胃癌干细胞中差异表达microRNA对其生物学特性的影响

Differential Expression of Micro RNAs and Its Relation to Biological Characteristics in Gastric Cancer Stem Cells

[目的]探讨胃癌干细胞中差异表达microRNA及其对胃癌干细胞生物学特性(自我更新、侵袭、耐药能力)的调控作用。[方法]采用无血清悬浮培养法和干细胞标志物流式分选术分离人胃癌干细胞;分别采用胃癌细胞球形成实验、体外侵袭、耐药实验鉴定胃癌干细胞的生物学特性;microRNA表达谱芯片检测人胃癌干细胞中差异表达microRNA;采用定量逆转录PCR(qRT-PCR)技术检测相关差异表达microRNA的表达情况,验证microRNA芯片结果;相关microRNA抑制剂干扰后,分别进行胃癌细胞球形成实验、体外侵袭与耐药实验检测microRNA对人胃癌干细胞自我更新、侵袭、耐药能力的影响。[结果 ]成功分离得到人胃癌干细胞CD44(+)亚群,其具有典型的肿瘤干细胞生物学特性:较强的自我更新能力、侵袭和耐药能力。与CD44(-)细胞亚群相比,人胃癌干细胞CD44(+)细胞亚群高表达miRNAs共有17个;低表达miRNAs共有33个。采用q PCR技术验证microRNA表达谱芯片相关结果,得到:CD44(+)亚群中miR-196a-5p、miR-155-5p、miR-30a、miR-30b、miR-30c表达上调,miR-1246、miR-195-5b表达下调。抑制相关高表达microRNA(miR-196a-5p、miR-155-5p、miR-30a、miR-30b、miR-30c)表达后,肿瘤干细胞的自我更新能力、侵袭和耐药能力均下降。[结论 ]胃癌干细胞中具有多种差异表达microRNA,microRNA对胃癌干细胞生物学特性具有调控作用。

[Purpose] To investigate the differential expression of microRNAs and its relation to biological characteristics in gastric cancer stem cells（GCSCs）.[Methods] The serum-free suspension culture and flow cytometry sorting technology were used to isolate human GCSCs.Biological characteristics of GCSCs were detected by sphere colony formation assay,invasion assay and drug resistance test in vitro.Mi RNA microarray was performed to analyze differentially expressed microRNAs in GCSCs.Quantitative real time-PCR was used to verify results of miRNA microarray.After transfection of miRNA inhibitor,the changes of biological characteristics of GCSCs were examined.[Results] Human GCSCs were successfully isolated by using the serum-free suspension culture and fluorescence-activated cell sorting.Functional studies revealed that FAC-sorted CD44（＋） cells had characteristics of cancer stem-like cells.CD44（＋） cells formed more sphere colonies,showed higher invasiveness and stronger drug resistance than CD44（-） cells.Mi RNA microarray analysis illustrated that compared with CD44（-） cells,there were 17 highly expressed microRNAs and 33 lowly expressed microRNAs in human GCSCs.Real-time PCR analysis verified the microarray data：the expressions of miR-196a-5p,miR-155-5p,miR-30 a,miR-30 b and miR-30 c were up-regulated,and the expressions of miR-1246 and miR-195-5b were down-regulated in CD44（＋） cells.Transfection of miRNA inhibitor suppressed the self-renewal capacity,invasion ability and drug resistance ability in CD44（＋） cells.[Conclusion] The differentially expressed microRNAs in human GCSCs may change its biological characteristics.