Studies On The Expression Of T-cadherin In Gastric Cancer And Its Effects On Biological Characteristics Of HGC-27Gastric Cancer Cell Line

Background:In the past few decades, gastric cancer incidence and mortality have declined sharply in the United States and other countries. However, gastric cancer as the fourth most common cancer remains a worldwide public health problem, and the second mortality rate for cancer. T-cadherin, also known as H-cadherin, cadherin protein-13or CDH13, is a non-classical cadherin. T-cadherin is a unique cadherin cell adhesion molecule, because of its lack of classical cadherin molecules without the transmembrane and cytoplasmic domain and attached to the membrane by glycosyl phosphatidylinositol (Glycosylphosphatidylinositol, GPI) T-cadherin was found first in the chick embryo brain tissue and subsequently cloned human T-cadherin. T-cadherin gene is located on the long arm of human chromosome16q24, a variety of tumors often occurred in the loss of alleles in this region. In most human tumor cell lines and tissues, T-cadherin gene and protein expression is down-regulated. T-cadherin down-regulation in tumors has at least three molecular mechanisms:(1) delete16q24sites which contains T-cadherin gene;(2) abnormal promoter methylation;(3) histone modifications. In the past decade, a lot of evidence has showed that the T-cadherin is down-regulated not only in breast cancer but also in a variety of other cancers and cancer cell lines, such as liver cancer, colorectal cancer, lung cancer, ovarian cancer, cervical cancer, breast cancer, skin squamous cell carcinoma and gastric cancer. T-cadherin is silent in many different types of tumors, indicating that it may play a role as a tumor suppressor gene. In addition, T-cadherin re-expression can inhibit tumor cell proliferation and invasion. Studies have shown that T-cadherin is closely associated with aggressive tumor biological characteristics, such as tumor malignant proliferation, apoptosis, invasion and metastasis. As research continues, the various functions of T-cadherin have been gradually recognized. Part I Decreased expression of T-cadherin is associated with gastric cancer prognosisObjective:To investigate the expression status of truncated-cadherin (T-cadherin) and its clinicopathological significance in gastric cancer.Meths:Expression status of T-cadherin was detected in a total of103surgical specimens of gastric cancer and34corresponding normal gastric tissues by real-time qPCR, western blotting and immunohistochemistry. Correlation between the expression of T-cadherin and clinicopathological factors of gastric cancer was analyzed.Results:T-cadherin mRNA levels were significantly reduced in29(85.3%) tumor tissue samples, compared with levels in the adjacent normal tissue samples (P<0.01). This finding was confirmed by western blotting analysis (P<0.01) and immunohistochemistry analysis (P<0.01). Larger tumor size (diameter>4cm), lymph node metastasis, invasion into surrounding tissues, poor-differentiation and higher TNM stage of carcinoma were significantly correlated with decreased expression of T-cadherin (P<0.05or P<0.01). Univariate Cox regression analysis showed that smaller tumor size (diameter<4cm), none lymph node metastasis, none invasion into surrounding tissues, well-differentiation, lower TNM stage and higher expression of T-cadherin were closely associated with increased overall survival (P<0.05or P<0.01). Multivariate Cox regression analysis showed that positive expression of T-cadherin was a most important independent risk factor in gastric cancer.Conclusion:Expression of T-cadherin is an important independent prognostic predictor, which can be used as a biomarker of progression and prognosis in gastric cancer. Part Ⅱ The effects of miR-155on biological characteristics in human renal carcinoma cell lineObjective:To find a suitable cell line to establish T-cadherin overexpression cell model in vitro and its validation.Methods:T-cadherin expression in human gastric cancer cell line HGC-27, SGC7901, MGC-803cells and normal gastric mucosal cell line GES-1were detected to find a suitable cell line as a T-cadherin overexpression cell model of gastric cancer in vitro. Construction of T-cadherin over-expression vector was used for transfection of the gastric cancer cell line HGC-27. The high expression of T-cadherin in gastric cancer cell HGC-27was detected by real-time fluorescence quantitative polymerase chain reaction (Real-time qPCR) and Western blot.Results:The detection of T-cadherin expression by Real-time qPCR in human gastric cancer cell line HGC-27, MGC-803, SGC7901and normal gastric cell line GES-1were1.000,6.774,16.761and65.662; The detection of T-cadherin protein expression by western blot were0.296,0.446,0.533and0.669. Select HGC-27to construct T-cadherin overexpression cell model in vitro (pG-CMV/EGFP/Neo. T-cad). The experiment was divided into three groups:normal group, the negative control group and overexpression group. After transfection, T-cadherin in gene and protein expression detected by Real-time qPCR and western blot in the normal group, negative control group, and overexpression group were1.000,1.291,150.018and0.306,0.312and0.974, respectively.Conclusion:1,HGC-27cells is suitable to build the T-cadherin overexpression cell model in vitro.2,pG-CMV/EGFP/Neo. T-cad can be effectively transfected with HGC-27cell, so that the T-cadherin in HGC-27cell is high. Part III The effects of T-cadherin overexpression on the biological characteristics of HGC-27Objective:To investigate the effects of T-cadherin overexpression on the biological characteristics of gastric cancer cell HGC-27.Methods:To investigate the T-cadherin overexpression on proliferation, apoptosis, invasion and metastasis ability of gastric cancer cell HGC-27by MTT assay, Annexin V-PE/7-AAD double staining flow cytometry, Transwell invasion assay and migration assay.Results:The relative survival rates of1,2,3,4, and5days after transfection of negative control group were97.6±2.9%,93.8±5.7%,98.1±3.0%,95.9±1.4%,92.3±1.6%, while over-expression group were93.5±2.1%,79.4±1.5%,72.7±1.2%,48.3±1.1%,30.0±1.0%. The relative survival of two groups2days after transfection is significantly different by t test (P<0.01). At24hours after transfection, the apoptosis rate of HGC-27cell in the overexpression group was21.76±0.86%, while in the normal group and negative control group, the HGC-27cell apoptosis rates were5.98±0.44%and7.16±0.67%. The overexpression group compared to the normal group and the negative control group, their apoptosis rats was significantly different (P<0.05). The cells number of overexpression group, normal group and negative controls in tumor invasion and migration assays were15.24±1.06,42.35±1.65,41.12±1.26and24.08±1.12,51.43±2.14,45.35±1.67. The cells number of overexpression group in tumor invasion and migration assays was significantly different compared to the normal group and the negative control group (P<0.05).Conclusion:1,The growth and proliferation of gastric cancer cell HGC-27with overexpression of T-cadherin is suppressed.2,T-cadherin overexpression induces an increased apoptosis rate in gastric cancer cell HGC-27.3,T-cadherin overexpression diminishs invasion and migration capacity of gastric cancer cell HGC-27.