人肝癌PLC/PRF-5细胞中干细胞样细胞的分离及其特异性miRNAs的筛选  

Isolation of stem-like cells from human hepatocellular carcinoma PLC/PRF-5 cells and screening for their specific miRNAs

作　　者：郑秋红[1] 许扬梅[1] 魏植强[1] 龚福生[1] 杨建伟[2] 谢云青[1] 应敏刚[3] 

机构地区：[1]福建医科大学附属教学医院福建省肿瘤医院肿瘤分子生物学研究室,福建福州350014 [2]福建医科大学附属教学医院福建省肿瘤医院肿瘤内科,福建福州350014 [3]福建医科大学附属教学医院福建省肿瘤医院肿瘤外科,福建福州350014

出　　处：《中国肿瘤生物治疗杂志》2011年第2期126-132,共7页Chinese Journal of Cancer Biotherapy

基　　金：福建省自然科学基金资助项目(No.2008J0077)~~

摘　　要：目的:分选及鉴定人肝癌PLC/PRF-5细胞中的肝癌干细胞样细胞,研究其microRNAs(miRNAs)表达谱。方法:以ABCG2为表面标志,免疫磁珠法分选、流式细胞术检测ABCG2+和ABCG2-PLC/PRF-5细胞,观察ABCG2+与ABCG2-PLC/PRF-5细胞的琼脂克隆形成能力和接种NOD/SCID小鼠的成瘤能力。应用miRNA芯片筛选ABCG2+和ABCG2-PLC/PRF-5细胞差异表达的miRNAs,real-time PCR验证部分差异表达的miRNAs。结果:免疫磁珠分选的ABCG2+PLC/PRF-5细胞纯度可达(84.20±4.52)%。ABCG2+PLC/PRF-5细胞比ABCG2-PLC/PRF-5细胞形成更多、更大的克隆集落(47.17±10.50 vs23.33±7.31,P<0.05);NOD/SCID小鼠接种1×104个ABCG2+PLC/PRF-5细胞即可成瘤,而ABCG2-PLC/PRF-5细胞至少需要5×105个才可成瘤;5×105个细胞时,ABCG2+PLC/PRF-5细胞组的肿瘤体积显著大于ABCG2-PLC/PRF-5细胞组[(3.73±1.19)cm3 vs(0.72±0.57)cm3,P<0.01]。ABCG2+PLC/PRF-5细胞和ABCG2-PLC/PRF-5细胞差异表达的miRNAs有20个:上调的13个,下调的7个;real-time PCR验证其中的hsa-miR-30a和hsa-miR-630的差异表达,其结果与miRNA芯片结果基本一致。结论:人肝癌细胞系PLC/PRF-5中ABCG2+细胞具有肿瘤干细胞的特性;ABCG2+和ABCG2-PLC/PRF-5细胞差异表达的miRNAs有20个,它们在肝癌发病中可能起重要的调控作用。Objective： To isolate stem-like cells from hepatocellular carcinoma cell line PLC/PRF-5 and to study their miRNA profile.Methods：ABCG2＋ and ABCG2-PLC/PRF-5 cells were isolated from the PLC/PRF-5 cell line by magnetic activated cell sorting（MACS） method,and further identified by flow cytometry.The colony formation ability in soft agar and tumor formation ability in NOD/SCID mice of ABCG2＋ and ABCG2-PLC/PRF-5 cells were observed.miRNA chip was adopted to screen the differentially expressed miRNAs between ABCG2＋ and ABCG2-PLC/PRF-5 cells;and real-time PCR assay was used to confirm the results of miRNA chip.Results：The purity of ABCG2＋PLC/PRF-5 cells isolated by MACS method was（84.20±4.52）%.The colony number and size formed by ABCG2＋ PLC/PRF-5 cells were more and larger than those formed by ABCG2-cells（47.17±10.50 vs 23.33±7.31,P〈0.05）.1×104 ABCG2＋ cells could form tumors with at least 5×105 cells needed for ABCG2-cells.The size of tumors generated by 5×105 ABCG2＋ cells was larger than that by the ABCG2-cells（[3.73±1.19] cm3 vs [0.72 ±0.57] cm3,P〈0.01）.There are 20 miRNAs differentially expressed between ABCG2＋ and ABCG2-cells,with 13 up-regulated and 7 down-regulated.Real-time PCR assay was applied to further verify the differential expression of hsa-miR-30a and hsa-miR-630,and the results were in accordance with those of miRNA chip.Conclusion： ABCG2＋ PLC/PRF-5 cells in hepatocellular carcinoma cell line PLC/PRF-5 have the properties of cancer stem cells.Twenty miRNAs are differentially expressed between ABCG2＋ and ABCG2-PLC/PRF-5 cells,which might play important roles in the carcinogenesis of hepatocellular carcinoma.

关 键 词：肝癌 肝癌干细胞 microRNA 表达谱 免疫磁珠分选 

分 类 号：Q279[生物学—细胞生物学] R392.12[医药卫生—免疫学]