The Roles And Mechanisms Of CHAF1A In Bladder Urothelial Carcinoma

【Background】As a malignant tumor with the highest incidence of the urinary system in China,bladder cancer（BCa）is still a major problem in clinical practice.Bladder cancer is divided into non-muscle-invasive bladder cancer（NMIBC）and muscle-invasive bladder cancer（MIBC）,according to the tumor infiltrating muscle layer or not.The pathological types of bladder cancer include urothelial carcinoma,squamous cell carcinoma,adenocarcinoma and small cell carcinoma.The most common of these is bladder urothelial carcinoma,which accounts for more than 90%of the total number of patients with bladder cancer.NMIBC accounts for 70-80%in new diagnosed BCa patients,who are always treated with transurethral resection of bladder tumor（TURBT）and other methods.About 30%of the NMIBC patients will continue to progress to MIBC after TURBT.The first choice of operation for MIBC patients is radical cystectomy（RC）and pelvic lymph node dissection（PLND）,but the prognosis is poor and the 3-year survival rate is less than 50%.There is still lack of effective molecular markers to predict the progression and prognosis of bladder cancer,and lack of effective targets and related drugs for the treatment of bladder cancer.Therefore,the discovery of effective molecular markers for the progression and prognosis of patients with bladder cancer is of great significance.The chaf1a protein encoded by the CHAF1A gene is the core component of the chromatin assembly factor 1（CAF-1）.CAF-1,a nuclear heterotrimeric complex comprised of subunits p150,p60,and p48,is well-known as the only histone chaperone protein that recruits histones H3 and H4 to newly synthesized DNA.In addition,CAF-1/CHAF1A participates in many important cell growth processes,such as cell cycle,chromatin assembly,DNA repair and DNA replication.In recent years,many studies have confirmed that the dysregulation of CHAF1A not only increases the instability of the genome,but also relates to the malignant phenotype and poor prognosis of the patients with malignant tumors.Nevertheless,the mechanisms underlying the involvement of CHAF1A in bladder cancer progression remain quite unknown.By analyzing the RNA Sequencing（RNA-seq）results of 10 pairs of cancerous tissues and corresponding adjacent normal bladder mucosae specimens,as well as the data about bladder cancer in the TCGA database,we found that the expression of CHAF1A in bladder cancer tissues was significantly increased（p<0.05）.We speculated that the overexpression of CHAF1A might be associated with clinicopathological features and poor prognosis of patients with bladder urothelial carcinoma.【Objective】The purpose of this study was to investigate the relationship between the over-expression of CHAF1A and the clinicopathological features and prognosis of patients with bladder urothelial carcinoma,and to predict the molecular mechanisms.【Materials and methods】1.We excavated the RNA-seq results of 10 pairs of cancerous tissues and corresponding adjacent normal bladder mucosae specimens of patients with bladder urothelial carcinoma.We had also analyzed the related data in the TCGA database.Real time quantitative PCR（real-time qPCR）was used to detect the relative expression of CHAF1A mRNA in 28 pairs of cancerous tissues and corresponding adjacent normal mucosa tissues,so as to verify the sequencing results.2.Immunohistochemical assay was used to detect the expression of CHAF1A in a tissue microarray,including 94 specimens of patients with bladder urothelial carcinoma and 29 adjacent normal bladder mucosa specimens.The different expression patterns of CHAF1A in bladder urothelial carcinoma and adjacent normal mucosa tissues was compared.The relationships between the CHAF1A expression and the clinicopathological parameters and the prognosis of patients with bladder urothelial carcinoma were analyzed.3.The lentivirus transfection method was used to explore the effects of silencing of CHAF1A on the proliferation and colony-formation ability of human bladder cancer cell lines T24 and J82.Flow cytometry analysis was used to detect the effects of down-regulation of CHAF1A on the cell cycle.4.GeneChip primeview human pathArray^TM was used to detect the changes of the downstream genes expressions after CHAF1A was down-regulated.The ingenuity pathway analysis（IPA）method was used to predict the key signaling pathways through which CHAF1A promoted the proliferation.【Results】1.By analyzing the RNA-seq results and the data about bladder cancer in the TCGA database,we found that the expression of CHAF1A of bladder cancer tissues was significantly increased（p<0.05）.In 22（78.57%）of the 28 pairs of tissues used for validation,the expression of CHAF1A in cancerous tissues was significantly higher than that in corresponding adjacent normal bladder mucosae specimens（p<0.05）.On the whole,the total expression of CHAF1A in the cancerous tissues was also significantly higher than that of the corresponding normal mucosa（p<0.05）.2.The positive expression rate of CHAF1A protein in cancerous tissues was 90.43%（85/94）,which was significantly higher than that of normal bladder mucosae specimens（20.69%,6/29）.According to the clinicopathological analysis,the high expression of CHAF1A is associated with smoking,high stage,high grade,lymph node invasion and metastasis（p<0.05）.The results of COX multivariate analysis showed that high expression of CHAF1A was an independent risk factor for overall survival（OS）and cancer specific survival（CCS）in patients with bladder urothelial carcinoma（p<0.05）.3.The results of CCK-8 test showed that the knockdown of CHAF1A significantly inhibited the proliferation of T24 and J82 cells in vitro（p<0.05）.In addition,the results of colony forming experiment also showed that the clonogenic ability of T24 and J82 cells decreased significantly after the knockdown of CHAF1A（p<0.05）.After the CHAF1A was down-regulated,the cell cycle of T24 and J82 cells was blocked in the G0/G1 phase.4.The results of GeneChip primeview human pathArray^TM revealed that after CHAF1A was knocked down,607 genes were up-regulated and 411 genes were down-regulated.After IPA analysis,we found that CHAF1A was involved in the regulation of multiple tumor-related signaling pathways,including cell growth and proliferation.In addition,the down-regulation of CHAF1A hindered the expression of multiple tumor-related genes,many of which had participated in the process of cell growth and proliferation.【Conclusions】CHAF1A was highly expressed in bladder urothelial carcinoma,and its expression was associated with smoking,high grade,high stage,lymph node invasion and metastasis.It was also an independent risk factor for the poor prognosis of patients with bladder urothelial carcinoma.The down-regulation of CHAF1A could inhibit the proliferation and clonogenic ability of bladder cancer cells.CHAF1A was speculated to participate in the regulation of multiple tumor-related signaling pathways,including cell growth and proliferation.The downregulation of the expression of CHAF1A hindered the expression of multiple tumor-related genes.CHAF1A was expected to be a potential biomarker for targeted therapy and prognosis evaluation of patients with bladder urothelial carcinoma.