微小RNA-200c对人胰腺癌干细胞上皮-间充质转化的作用

Effect of microRNA-200c on epithelial-mesenchymal transition of human pancreatic cancer stem cells

目的观察微小RNA-200c(miR-200c)在人胰腺癌干细胞上皮-间充质转化(EMT)中的作用。方法应用流式细胞术(FACS)在人胰腺癌细胞株PANC-1中分选出以CD24^(+)CD44^(+)ESA+作为标志物的胰腺癌干细胞。将miR-200c前体片段和阴性对照片段分别转染到胰腺癌干细胞中。应用反转录-聚合酶链反应(RT-PCR)检测细胞中E-钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)、E盒结合锌指蛋白1(ZEB1)和miR-200c的表达。Transwell试验检测细胞的侵袭能力。采用t检验。结果人胰腺癌细胞株PANC-1中分选出CD24^(+)CD44^(+)ESA+细胞(0.8%)。miR-200c在胰腺癌干细胞中的相对表达值(0.15±0.01)显著低于PANC-1细胞(1.00±0.09),差异有统计学意义(t=3.306,P<0.05)。胰腺癌干细胞平均穿膜细胞数[(321.00±7.62)个]明显高于PANC-1细胞[(70.00±16.47)个],差异有统计学意义(t=2.152,P<0.05)。转染miR-200c前体片段24 h后的胰腺癌干细胞中miR-200c的相对表达值(3.70±0.42)明显高于转染阴性对照片段的胰腺癌干细胞中miRNA-200c的相对表达值(0.22±0.21),差异有统计学意义(t=2.073,P<0.05)。E-cadherin、vimentin、ZEB1 mRNA在转染miRNA-200c前体片段的胰腺癌干细胞中的表达分别为(3.12±0.02)×10^(-4)、0.36±0.15、(0.38±0.07)×10^(-4),在转染阴性对照片段的胰腺癌干细胞中的表达分别为(1.31±0.05)×10^(-4)、1.16±0.08、(1.13±0.03)×10^(-4),两组比较差异有统计学意义(t=1.941、2.165、2.308,P<0.05)。转染miR-200c前体片段后的胰腺癌干细胞的平均穿膜细胞数[(80.00±5.35)个]明显低于转染阴性对照片段的干细胞[(310.00±19.41)个],两组比较差异有统计学意义(t=2.613,P<0.05)。结论miR-200c过表达可以抑制胰腺癌干细胞的上皮-间充质转化,逆转其侵袭能力。

Objective To explore the effects of microRNA-200c(miR-200c)on epithelial-mesenchymal transition(EMT)of human pancreatic cancer stem cells.Methods CD24^(+)CD44^(+)ESA+cells were sorted from PANC-1 cell line by flow cytometry.The miR-200c precursor fragment and negative control fragment were transfected into pancreatic cancer stem cells.The reverse transcriptase-polymerase chain reaction(RT-PCR)was used to detect the expression of E-cadherin,vimentin,zinc finger E-box binding protein 1(ZEB1)and miR-200c.Transwell invasion assay was used to detect the invasiveness.Results CD24^(+)CD44^(+)ESA+cells(0.8%)were isolated in PANC-1 cells.The relative miR-200c expression level in pancreatic cancer stem cell line(0.15±0.01)was significantly lower than that in PANC-1 cell line(1.00±0.09,t=3.306,P<0.05).The transmembrane number of pancreatic cancer stem cells[(321.00±7.62)cells]was greater than that of PANC-1 cells[(70.00±16.47)cells,t=2.152,P<0.05].The relative miR-200c expression level in pancreatic cancer stem cell line in miR-200c precursor transfected group(3.70±0.42)was significantly higher than that in negative control group(0.22±0.21,t=2.073,P<0.05).The mRNA expression of E-cadherin,vimentin,ZEB1 in miR-200c precursor transfected group was(3.12±0.02)×10^(-4),0.36±0.15,(0.38±0.07)×10^(-4),and that in the negative control group was(1.31±0.05)×10^(-4),1.16±0.08,(1.13±0.03)×10^(-4).There was significant difference between the two group(t=1.941,2.165,2.308,P<0.05).The transmembrane number in miR-200c precursor transfected group[(80.00±5.35)cells]was less than that in negative control group[(310.00±19.41)cells,t=2.613,P<0.05].Conclusion Overexpression of miR-200c in the PCSCs could inhibite EMT and reverse invasiveness.