Expression And Effect On Cell Proliferation Of ARNT2 In Human Gastric Carcinoma

Gastric cancer is one of the most common form of malignant tumor all over the world. Although its morbidity has decreased in some developed countries, its incidence rate and mortality still in the top three in malignant tumor of our country. The abnormal changes of transcription factors often occurred in the occurrence and development of gastric carcinoma, and regulation to gene’s expression of transcription factors play a very important role in the occurrence and development of carcinoma. Therefore, we can improve the diagnosis and treatment of gastric cancer in gene level through in-depth study of this phenomenon.Aryl hydrocarbon-receptor nuclear translocator (ARNT2) is a kind of very important transcription factor in cell nucleus. It belongs to bHLH-PAS superfamily, which is mainly expressed in kidney and nervous system. ARNT2 has been shown to be involved in angiogenesis and nerve development of embryo. Recently, some studies indicate that ARNT2 is associated with the occurrence and development of carcinoma. The high expression level of ARNT2 in breast cancer is a protective factor for survival. The expression level of ARNT2 in non-small cell lung cancer is much lower than corresponding adjacent non-tumor tissues. Otherwise, the results of research in hepatocellular carcinoma is in the opposite. Thus the expression of ARNT2 in carcinoma has tissue specificity. As a result, it is necessary to study its expression in gastric cancer and specific function in the occurrence and development of gastric cancer.Objective1. To detect the expressions of ARNT2 in human normal gastric mucosal epithelial cell line and several gastric cancer cell lines, and compare the difference. To detect the expressions of ARNT2 in human gastric cancer and normal gastric mucosal tissue. To compare the difference and analyze the relationship between the expression level of ARNT2 in gastric cancer and the clinical pathological parameters and prognosis. To discuss its value in clinic application.2. To detect the change of the cell proliferation and expression of anti-apoptotic Survivin and bcl-2 protein of gastric cancer cells over-expressed ARNT2. To definite the effect of ARNT2 on gastric cancer cells’ proliferation and discuss its specific action mechanism.Methods1. qRT-PCR and western blot were used respectively to measure the mRNA and protein expression levels of ARNT2 in normal gastric mucosal epithelial cell line and several gastric cancer cell lines.2. Immunohistochemistry was used to detect the expression level of ARNT2 protein in 89 cases of gastric cancer tissues and corresponding adjacent non-tumor tissues.3. Construct over-expressed ARNT2 plasmid, and it was transfected respectively into two kinds of gastric cell lines, BGC-823 and MGC-803. Fluorescence microscope was used to detect the transfection efficiency. qRT-PCR and western blot was used to detect the over-expression level of ARNT2.4. CCK-8 was performed to examine the effect of over-expressed ARNT2 on the gastric cell proliferation.5. Western blot were used to detect the expression level’s change of anti-apoptotic Survivin and bcl-2 protein in gastric cancer cell over-expressed ARNT2.Result1. The results of qRT-PCR and western blot show that the expression levels of ARNT2 in four different gastric cancer cell lines was significantly lower than normal gastric mucosal epithelial cell line (P< 0.05).2. The results of immunohistochemistry show that the positive expression rates of ARNT2 in the gastric cancer tissues was 44.94%, which was lower than that in adjacent non-tumor tissues (79.78%, P＜0.05). The expression level of ARNT2 in the gastric cancer tissues was correlated with gender of patients, histological type, differentiated degree and depth of invasion of tumor (P＜0.05). However, other clinical characteristics, including age, tumor size, lymphatic metastasis and TNM stage were not directly related to the expression level of ARNT2 in gastric tumor (P>0.05). And overall survival of patient with high ARNT2 expression level was significantly longer than that with low ARNT2 expression level (P＜0.05).3. The results of fluorescence microscope show that the transfection efficiency of control and over-expressed ARNT2 plasmid in two kinds of gastric cancer cell lines, BGC-823 and MGC-803, are both more than 70%. The results of qRT-PCR and western blot show that the expression levels of ARNT2 in two kinds of gastric cancer cell lines transfected with over-expressed ARNT2 plasmid have obvious increase (P＜0.05).4. The results of CCK-8 show that the cell proliferation of gastric cancer cell over-expressed ARNT2 was lower than transfected control plasmid group (P＜0.05). This claim that ARNT2 could restrain the proliferation of gastric cancer cell. 5. The results of Western blot show that the expression levels of anti-apoptotic Survivin and bcl-2 in gastric cancer cell over-expressed ARNT2 were lower than transfected control plasmid group (P＜0.05).ConclusionARNT2 mRNA and protein expression level in different gastric cancer cell lines were all lower than normal gastric mucosal epithelial cell line. And ARNT2 protein expression level in gastric cancer tissue was significantly lower than corresponding adjacent non-tumor tissues, which is in concordance with the result of cell lines. The gastric cancer expressed higher ARNT2 has higher differentiated degree, lower depth of invasion and longer overall survival. The cell proliferation of gastric cancer cell over-expressed ARNT2 receded obviously, and the expression level of anti-apoptotic Survivin and bcl-2 reduced. All the above results reminded that ARNT2 might effect occurrence and development, as an anti-oncogene.