关键词: 膀胱肿瘤, 蛋白激酶D2, 肿瘤免疫微环境, 预后标志物, 生物信息学分析

Abstract:

Objective To investigate the expression of protein kinase D2 （PRKD2） in bladder cancer （BLCA） tissue using bioinformatics analysis method and its effect on the prognosis of BLCA patients， and to clarify the role of PRKD2 in the occurrence and development of BLCA. Methods The data from 9 normal bladder samples， 19 BLCA paracancerous samples， and 407 BLCA tumor samples were downloaded from the UCSC Cancer Genome Database. The Mann-Whitney U test was applied to analyze the difference in expression of PRKD2 mRNA in BLCA tumor and normal bladder tissues， and the Human Protein Atlas （HPA） database was used for proteomic validation. DESeq2 package in R software was applied to screen the differentially expressed genes （DEGs） in BLCA tissue in PRKD2 low- and high-expression groups. The co-expression heatmaps of PRKD2 were plotted using the ggplot2 package， Gene Ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） were used for functional annotation analysis and pathway enrichment analysis of DEGs， and Gene Set Enrichment Analysis （GSEA） was used to obtain the gene sets that were significantly enriched for DEGs. The BLCA samples were divided into low- and high-expression groups according to the expression level of PRKD2， and the correlations between PRKD2 expression and immune cell infiltration in the BLCA patients were analyzed with GSVA package. The relationship between PRKD2 and prognosis of BLCA patients was further analyzed using the survival package and the survminer package. The PRKD2 gene mutations in BLCA tissue were analyzed using the cBioPortal database. The cystitis， bladder polyp and BLCA tissues were collected， and the expression levels of interleukin-17F （IL-17F） protein in BLCA and control tissues were detected using immunohistochemical staining technique. Results PRKD2 was highly expressed in a variety of malignant tumors， and the expression levels of PRKD2 mRNA and protein in BLCA tissue were significantly increased compared with those in normal bladder tissue （P<0.05）. Single gene differential analysis of PRKD2 yielded a total of 1 058 DEGs， of which a total of 29 genes were up-regulated and 1 029 were down-regulated. The results of GO functional enrichment analysis showed that DEGs were mainly enriched in the biological process （BP）， such as chemical stimuli involved in sensory perception， Cajal body， and endopeptidase inhibitor activity. The results of KEGG pathway analysis showed that DEGs were mainly enriched in the pathway of Staphylococcus aureus infection and the pathway of maturity onset diabetes of the young. GSEA analysis showed that DEGs were mainly enriched in the Notch signaling pathway， the retinoic acid-inducible gene-Ⅰ（RIG-Ⅰ）-like receptor signaling pathway， the cytoplasmic DNA screening pathway， the base excision repair signaling pathway， natural killer （NK） cell-mediated cytotoxicity signaling pathway and T cell receptor signaling pathway. The results of immune infiltration analysis indicated that the expression of PRKD2 was positively correlated with five types of cells， such as activated dendritic cells （aDC）， NK CD56dim cells and central memory T cells （Tcm） （P<0.05）， and negatively correlated with three types of immune cells， including macrophages， effector memory T cells （Tem） and plasmacytoid dendritic cells （pDC） （P<0.05）.The clinical characteristic subgroup analysis results showed that the expression levels of PRKD2 mRNA in BLCA patients who were over 70 years old and developed lymphovascular invasion were decreased （P<0.05）； the overall survival （OS）， disease-specific survival （DSS） and progression-free interval （PFI） in the BLCA patients with PRKD2 high expression were significantly longer than those with PRKD2 low expression （P<0.05）. The univariate and multivariate Cox analyses indicated that distant metastasis， primary therapy outcome and clinicopathologic stage were the important factors affecting BLCA prognosis. About 9% patients had PRKD2 gene mutations， including missense mutation， gene amplification， mRNA low or high expression， and multi-motif mutation. The immunohistochemistry results showed that the expression level of IL-17F protein in BLCA tissue was significantly higher than that in cystitis tissue （P<0.05）. Conclusion The expression level of PRKD2 in BLCA tissue is obviously increased， which could up-regulate the expression of IL-17F protein， and the decrease of PRKD2 protein expression may be a potential factor for the poor prognosis of BLCA patients.

Key words: Bladder neoplasm, Protein kinase D2, Tumor immune microenvironment, Prognostic marker, Bioinformatics analysis