关键词: 胰腺导管腺癌, 烟碱型乙酰胆碱受体α5亚基, 神经递质, 细胞周期

Abstract:

Objective To investigate the effects of cholinergic receptor nicotinic alpha 5 subunit（CHRNA5） on the invasion and proliferation of pancreatic ductal adenocarcinoma （PDAC） using bioinformatics approaches and cellular experiments， and to analyze its regulatory pathways and provide foundation for the identification of novel therapeutic targets for pancreatic cancer. Methods Transcriptome， gene variation， and clinical data of pan-tumor and normal tissues were downloaded from public databases including The Cancer Genome Atlas （TCGA） and Genotype-Tissue Expression （GTEx）. The expression levels of CHRNA5 mRNA between pan-tumor patients and healthy individuals were compared using the Wilcoxon test. The expression levels of CHRNA5 mRNA between PDAC and para-cancer tissues were further compared using datasets from the Gene Expression Omnibus （GEO） database. The data were divided into three groups according to the different expression levels of CHRNA5 mRNA using the tertile method： CHRNA5 high-expression group， CHRNA5 medium-expression group， and CHRNA5 low-expression group. Survival analysis was performed in CHRNA5 high-expression and low-expression groups to assess the prognostic impact of CHRNA5 in pancreas cancer. Real-time fluorescence quantitative PCR （RT-qPCR） and immunohistochemistry methods were used to detect the expressions of CHRNA5 mRNA and protein in tumor tissue， paracancer tissue， tumor cells and normal cells. Differential analysis of the transcriptome data and enrichment analysis of Hallmark genes and Reactome pathways were then conducted. Immune cell infiltration analysis was performed on PDAC based on transcriptome data. Tumor mutation burden （TMB） analysis， single nucleotide variation （SNV）， and copy number variation （CNV） analysis were also conducted. The si-NC and si-CHRNA5 were transfected into the MIA PaCa-2 and Capan-2 cells， and the samples were divided into NC， siRNA-1 and siRNA-2 groups according to the transfected siRNA. The proliferation activities of the cells in various groups under acetylcholine （Ach） （μmol·L^-1） and non-ACh conditions were detected using the CCK-8 method， and the expression of c-Myc protein in the cells in various groups were detected using Western blotting method. Results Compared with normal tissue， the expression levels of CHRNA5 mRNA were elevated in 28 types of tumor tissues， including pancreatic cancer （adjusted P<0.05）. The results of RT-qPCR and immunohistochemistry revealed that CHRNA5 was highly expressed in tumor tissue and pancreatic ductal adenocarcinoma （PDAC） cells compared with paracancer tissue and normal pancreatic ductal cells. Survival analysis showed that PDAC patients with high-expression of CHRNA5 had worse prognosis compared with those with low-expression of CHRNA5. A total of 994 differentially expressed genes （DEGs） were identified between PDAC and control samples， with 381 up-regulated genes and 613 down-regulated genes. Gene Set Enrichment Analysis （GSEA） indicated that CHRNA5 was highly associated with oxidative phosphorylation， cell proliferation， immune infiltration， and cell cycle. Immune analysis showed that the samples with high expression of CHRNA5 had poorer immune infiltration compared with those with low expression of CHRNA5. Compared with CHRNA5 low-expression group， the mutation rates of pancreatic cancer progression-related genes KRAS， TP53， and SMAD4 in CHRNA5 high-expression group were inereased. In cell experiments， compared with NC+Ach group， the proliferation activities of cells in siRNA-1+Ach group and siRNA-2+Ach group were significantly decreased （P<0.05）. The results of Western blotting revealed that the expression amounts of c-Myc protein in siRNA-1 and siRNA-2 groups were significantly lower than those in NC group. Conclusion CHRNA5 modulates the cell cycle via myelocytomatosis viral oncogene （MYC） family， thereby influencing the proliferation of pancreas cancer cells and promoting the progression of PDAC， suggesting that CHRNA5 is a potential therapeutic target for pancreas cancer.

Key words: Pancreatic ductal adenocarcinoma, Cholinergic receptor nicotinic alpha 5 subunit, Neurotransmitter, Cell cycle