沉默FOXC1基因对人胰腺癌细胞增殖的影响

Effect of si RNA-mediated FOXC1 gene silencing on proliferation of pancreatic cancer cells

目的:研究小干扰RNA(small interfering R N A,s i R N A)沉默人胰腺癌Capan-2、PANC-1细胞中叉头框蛋白C1(fork head box C1,FOXC1)基因对胰腺癌细胞增殖能力的影响及作用机制.方法:以胰腺癌细胞、原发胰腺癌组织为研究对象,实时定量逆转录聚合酶链反应(quantitative real-time reverse transcription polymerase chain reaction,q RT-PCR)检测FOXC1 m RNA在胰腺癌细胞及组织中的表达情况;将人胰腺癌细胞分为2组:FOXC1s i R N A组(实验组)、N C s i R N A组(阴性对照组),脂质体转染法将FOXC1 si RNA转染入胰腺癌细胞,q RT-PCR、Western blot技术检测胰腺癌细胞FOXC1 m RNA及蛋白表达变化,5-乙炔基-2'-脱氧尿苷(5-ethynyl-2'-deoxyuridine,Ed U)细胞增殖法检测各组胰腺癌细胞增殖情况;流式细胞技术(flow cytometry,FCM)检测各组胰腺癌细胞的周期分布,Western blot技术分析周期相关蛋白P21、P53、Cyclin D1蛋白表达情况.结果:q RT-PCR结果提示:相比正常胰腺上皮细胞和癌旁胰腺组织,FOXC1 m RNA在胰腺癌细胞及胰腺癌组织中表达较高(P<0.05);q RT-P C R及Westernblo t结果显示FOXC1 si RNA有效沉默了胰腺癌细胞F O X C1基因的转录和表达,E d U细胞增殖实验提示:沉默了胰腺癌细胞F O X C1基因表达后,胰腺癌细胞胞的增殖能力明显下降,较阴性对照组,差异有统计学意义(P<0.05);F C M结果显示:沉默了胰腺癌细胞FOXC1基因表达后胰腺癌细胞被阻滞在G0/G1期,较阴性对照组,差异有统计学意义(P<0.05);Western blot结果显示:较阴性对照组,P21、P53表达水平无明显变化,Cyclin D1表达水平下降,差异有统计学意义(P<0.05).结论:FOXC1 si RNA能够有效沉默人胰腺癌Capan-2、PANC-1细胞FOXC1基因的表达,抑制其增殖,将细胞周期阻滞于G0/G1期,提示FOXC1影响细胞增殖可能是通过调控细胞周期实现的,其机制可能是部分通过调控细胞周期蛋白Cyclin D1的表达而实现.

AIM： To investigate small interfering RNA（si RNA）-mediated silencing of fork head box protein C1（FOXC1） gene on the proliferation human pancreatic cancer Capan-2 and PANC-1 cells and to explore the possible underlying mechanisms.METHODS： Quantitative real-time reverse transcription-polymerase chain reaction（q RTPCR） was used to examine the expression of FOXC1 m RNA in pancreatic cancer cell lines and primary carcinoma tissues from human patients. The human pancreatic cancer cells were divided into two groups： an FOXC1 si RNA group（experimental group） and an NC si RNA group（negative control group）. q RT-PCR and Western blot were used to detect FOXC1 m RNA and protein expression in pancreatic cancer cells and to determine proliferation rate after transfection with 5-ethynyl-2’-deoxyuridine（Ed U）. Flow cytometry was used to examine cell cycle distribution. Western blot analysis was used to detect the expression of cell cycle related proteins P21, P53, and Cyclin D1.RESULTS： FOXC1 m RNA expression in pancreatic cancer cells and pancreatic cancer tissues were significantly higher than in normal epithelial cells and matched tumor adjacent pancreatic tissue, respectively（P 〈 0.05）. q RT-PCR and Western blot analysis showed that FOXC1 si RNA effectively silenced the transcription and expression of FOXC1 in pancreatic cancer cells. Ed U cell proliferation experiments showed that compared with the control group, silencing FOXC1 gene expression in pancreatic cancer cells significantly decreased cell proliferation（P 〈 0.05）. FCM results showed that compared with the control group, FOXC1 silencing arrested the tumor cells in G0/G1phase（P 〈 0.05）. Western blot analysis showed that compared with the control group, Cyclin D1 expression was significantly decreased（P 〈 0.05）, while P21 and P53 expression was unchanged.CONCLUSION： FOXC1 si RNA can effectively silence the FOXC1 gene expression in human pancreatic cancer Capan-2 PANC-1 cells. FOXC1 silencing inhibits pancreatic cancer cell proliferation and arrests cell cycle in G0/G1 phase, suggesting that FOXC1 alters cell proliferation possibly by regulation of the cell cycle through, in part, regulating the expression of Cyclin D1.