The Effect Of NDRG1 On Biological Properties Of Colorectal Cancer Cell Line Caco2 In Vitro

Objective:To investigate the effect of NDRG1 on biological properties of colorectal cancer cell line Caco2 in vitro.Methods:NDRG1 stably overexpressed Caco2 cell line was established by lentiviral infection,and NDRG1 knock-out Caco2 cell line was established by CRISPR/Cas9.Furthermore,the mRNA and protein levels of NDRG1 in Caco2 cells after overexpression and knock-out were detected by Real-time PCR and Western Blot.The cell proliferation rate was measured by CCK8 method;cell cycle and apoptosis were detected by flow cytometry;invasion and migration ability were detected by 24-Transwell method.Results:The results of qPCR had shown that the relative expression level of NDRG1 mRNA in the overexpressed group(3.218±0.6756)raised 3 times compared with the empty plasmid group(1±0),and the difference is statistically significant(P=0.0006),the relative expression level of NDRG1 mRNA in the knockout group(0.07102 ± 0.001901)was obviously reduced,the difference is statistically significant(P<0.0001)comparing with the empty plasmid group(1±0)after NDRG1 gene knockout.Western Blot results revealed that the protein expression level in the overexpressed group raised about two times(mean of overexpression group:1.92±0.06285;mean of empty plasmid group:0.9279±0.03871),the difference is also statistically significant(P<0.0001).After NDRG1 gene knockout,the protein expression level reduced significantly(knockout group mean:0.3743±0.05146;empty plasmid group mean:1.108±0.05031),the difference is statistically significant(P<0.0001).CCK8 data showed that the proliferation rate of Caco2 cells in NDRG1 overexpression group decreased at various time points except 24-hour points,and the difference was statistically significant(P<0.01);the proliferation rate of Caco2 cells in NDRG1 gene knockout group raised at various time points,and the difference was statistically significant(P<0.01);Cell cycle was detected by Flow cytometry,G1 phase cells increased significantly(P<0.0001)after NDRG1 overexpression,S and G2 phase cells decreased,which indicated that the cell cycle was arrested at G1/S phase.There was no difference in the proportion of cells in G11 phase(P=0.883 7)after NDRG1 gene knockout,S phase cells decreased,and G2 phase cells obviously increased(P<0.0001),it was showed that cell proliferation ratio increased,and cell growth is active.The results of flow cytometry showed that the rate of early(P=0.0002)and total apoptosis(P=0.0004)of Caco2 cells was decreased after NDRG1 overexpression,the rate of late apoptosis was not statistically significant(P=0.9296).The rate of early(P=0.0013)and total apoptosis(P=0.0002)of Caco2 cells was increased after NDRG1 gene knockout,and the rate of late apoptosis was not statistically significant(P=0.3330),it was indicated that NDRG1 inhibited cell apoptosis.The ability of Caco2 cell invasion and migration was detected by 24-Transwell method.The ability of Caco2 cells invasion and migration was reduced at 48h after NDRG1 overexpressed,while when NDRG1 gene knockout,the ability of Caco2 cell invasion and migration was increased.Conclusions:1.The inhibition mechanism of NDRG1 for Caco2 cells was associated with arresting cell cycle at G1/S phase;2.NDRG1 promotesed apoptosis of Caco2 cells;3.NDRG1 inhibited the invasion and migration of Caco2 cells.