体外RNA干扰抑制REG4基因表达对胃癌细胞增殖及凋亡的影响

INFLUENCE OF DOWN-REGULATION OF REG4 EXPRESSION BY SMALL INTERFERING RNA ON PROLIFERATION AND APOPTOSIS OF GASTRIC CANCER CELLS

目的探讨体外RNA干扰抑制REG4基因表达对胃癌细胞增殖及凋亡的影响。方法应用实时定量PCR方法检测七株胃癌细胞株中REG4基因的mRNA表达。针对REG4基因设计三条小干扰RNA片段(siRNA1、siRNA2、siRNA3),瞬时转染高表达胃癌细胞株。用RT-PCR方法检测转染后REG4基因的mRNA表达水平、MTT法检测细胞增殖能力、AnnexinV-PI法检测细胞凋亡水平。结果以永生化正常胃粘膜细胞株GES-1作为参照。MKN-45和AGS胃癌细胞株中REG4表达水平升高200倍以上,遂选用AGS细胞进行RNA干扰。RT-PCR结果证实siRNA2、siRNA3均有干扰效果,尤以siRNA3效果明显。选用siRNA3干扰AGS细胞株,MTT实验结果证实干扰AGS后,细胞增殖受到明显抑制(P<0.05)。AnnexinV-PI实验结果显示细胞凋亡率明显增加(P<0.05)。结论干扰REG4基因具有抑制胃癌细胞增殖、促进凋亡的作用,REG4基因可能成为胃癌基因靶向治疗的新靶点。

Objective To investigate REG4 expression level in human gastric cancer cell lines and the effect of RNA interfering the REG4 on proliferation and apoptosis of gastric cancer cells.Methods The expression levels of REG4 in seven gastric cancer cell lines were examined by real time PCR,and 3 small interfering RNA（siRNA1,siRNA2,siRNA3） targeting REG4 were designed and transfected into gastric cancer cell lines.The changes of REG4 mRNA expression level after siRNA interfering were detected by RT-PCR.The proliferation was assayed by MTT method.Flow cytometry was used to detect apoptosis of gastric cancer cells.Results Compared with immortalizad gastric mucosa cell line GES-1,the expression level of REG4 in gastric cancer cells in MKN45 and AGS were much higher（more than 200 hundred fold） than that in GES-1.Then AGS cell was used for siRNA experiment.SiRNA2 and siRNA3 showed notably down-regulated expression of REG4 mRNA levels respectively in comparison with that in control group.So siRNA3 was used to carry out do cell proliferation assay.After 48 hours＇ transfection of siRNA3,the proliferation of AGS cell significantly decreased compared with the control group（P0.05）.The results of flow cytometry revealed that transfection with siRNA3 after 36h,the apoptosis rate of AGS significantly increased（P0.05）.Conclusion Interfering and down regulating REG4 gene can inhibit proliferation and promote apoptosis of gastric cancer cells,indicating that REG4 gene is probably a target for gastric cancer gene therapy.