[Effect of 53 Targeted Co-Inhibitory Pathways Based on Comprehensive Bioinformatics Analysis in Treating Squamous Cell Carcinoma of the Head and Neck].

OBJECTIVE

To investigate the association between WD40-encoding RNA antisense to 53 ( 53 ), 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 53 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 53 expression at the single cell level. Analysis of the small molecule inhibitors potentially targeting 53 was carried out by Computational Analysis of REsistance (CARE). In the verification experiment, recombinant lentiviral particles with the sh 53 sequence were synthesized. Then, the oral squamous cell carcinoma cell line Cal27 (the sh 53 group) stably expressing sh 53 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 53 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 , 23 and 28 passages of the sh 53 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 53 in HNSC tissues was significantly higher than that in normal tissues. There was a significant positive correlation between 53 expression and multiple genes in the 53 pathway, including 1, 2 and 1. Although no significant correlation between 53 expression and infiltrating immune cells was found, 53 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 53 in malignant cells, and very low or zero expression in immune cells. According to the CARE scores, the most potent 53 co-inhibitory drugs were , 1 and 4 targeted inhibitors. cell experiments showed that the proliferation ability of Cal27 cells decreased significantly in the sh 53 group as compared with that of the control group between Day 5 and Day 7 ( <0.05). Furthermore, the expression of P53 decreased significantly in the sh 53 group. As compared with the control group, the expression of WRAP53β in sh 53 group significantly decreased in the 18 , 23 and 28 passages ( <0.05), while γ-H2AX expression only decreased in the 18 and 28 passages ( <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

53 showed significantly higher expression level in HSNC, and was significantly associated with 53 pathway genes. , 1 and 4 inhibitors may be potential 53 co-inhibitory agents. RNA interference of 53 expression may cause inhibition of DNA damage, thereby indicating therapeutic potential for HNSC.