浸润与非浸润性膀胱癌中细胞起源的比较

Comparative research on the origin of muscle invasive bladder cancer and non-muscle invasive bladder cancer cells

目的鉴定膀胱癌的细胞起源,阐明膀胱组织中具有干细胞特性的细胞异常分化引起癌变的机制.方法依托深圳市罗湖医院集团罗湖人民医院泌尿外科于2018年收集膀胱癌前病变组织(Ta)1例和肌层浸润性膀胱癌组织(T2期以上)2例,制作石蜡组织切片并培养原代细胞.利用t-SNE算法,对肌层浸润性膀胱癌(MIBC)和非肌层浸润性膀胱癌(NMIBC)样本RNA表达谱进行无监督聚类后;在GO数据库中对差异基因进行通路注释;使用方差分析对MIBC进程中的分化特征进行研究.在肿瘤组织石蜡切片染色和膀胱癌细胞株中进行初步验证,并通过分化抑制剂进行干预.结果通过聚类分析发现膀胱上皮分化相关的基因表达方面,MIBC和NMIBC有区别.利用GO数据库对筛选出的1 502个基因进行通路注释,找到差异表达基因中与干性、癌症转移、分化、尿路上皮相关的标志物(P<0.01);随着膀胱癌进展,干性相关标志物CD29、CD90、CD166、癌胚抗原(CEA)和细胞角蛋白5(CK5)的表达水平逐渐下降,而上皮细胞标志物细胞角蛋白18(CK18)逐渐升高(t=3.170,P<0.05).免疫荧光检测结果显示,比较NMIBC细胞株RT4,MIBC细胞株5637细胞的肿瘤相关标志物(ABCG2)、干细胞相关的基因(BMI1)、尿路上皮基底层标志物(KRT5)表达增加,分布范围更广泛.免疫组织化学染色结果表明,与癌旁组织比较,随着MIBC的进展,上皮分化标志物尿溶蛋白3B(UPK3B)、分化相关标志物酪氨酸磷酸酶受体C(PTRPC)表达强度升高,分布范围逐渐扩大;与干细胞特性相关的基因(CD90、CEA1)表达强度下降,分布范围逐渐缩小.尿路上皮分化抑制剂处理两种膀胱癌细胞株后,实时定量PCR检测干性相关的基因,处理后的UM-UC-3细胞中各干性相关基因的表达有不同程度的升高(t=3.650,P<0.01),而RT4细胞处理前后则差异无统计学意义(t=2.050,P>0.05).结论MIBC的进展是一个分化的过程,其肿瘤细胞处于低分化状态,这可能成为膀胱内的组织干细胞作为MIBC起源细胞的证据.

Objective To identify the cell origin of bladder cancer and to elucidate the mechanism of cancer caused by abnormal differentiation of cells with stem cell characteristics in bladder tissue.Methods Relying on Department of Urology,Shenzhen Luohu Hospital Group Luohu People’s Hospital,the following tissues were collected in 2018:bladder precancerous lesion(Ta)1 case and muscle invasive bladder cancer(≥T2)2 case in bladder cancer patients.And then,paraffin tissue sections were prepared and then the primary cells were cultured.Unsupervised clustering of RNA expression profiles of muscle invasive bladder cancer(MIBC)and non muscle invasive bladder cancer(NMIBC)samples using t-SNE algorithm.Pathway annotations of differential genes in GO database.Analysis of differentiation characteristics in MIBC process by analysis of variance.Preliminary validation was performed in paraffin section staining of tumor tissue and bladder cancer cell lines.Results Cluster analysis revealed differences in gene expression associated with bladder epithelial differentiation between MIBC and NMIBC.Pathway annotations were performed in GO database to identify markers associated with stem,cancer metastasis,differentiation and urothelium in differentially expressed genes(P<0.01);As bladder cancer progressed,the expression levels of the stemness markers CD29,CD90,CD166,carcinoembryonic antigen(CEA)and creatine kinase 5(CK5)gradually decreased,while the epithelial cell marker creatine kinase 18(CK18)gradually increased(t=3.170,P<0.05).Immunofluorescence assay showed that compared to NMIBC cell line RT4,the expression of tumor-associated markers(ABCG2),stemness-associated gene(BMI1)and urothelial basal layer marker(KRT5)of MIBC cell line 5637 cells are increased,which also distributed wider.Immunohistochemical staining showed that the expression of epithelial differentiation markers urolysin 3B(UPK3B)and differentiation-related markers tyrosine phosphatase receptor C(PTRPC)increased with the progress of MIBC,and the distribution range gradually expanded.The expression of stemness-associated genes(CD90,CEA1)decreased,and the distribution range gradually decreased.After urothelial differentiation inhibitors were treated to bladder cancer cell lines,the stemness-related genes were detected by real-time quantitative polymerase chain reaction(PCR).The expression of stemness-related genes in UM-UC-3 cells was increased to varying degrees(t=3.650,P<0.01),while there was no significant difference in RT4 treatment(t=2.050,P>0.05).Conclusion The progression of MIBC is a process of differentiation,whose tumor cells are in a poorly differentiated state.It may be a strong evidence that stem cells in the bladder tissue act as the cell origin of MIBC.