Inhibition Effects Of RNA Interference On Expression Of Glucose-regulated Protein 78 In Human Colorectal Carcinoma Cell Line RKO

Objective:To study the expression of GRP78 in human colorectal carcinoma specimens and its clinical significance.And to observe silencing effect on GRP78 expression in human colorectal carcinoma cell line with the eukaryotic expression vector of RNA interference specific for it(PGPU6/GFP/Neo-GRP78).Methods:1.Immunohistochemistry PV method and reverse transcription-polymerase chain reaction(RT-PCR) method were used to detect the expression of GRP78 of colorectal carcinoma specimens,colorectal adenoma specimens and normal colorectal specimens.2.Human colorectal carcinoma cell line RKO was transfected with the recombinant plasmid(PGPU6/GFP/Neo-GRP78) by Lipofectamine 2000.The expression of GRP78 was detected both at mRNA and protein level by reverse transcription-polymerase chain reaction(RT-PCR) and immunoblotting,respectively.Results:1.Immunohistochemical analysis showed higher expression of GRP78 in colorectal carcinoma compared with that in normal specimens(P＜0.05) and its expression in colorectal adenoma was significantly higher than that in normal specimens but lower than that in colorectal carcinoma(P＜0.05).Whereas no significant difference was detected between the expression of GRP78 and the clinical factors(P＞0.05). RT-PCR analysis showed there was no difference in the relative mRNA expression level of GRP78 between normal specimens and colorectal tumors(P＞0.05).2.The expression of GRP78 was obviously down-regulated both at mRNA and protein level by the recombinant plasmid(PGPU6/GFP/Neo-GRP78) transfection.The analysis for the relative level of GRP78 mRNA showed a gradual and stable decrease with the prolonging of time.The expression of GRP78 mRNA was significantly lower in the cells after transfection than that before or without transfection(P＜0.05).There was no difference between the negative control group and pre-transfection group(P＞0.05).3. Immunoblotting analysis showed that the protein expression of GRP78 had been significantly decreased in the cells after transfection than that before transfection(P＜0.05).There was also no difference between the negative control group and pre-transfection group(P＞0.05).Conclusions:1.The levels of GRP78 were elevated gradually in the human normal colorectal specimens,adenoma specimens and carcinoma specimens.GRP78 may play some important roles in the development of colorectal carcinomas.Whereas no significant difference in the relative mRNA expression level of GRP78 was detected among colorectal carcinoma specimens,colorectal adenoma specimens and normal colorectal specimens.2.The shRNA eukaryotic expression vector PGPU6/GFP/Neo-GRP78 specific for GRP78 can evidently inhibit the expression of GRP78 both at protein and mRNA level.