Abstract:

Objective To screen for differentially expressed genes （DEGs） between squamous cell carcinoma （abbreviated as squamous carcinoma） and adenocarcinoma based on The Cancer Genome Atlas （TCGA） database， and to investigate the role of nerve growth factor receptor （NGFR） gene in promoting apoptosis of squamous carcinoma and adenocarcinoma cells by T lymphocytes activated by programmed death receptor 1 （PD-1） inhibitor through in vitro cell experiments. Methods RNA sequencing （RNA-Seq） data of squamous carcinoma and adenocarcinoma tissues from human esophagus， lung and cervix were downloaded from TCGA database. R software DESeq2 package was used to analyze DEGs. Gene Ontology （GO） functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） signaling pathway enrichment analysis were used to screen for genes related to phosphatidylinositol 3?kinase （PI3K）/protein kinase B （AKT） and Janus kinase （JAK）/signal transducer and activator of transcription （STAT） signaling pathways， and survival prognosis analysis was performed to determine NGFR gene as the outcome gene. For in vitro cell experiments， lung adenocarcinoma （LUAD） A549 cell line， cervical adenocarcinoma （CEAD） HeLa cell line， lung squamous carcinoma （LUSC） SK-MES-1 cell line and cervical squamous carcinoma （CESC） SiHa cell line were selected. The cells were divided into control group （untreated tumor cells）， OE-NC group （tumor cells 48 h after empty vector plasmid transfection）， si-NC group （tumor cells 48 h after negative siRNA transfection）， OE-NGFR group （tumor cells 48 h after NGFR gene overexpression plasmid transfection）， and si-NGFR group （tumor cells 48 h after NGFR gene siRNA transfection）. The co-culture system of tumor cells and T lymphocytes was established and divided into control group， Control+T lymphocytes group， Control+T lymphocytes+anti-PD-1 group， OE-NC/si-NC group， OE-NC/si-NC+T lymophocytes group， OE-NC/si?NC+T lymophocytes+anti-PD-1 group， OE-NGFR/si-NGFR group， OE-NGFR/si-NGFR+T lymophocytes group， and OE-NGFR/si-NGFR+T lymophocytes+anti-PD-1 group. Overexpression plasmid was used to increase the expression of NGFR gene in adenocarcinoma cells， and siRNA was used to knock down the expression of NGFR gene in squamous carcinoma cells. Real-time fluorescence quantitative PCR （RT-qPCR） was used to detect the NGFR mRNA expression level in tumor cells in various groups； Western blotting method was used to detect the NGFR protein expression level in tumor cells in various groups； flow cytometry was used to detect the apoptotic rates of tumor cells in various groups， and the relative increase folds of apoptie rates were calculated. Results The TCGA database analysis results showed that the expression level of NGFR gene in squamous carcinoma tissue was significantly higher than that in adenocarcinoma tissue （P<0.05）. The in vitro cell experiment results showed that after transfection with NGFR gene overexpression plasmid， the NGFR mRNA and protein expression levels in A549 cells and HeLa cells were significantly increased （P<0.05）； compared with Control+T lymophocytes+anti-PD-1 group and OE-NC+T lymophocytes+anti-PD-1 group， the relative increase fold in apoptotic rate of adenocarcinoma cells in OE-NGFR+T lymophocytes+anti-PD-1 group was significantly increased （P<0.05）. After transfection with NGFR gene siRNA， the NGFR mRNA and protein expression levels in SK-MES-1 cells and SiHa cells were significantly decreased （P<0.001）； compared with Control+T lymophocytes+anti-PD-1 group and si-NC+T lymophocytes+anti-PD-1 group， the relative increase fold in apoptotic rate of squamous carcinoma cells in si-NGFR+T lymophocytes+anti-PD-1 group was significantly decreased （P<0.001）. Conclusion The expression level of NGFR gene can positively regulate the pro-apoptotic effect of T lymphocytes activated by PD-1 inhibitor on the squamous carcinoma and adenocarcinoma cells. High expression of NGFR gene can enhance the sensitivity of squamous carcinoma and adenocarcinoma cells to PD-1 inhibitor， and it may serve as a potential biomarker for predicting the efficacy of PD?1 inhibitor.

Key words: Nerve growth factor receptor, Squamous cell carcinoma, Adenocarcinoma, Differentially expressed genes, Apoptosis, Programmed death receptor 1