification experiment, recombinant lentiviral particles with the shWRAP53β sequence were synthesized. Then, the oral squamous cell carcinoma cell line Cal27 (the shWRAP53β group) stably expressing shWRAP53β 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 shWRAP53β 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 shWRAP53β 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 WRAP53β in HNSC tissues was significantly higher than that in normal tissues. There was a significant positive correlation between WRAP53β expression and multiple genes in the p53 pathway, including CCNB1, CCNB2 and CDK1. Although no significant correlation between WRAP53β expression and infiltrating immune cells was found, WRAP53β 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 WRAP53β in malignant cells, and very low or zero expression in immune cells. According to the CARE scores, the most potent WRAP53β 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 shWRAP53β 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 shWRAP53β group. As compared with the control group, the expression of WRAP53β in shWRAP53β 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 results of Western blot. Clinical specimens showed rather high positive expression rate of γ-H2AX in oral squamous cell carcinoma tissues (12/13), while the expression of WRAP53β was not detected in oral mucositis samples (0/7).

  Conclusions  WRAP53β showed significantly higher expression level in HSNC, and was significantly associated with p53 pathway genes. ATM, CDK1 and MDM4 inhibitors may be potential WRAP53β co-inhibitory agents. RNA interference of WRAP53β expression may cause inhibition of DNA damage, thereby indicating therapeutic potential for HNSC.