N-myc下游调节基因家族成员3在胃癌细胞增殖和侵袭中的作用

目的探讨N-myc下游调节基因家族成员3(NDRG3)对胃癌细胞生物学行为的影响及其机制。方法实验分NDRG3敲减组和对照组,AGS-NDRG3小发夹状RNA(shRNA)组采用shRNA技术干扰AGS细胞中NDRG3表达,同时设置AGS-Vector对照组转染空白对照病毒;通过反转录-聚合酶链反应(RT-PCR)和蛋白质印迹法(Western blot)检测两组细胞中NDRG3 mRNA和蛋白表达以检查AGS-NDRG3 shRNA组中NDRG3敲减效果,利用细胞计数试剂盒(CCK-8)法和Transwell实验分别检测两组细胞中细胞增殖和侵袭能力,利用流式细胞仪分析两组细胞中细胞周期分布情况,并通过Western blot检测敲减两组细胞中细胞核增殖抗原(Ki-67)和p53蛋白表达水平,组间比较采用单因素方差分析。结果AGS-NDRG3 shRNA组NDRG3 mRNA和蛋白表达水平均显著低于对照组(NDRG3 mRNA,0.220±0.035比1.020±0.078,t=14.713,P<0.01;NDRG3蛋白,0.140±0.018比0.820±0.025,t=7.892,P<0.01),差异均有统计学意义。CCK-8实验结果显示,在培养24、48、72 h后,AGS-NDRG3 shRNA组细胞的增殖水平(0.388±0.025、0.576±0.044、0.873±0.051)明显低于对照组(0.457±0.033、0.674±0.047、1.125±0.062),差异均有统计学意义(t=4.983、5.852、8.082,P<0.01);Transwell迁移实验结果表明,AGS-NDRG3 shRNA组侵袭穿过Transwell小室膜的细胞数(474.5±27.6)个]明显低于对照组(1127.0±48.8)个],差异有统计学意义(t=12.380,P<0.01);细胞周期分析显示,敲减NDRG3表达的AGS细胞周期阻滞于S期,其S期占49.33%,对照组S期占30.29%(t=4.634,P<0.01),差异均有统计学意义;Western blot检测结果显示,敲减NDRG3表达后AGS细胞中Ki-67蛋白表达低于对照组(0.380±0.021比0.860±0.036,t=5.163,P<0.01)、野生型p53蛋白表达高于对照组(0.720±0.018比0.160±0.013,t=11.354,P<0.01),差异均有统计学意义。结论NDRG3可能通过调控p53通路促进AGS细胞增殖及侵袭能力。

The effects of N-myc downstream-regulated gene 3 on the proliferation and invasion of gastric cancer cells

Objective To investigate the effects of N-myc downstream-regulated gene 3(NDRG3)on the cellular malignant phenotype of human gastric cancer cells,and its possible mechanism.Methods The experiment was divided into NDRG3 knockdown groupThe expression of NDRG3 in AGS-NDRG3 small hair RNA(shRNA)cells was interfered with shRNA],and the control group(AGS-vector was only transfected into blank control virus).The expression level of NDRG3 mRNA and protein in AGS-NDRG3 shRNA cells and AGS-vector cells was detected by real-time RT-PCR and Western blotting,respectively.The proliferation and invasion of AGS-NDRG3 shRNA cells and AGS-vector cells were detected by cell counting kit-8(CCK-8)and Transwell assay,respectively,and the cell cycle distribution of AGS-NDRG3 shRNA cells and AGS-Vector cells was analyzed by flow cytometer.The expression levels of proliferation cell nuclear antigen(Ki-67)and p53 protein were detected by Western blotting.Results The expression of NDRG3 mRNA and protein in AGS-NDRG3 shRNA group was significantly lower than that in control group(NDRG3 mRNA:0.220±0.035 vs.1.020±0.078,t=14.713,P<0.01;NDRG3 protein:0.14±0.018 vs.0.820±0.025,t=7.892,P<0.01).After down-regulating the expression level of NDRG3,the proliferation activities of AGS cells at 24,48 and 72 h were significantly lower than those in the control cells(0.388±0.025 vs.0.457±0.033,t=4.983,P<0.01;0.576±0.044 vs.0.674±0.047,t=5.852,P<0.01;0.873±0.051 vs.1.125±0.062,t=8.082,P<0.01),and the invasion abilities of AGS cells were also significantly lower than those in the controls(474.5±27.6 vs.1127±48.8,t=12.380,P<0.01).Cell cycle analysis showed that NDRG3-shRNA AGS cells were blocked at the S stage,and the percentage of NDRG3-shRNA AGS cells and controls at the S stage was 49.33%and 30.29%respectively(t=4.634,P<0.01).Western blotting results showed that the down-regulation of NDRG3 significantly decreased the expression of Ki-67(0.380±0.021 vs.0.860±0.036,t=5.163,P<0.01),and significantly increased the expression of p53(0.720±0.018 vs.0.160±0.013,t=11.354,P<0.01).Conclusion NDRG3 may promote the cell proliferation and invasion of AGS cells by regulating p53 pathways.