Effect Of NDRG2 Gene On Gastric Cancer Cells Malignant Phenotype

Gastric cancer is one of the most common malignant tumors in the world, which morbidity and mortality list second in China, far more than the world level. Most patients have been in the late period, when they see the doctor, lead to the lost of chance of operation. This is mainly because the mechanism of gastric cancer is still unknown. The effective clinical early diagnosis index and effective treatment is lacked. Local invasion and distant metastasis of malignant tumor, relapse again and multi-resistant is the leading cause of death in patients with late gastric cancer. Therefore it is necessary to study of the mechanism of its malignant phenotype. Additionally, it is important for the early diagnosis and treatment of gastric cancer. Human NDRG family consists of NDRG1, NDRG2, NDRG3 and NDRG4, about 57%- 65% of the amino acid composition between each member has a homology. The NDRG2 is firstly cloned and reported by the fourth military medical university biochemistry and molecular biology teaching and research section. Previous studies have shown that the expression of NDRG2 decrease in most tumor, and it has similar reports in gastric cancer, but its effect on gastric cancer cytology level and its related molecular mechanism is not clearly understand, which need deep study.Purpose:NDRG2 is a potential tumor suppressor gene, with the low expression in gastric cancer tissue. Our study aim at exploring the effects of NDRG2 gene on gastric cancer cells malignant phenotype.Methods:1. To test the expression level of NDRG2 in Immortalized normal gastric epithelial cells and gastric cancer cell line(MKN-28、MKN-45、AGS、SGC-7901、BGC-823) with Western Blot、Real-Time PCR methods.2. To get the pcDNA3.1-NDRG2、pcDNA3.1、siRNA-NDRG2、siRNA-negetive into the AGS cell with transient transfection method, verify the transfection rate by Western Blot、Real-Time PCR. We set 4 groups including pc DNA3.1-NDRG2(Higher expression vector group)、pc DNA3.1(control group 1)、si RNANDRG2(Lower expression vector group)、si RNA-negetive(control group 2). Detecting proliferation of cell in vitro by using MTT and flat cloning expreriments; Detecting apoptosis of cell by using flow cytometry analysis.3. To test the relation between NDRG2 and Bcl-2 with Western Blot method after transfect pc DNA3.1-NDRG2、pc DNA3.1、si RNA-NDRG2、si RNA-negetive. By purchasing organization preliminary detection NDRG2 chip using the immunohistochemical technique to test the Bcl-2 and NDRG2 in gastric cancer tissue expression pattern, Then by collecting clinical specimens and pathological information, enlarge the sample size by using immunohistochemical technique to detect expression pattern of the Bcl-2 and NDRG2 in the tissue of gastric cancer. The relationship between NDRG2 and Bcl-2 and clinical pathological data are analysis by statistical software.Results:1. We found that the NDRG2 have a lower expression in gastric cancer cells,centered in AGS cells expressing quantity with Western Blot、Real-Time PCR methods, and it could be used in the construction of over expression vector and low expression vector.2. After AGS cell transfected with pcDNA3.1-NDRG2 、 siRNA-NDRG2 with RT-PCR and Western Blot method, we found that compared with control group, m RNA and protein levels of NDRG2 will be increased and decreased respectively. The cell reproductive ability of pc DNA3.1-NDRG2 group was lower than pc DNA3.1, this ability of si RNA-NDRG2 group was higher than si RNA-negetive group with MTT method. The clonality of pc DNA3.1-NDRG2 group was lower than pc DNA3.1, this ability of si RNA-NDRG2 group was higher than si RNAnegetive group with MTT method. The cell apoptosis of pc DNA3.1-NDRG2 group was higher than pc DNA3.1, the apoptosis of si RNA-NDRG2 group is lower than si RNA-negetive group with tablet cloning experiments.3. After transfected with pc DNA3.1-NDRG2, the expression of Bcl-2 decreased with Western Blot method. However, after transfected with si RNA-NDRG2, the expression of Bcl-2 increased. The NDRG2 got a lower expression in gastric cancer tissue, but Bcl-2 get a higher level of expression, and it is a negative correlation. With analysis the clinical pathological data, we found that NDRG2 and Bcl-2 expression in gastric cancer tissue positive rate has nothing to do with age, gender, but with the tumor differentiation degree, clinical stage, with and without lymph node metastasis.Conclusion:NDRG2 could inhibit gastric cancer cell proliferation, and promote apoptosis of gastric cancer. We preliminary found tumor suppressor of NDRG2 in gastric cancer may have association with the Bcl-2, but the detailed mechanism remains to be further study.