小分子干扰RNA沉默鸟嘌呤核苷酸解离抑制因子2基因表达对胰腺癌细胞迁移侵袭的影响

Effect of small interfering RNA silencing guanine nucleotide dissociation inhibitory factor 2 gene expression on migration and invasion of pancreatic cancer cells

目的探讨小分子干扰RNA(siRNA)沉默鸟嘌呤核苷酸解离抑制因子2(RhoGDI2)基因表达对胰腺癌细胞迁移侵袭的影响。方法选取2016年1月至2019年12月于张家港市第一人民医院进行手术的60例患者的胰腺癌肿瘤组织标本作为研究对象。应用Western blot检测RhoGDI2在人胰腺癌细胞株CFPAC-1、HPAF-Ⅱ、MIA PaCa-2、PANC-1、SW1990中的表达,利用siRNA沉默CFPAC-1细胞中RhoGDI2的表达,细胞划痕实验和Transwell实验检测细胞体外迁移能力和侵袭能力,Western blot检测P21蛋白激活激酶1(Pak1)表达情况,免疫组织化学染色检测胰腺癌组织标本中RhoGDI2和Pak1的表达情况。结果Western blot检测结果显示,RhoGDI2蛋白在胰腺癌细胞株中呈不同程度的表达水平,表达水平由高到低的细胞顺序依次为CFPAC-1、SW1990、PANC-1、HPAF-Ⅱ、MIA PaCa-2。WT、si-NC及si-RhoGDI2组细胞的RhoGDI2蛋白表达量分别为(1.25±0.09)、(1.19±0.11)、(0.38±0.07);转染si-RhoGDI2后RhoGDI2蛋白的表达显著下降,3组蛋白表达比较差异有统计学意义(P<0.05)。WT、si-NC及si-RhoGDI2组胰腺癌细胞的48 h划痕迁移率分别为(62.33±5.29)%、(62.27±3.37)%、(17.60±6.16)%,3组比较差异有统计学意义(P<0.05)。WT、si-NC及si-RhoGDI2组胰腺癌细胞的48 h划痕迁移率分别为(62.33±5.29)%、(62.27±3.37)%、(17.60±6.16)%,3组比较差异有统计学意义(P<0.05)。WT、si-NC及si-RhoGDI2组胰腺癌细胞穿膜细胞数分别为(132.3±10.3)、(120.7±16.5)、(24.3±9.5)个,3组比较差异有统计学意义(P<0.05)。WT、si-NC及si-RhoGDI2组细胞的Pak1蛋白表达量分别为(0.82±0.06)、(0.73±0.07)、(0.23±0.03),3组蛋白表达量比较差异有统计学意义(P<0.05)。免疫组织化学染色检测结果显示,RhoGDI2阳性47例,阳性率为78.3%,Pak1阳性45例,阳性率为75.0%。Spearman等级相关分析结果显示,RhoGDI2与Pak1的表达水平呈正相关(r>0,P<0.05)。结论沉默RhoGDI2基因表达能够抑制胰腺癌细胞的迁移和侵袭,RhoGDI2可能在胰腺癌发生发展过程中具有重要作用。

Objective To investigate theEffect of small interfering RNA(siRNA)silencing guanine nucleotide dissociation inhibitory factor 2(RhoGDI2)gene expression on migration and invasion of pancreatic cancer cells.Methods The pancreatic cancer tissue specimens of 60 patients who underwent surgery in the First People's Hospital of Zhangjiagang from January 2016 to December 2019 were selected as the research subjects.The expression of RhoGDI2 in human pancreatic cancer cell lines CFPAC-1,HPAF-Ⅱ,MIA PaCa-2,PANC-1 and SW1990 was detected by Western blot,cell scratch test and Transwell test were used to detect cell migration and invasion ability in vitro,the expression of P21 protein activates kinase 1(Pak1)was detected by Western blot,immunohistochemical staining was used to detect the expression of RhoGDI2 and Pak1 in pancreatic cancer tissue samples.Results Western blot results showed that,RhoGDI2 protein was expressed in different degrees in pancreatic cancer cell lines,the order of expression level from high to low was CFPAC-1,SW1990,PANC-1,HPAF-Ⅱ,and MIA PaCa-2.The expression levels of RhoGDI2 protein in WT,si-NC and si-RhoGDI2 groups were(1.25±0.09),(1.19±0.11)and(0.38±0.07);after transfection with si-RhoGDI2,the expression of RhoGDI2 protein decreased significantly,and there were significant differences in the expression of RhoGDI2 protein among the three groups(P<0.05).The 48 h scratch migration rates of WT,si-NC and si-RhoGDI2 groups were(62.33±5.29)%,(62.27±3.37)%and(17.60±6.16)%,there were significant differences among the three groups(P<0.05).The number of penetrating cells in WT,si-NC and si-RhoGDI2 groups were(132.3±10.3),(120.7±16.5)and(24.3±9.5),there were significant differences among the three groups(P<0.05).The Pak1 protein expression levels of WT,si-NC and si-RhoGDI2 groups were(0.82±0.06),(0.73±0.07)and(0.23±0.03),there were significant differences in protein expression among the 3 groups(P<0.05).Immunohistochemical staining results showed that,47 cases were positive for RhoGDI2,the positive rate was 78.3%,45 cases were positive for Pak1,the positive rate was 75.0%.Spearman rank correlation analysis showed that the expression levels of RhoGDI2 and Pak1 were positively correlated(r>0,P<0.05).Conclusion Silencing RhoGDI2 gene expression can inhibit the migration and invasion of pancreatic cancer cells.RhoGDI2 may play an important role in the occurrence and development of pancreatic cancer.