p53和DNA损伤调控基因1促进结直肠癌细胞转移及上皮-间充质转化的作用及其机制

Effects and mechanism of p53 and DNA damage-regulated gene 1 promotes metastasis and epithelial-mesenchymal transition in Colorectal cancer cells

目的检测p53和DNA损伤调控基因1(PDRG1)在结直肠癌细胞中的表达,探讨PDRG1在结直肠癌细胞增殖、侵袭及迁移过程中的作用及其机制。方法实时定量反转录聚合酶链反应法(RT-qPCR)和蛋白质印迹法(Western blot)检测结肠癌(SW620)和正常结肠(NCM460)2种细胞中PDRG1 mRNA及蛋白的表达。转染PDRG1过表达载体和小干扰RNA(siRNA)感染序列至SW620细胞,实验分为3组,过表达转染组,阴性转染对照组,抑制组。Western blot检测PDRG1、锌指蛋白转录因子1(Snail1)、上皮型钙黏附蛋白(E-Cadherin)、波形蛋白(Vimentin)和甘油醛-3-磷酸脱氢酶(GAPDH)蛋白的表达。细胞计数试剂盒(CCK-8),Transwell小室检测细胞增殖、迁移及侵袭能力。检测转染Snail1 siRNA感染序列对过表达PDRG1 SW620的影响。计数数据采用χ^(2)检验或者Fisher′s精确检验,采用t检验比较各组间差异。结果PDRG1 mRNA和蛋白在SW620的相对表达量分别为1.853±0.118、1.563±0.064,明显高于NCM460中的1.00±0.00、1.00±0.00(t=12.51、15.08,P<0.01)。SW620细胞转染PDRG1过表达载体后mRNA和蛋白的相对表达量较对照组(t=20.073、16.842,P<0.01)明显升高,转染72 h后细胞增殖水平高于对照组(t=4.857,P<0.01),侵袭和迁移细胞数122.3±18.1、174.6±13.2,较对照组99.5±16.2、110.5±12.2明显增加(t=2.956、11.271,P<0.01)。Snail1、Vimentin的表达明显升高(t=9.756、10.950,P<0.01),E-Cadherin的表达明显降低(t=-6.060,P<0.01)。SW620细胞转染PDRG1 siRNA后mRNA和蛋白的表达较对照组明显降低(t=8.072、12.704,P<0.01),转染48、72 h后增殖水平低于对照组(t=5.557、-4.782,P<0.01),侵袭和迁移细胞数(30.8±8.6、37.5±8.9),较对照组(117.6±12.8、126.9±11.9)明显降低(t=-17.734、-18.873,P<0.01)。Snail1、Vimentin蛋白的表达明显降低(t=-7.588、-7.187,P<0.01),E-Cadherin的表达明显升高(t=13.239,P<0.01)。转染Snail1 siRNA感染序列逆转了PDRG1调节的上皮-间充质化(EMT)过程和细胞迁移和侵袭。结论PDRG1可通过调控Snail1的表达来影响EMT过程,从而促进肿瘤细胞的增殖,侵袭和迁移。

Objective To investigate the expression of p53 and DNA damage-regulated gene 1(PDRG1)and to explore the effects and mechanism of PDRG1 promotes metastasis and epithelial-mesenchymal transition(EMT)in Colorectal cancer cells.Methods The mRNA and protein expression levels of PDRG1 were detected by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)and Western blotting in two cells(SW620 and NCM460).The PDRG1 over-expression vector and small interfering RNA(siRNA)were transfected into SW620.The experiment was divided into three groups:overexpression transfection group,negative transfection control group and inhibition group.The expression of PDRG1,Snail1,E-Cadherin,Vimentin and glyceraldehyde-3-phosphate dehydrogenase(GAPDH)were detected by Western blotting.Cell proliferation was detected by cell counting kit-8(CCK-8)assay,invasion and migration were detected by Transwell.The counting data were tested byχ^(2) test or Fisher′s exact test and the differences between groups were compared by t test.Results The expression of PDRG1 mRNA and protein in SW620(1.853±0.118,1.563±0.064)were significantly higher than those in NCM460(1.00±0.00,1.00±0.00)(t=12.51,15.08,P<0.01).The relative expression of PDRG1 mRNA and protein were significantly higher(t=20.073,16.842,P<0.01),cell proliferation after transfected for 72 h was significantly increased(t=4.857,P<0.01),the number of invasion and migration cells(122.3±18.1,174.6±13.2 vs.99.5±16.2,110.5±12.2)were significantly increased(t=2.956,11.271,P<0.01),Snail1,Vimentin expression were higher(t=9.756,10.950,P<0.01),E-Cadherin expression was lower(t=-6.060,P<0.01)in the PDRG1 over-expression group.The relative expression of PDRG1 mRNA and protein were significantly lower(t=8.072,12.704,P<0.01),cell proliferation after transfected for 48 h and 72 h were significantly decreased(t=5.557,-4.782,P<0.01),the number of invasion and migration cells(30.8±8.6,37.5±8.9 vs.117.6±12.8,126.9±11.9)were significantly decreased(t=-17.734,-18.873,P<0.01),Snail1,Vimentin expression were lower(t=-7.588,-7.187,P<0.01),E-Cadherin expression was higher(t=13.239,P<0.01)in the PDRG1 knockdown group.The migration and invasion abilities of PDRG1-overexpressed cells could be reversed by silencing Snail1 expression.Conclusion PDRG1 promotes CRC cell EMT process through a Snail1-dependent pathway.