| BackgroundGastric cancer is one of the most common malignant tumor of digestive system worldwide, especially in China, the incidence rate was significantly higher than that in Europe and the United States, seriously endangering the human health. China’s annual new gastric cancer cases up to 400000 people, the death toll reached 300000 people and causes of high mortality of gastric carcinoma mainly lies in early gastric cancer usually has no specific clinical manifestations. Most patients with newly diagnosed is advanced gastric cancer and lost the opportunity of surgical curative resection. Gastric cancer is a multifactorial disease involved in intricate web of signaling pathways that results from multiple exposures to environmental factors, including biological factors, such as Helicobacter pylori (HP) infection, life-style risk factors, and individual genetic predisposition. So it is of practical significance to identify new genes related to gastric cancer development, and to provide new therapeutic targets for clinical treatment.Vitamin D receptor (VDR) is a member of the nuclear receptor family of transcription factors. VDR can be activated by activation form of Vitamin D. After binding to that ligand, VDR formed a heterodimer with another nuclear receptor RXR. By identifying and binding to the specific binding sites in target gene regulatory sequence which called vitamin D response elements (VDREs), to regulate the transcription of the downstream target genes, producing a series of biological effects, such as cell proliferation suppression, apoptosis induction, cell signal disturbing, tumor suppression gene activity enhancing and oncogene expression suppression etc. not only to regulate the transcriptional responses, VDR is also involved in post transcriptional regulation by microRNA. In humans, the vitamin D receptor is encoded by VDR gene, a member of the trans-regulatory factor family, similar to the sequence of the steroid and thyroid hormone receptors. Downstream of the receptor is mainly involved in mineral metabolism, immune response and carcinogenesis. The polymorphism of VDR gene is significantly. So far, at least 25 single-nucleotide polymorphisms (SNP) have been identified. Most studies have focused on the Fok I restriction enzyme sites which located in the transcription initiation site. This polymorphism can change the amino acid sequence length due to different translation initiation codon, eventually produce the same protein of different variants. Previous studies have confirmed that the Fok I polymorphism of VDR gene is associated with the incidence of prostate cancer, breast cancer, melanoma and colorectal cancer. But few research focus on the relationship between VDR gene and gastric cancer. There is no research data show the correlation of Fok I restriction enzyme polymorphism with the pathogenesis and prognosis of Chinese gastric cancer. Mechanism of vitamin D receptor modulating the proliferation and invasion of gastric cancer is unknown. Our previous studies confirmed the VDR expression in gastric cancer tissues was lower than in normal gastric tissue; and the high level of VDR expression in gastric cancer tissues forebode a worse pathological differentiation; Compared with negative on VDR, the one with positive VDR expression has an extended progression free survival and overall survival were. This subject then will discuss the relationship between Han population VDR gene Fok I polymorphism and susceptibility to gastric cancer, as well as the pathological factors and biochemical indexes.The work is expected to provide guidance for the clinical diagnosis and treatment of gastric cancer.We further study the role of VDR in the proliferation and invasion of gastric cancer cells through cell experiments. At the same time, in order to explore the intracellular signal transduction pathway modulated by VDR in the development of gastric cancer, we applied of the TargetScan software to forecast the possible target genes, and discovered that β-catenin are likely to be the potential target of VDR.β-catenin is a signaling protein discovered in recent years. The effect of p-catenin on the development of malignant tumor has been of special concern in medical field, believed to be involved in and mediated a series process of tumor cell proliferation, apoptosis, invasion and metastasis. Wnt signaling pathway is a highly conserved signaling pathway that is widely found in invertebrates and vertebrates. It plays an important role in the early development, organ formation, tissue regeneration and other physiological processes of the animal embryonic development. If the key protein in this signal pathway mutations, resulting in abnormal signal activation, it is possible to induce the occurrence of cancer. Wnt signaling pathway is a group of signal transduction pathways from the cell surface to the inside of the cell. It has three branches:(1) the β-catenin pathway (canonical Wnt pathway), which activates target genes in the nucleus; (2) the planar cell polarity pathway, which involves Jun N-terminal kinase and cytoskeletal rearrangements; and (3) the Wnt/Ca2+pathway. Wnt is a secreted glycoprotein that binds to Frizzled receptors (FZD). In the absence of Wnt binding, β-catenin is down-regulated via a degradation complex including APC, CK1, Axin, and GSK-3. Processive phosphorylation by CK1 and GSK-3 leads to ubiquitination and proteasomal degradation. In the presence of Wnt binding, dishevelled (DVL) is activated. Activated DVL is part of a protein complex that recruits GSK-3 away from the degradation complex, allowing the dephosphorylation and nuclear import of β-catenin and subsequent gene induction via binding to LEF/TCF. Then start the transcription of downstream target genes such as c-myc and cyclin D1, which leads to the occurrence of abnormal cell proliferation and tumor.Previous studies have indicated that Wnt/β-catenin signaling pathway plays an important role in the occurrence and development of benign and malignant breast tumors. The expression level of P-catenin was increased both in tumor cell nucleus and cytoplasm, and its high expression was closely related to the poor prognosis of patients with breast cancer. Researches on metastasis breast cancer had suggested that Wnt is involved in epithelial-mesenchymal transition (EMT) in breast cancer cells. Reduced Wnt/beta-catenin signal can prevent the occurrence of EMT, thus inhibiting the neoplasm metastasis. Wnt signaling pathway is not only an important factor in the development of breast cancer, but also involved in the development of colorectal cancer, melanoma, prostate cancer, lung cancer and several other types of malignant tumors. Increased expression of Wnt ligand protein was found in glioblastoma, esophageal and ovarian cancers respectively. In view of the important role Wnt pathway had played in malignant tumor formation process, we have reason to believe that Wnt/β-catenin signaling pathway is one of the mechanisms that VDR can do to the development of gastric cancer. Our study further researched the relationship of VDR and Wnt/β-catenin signaling pathway and the function mechanism in evolution process of gastric cancer, in expect to providing clues for the follow-up studies. We hope the frame elaborated in this research can be regarded as a reference for the early diagnosis, prognosis estimation and targeted therapy in gastric cancer.CHAPTER I A case-control study of the correlation between Fok I polymorphism of Vitamin D Receptor gene and susceptibility of gastric cancerObjective1. To identify the correlation between VDR Fok I polymorphism and the susceptibility of gastric cancer in Han People.2. To evaluate the role which vitamin D receptor Fok I restriction endonuclease polymorphism had played in the pathological differentiation and Biochemical index.MethodsThe study was carried out in Shandong Provincial Hospital affiliated to Shandong University.187 newly diagnosed cases of gastric cancer from July 2009 to March 2014 and 212 healthy controls were investigated by case-control study. All cases and controls completed a questionnaire to obtain information on demographic characteristics (age, sex, body mass index, and occupation), history of smoking and alcohol drinking, family history of cancer and frequency of sunshine exposure. The VDR Fok I genotype was analyzed using the PCR-RFLP method. The serum vitamin D levels of gastric cancer patients and healthy people are determinated by enzyme-linked immunosorbent assay (ELISA). Calculate the VDR Fok I restriction site genotype and f-allele frequency both in case group and normal control group. Statistical analysis was performed by the SPSS 17.0 statistical software package. Univariate analysis for categorical variable was performed using the chi-squared test or Fisher’s exact test as appropriate. Differences in numerical variables among the two groups were analyzed by an unpaired Student’s t-test. Significant factors produced by the above methods were analyzed using multivariate logistical regression analysis. Logistic regression analysis was also used to calculate the odds ratios (ORs), 95% confidence intervals (CIs) and corresponding P-values. A P-value of< 0.05 was considered statistically significant.Results1. The Hardy-Weinberg equilibrium was evident for the gene polymorphism of VDR Fok I gene in both the case and control groups. The genotypes of FF, Ff and ff were 22.5%,51.9%,25.6% in gastric cancer cases and 32.1%,48.6%,19.3% in healthy controls respectively.2. The f allele frequency in patients and controls were significantly different (51.6% and 43.6%, P< 0.05). No significant differences were found between the two groups in age, sex, BMI, smoking history, alcohol consumption, frequent NSAID intake, and sunshine exposure time per day. In addition, the frequency of a family history of tumors was higher in the case group than the control group (14.9% vs. 5.7%, P< 0.05).3. A worse histological differentiation (poorly rather than moderately or well-differentiated) was shown in the Ff+ff group than that in the FF group. In addition, the CRP level on admission was higher in the Ff+ff group compared with the FF group (5.5±2.4 mg/L vs.3.4±1.3 mg/L, P<0.05).4. There were no statistical differences between the two groups in the serum concentration of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), CEA, CA-125, Uric acid, TNM staging (stage I to IV), serum vitamin D and calcium levels (P> 0.05).ConclusionIn this study we investigated the relationship of VDR Fok I gene polymorphism and the susceptibility to gastric cancer in a Chinese Han population. The F allele of VDR Fok I may be a protective factor against gastric cancer. Compared with the subjects without the f allele (FF), the risk of gastric cancer increased 2.73-fold in subjects with the f allele (FF+Ff). There was no difference between the case and control groups for frequent NSAID intake or sunshine exposure time, but the subjects with a family history of tumors in case group was more than that in control group, which indicated that the Fok I polymorphism may affect the genetic susceptibility to gastric cancer independently of the environmental agents. In addition, we found the patients with f allele (Ff and ff genotype) were associated with a poorer histological differentiation and a higher level of CRP, indicating a more severe inflammation condition and worse prognosis of gastric cancer.CHAPTER Ⅱ Molecular mechanism of Vitamin D Receptor modulating carcinogenesis and progression of gastric carcinoma via Wnt/p-catenin signaling pathwayObjective1. To determine the effect of different VDR genotypes on proliferation, invasion and metastasis of gastric cancer cell.2. To explore the relationship between VDR and Wnt/β-catenin signaling pathway during the occurrence and development of gastric cancer.Methods1. The VDR gene was detected by PCR-RFLP in different gastric cancer cell lines, and the expression of VDR in different gastric cancer cell lines was determined by SDS-PAGE and Western blotting.2. The expression of VDR in gastric cancer cell line was interfered by plasmid transfection and RNA interference technology. Cell viability and invasive ability were analyzed by MTT assay, colony formation assay and transwell migration assay.3. The wild-type and mutant VDR expression plasmids were transfected into cells by Lipofectamine2000, and the biological behaviors of gastric cancer cells with different VDR genotypes were observed.4. VDR was stimulated with activated vitamin D3 in different gastric cancer cells, and VDR expression was interfered by the application of siRNA and plasmid transfection. Then detected the level of P-catenin, c-myc protein and E-cadherin by Western blot analysis, to clear the links between VDR and Wnt/β-catenin signaling pathway.Results1. VDR gene were homozygous wild type in gastric carcinoma cell lines AGS, MGC803, N87, MKN-28 and SGC7901. Protein expression of VDR was detected in all this 5 kinds of cell lines, and was significantly reduced in cell line N87. VDR expression in cell line M28 was the highest.2. When VDR plasmid was transfected into N87 cell line, which natural state of VDR expression was the lowest, the overexpression of VDR can significantly inhibit the growth of cells; on the contrary, when VDR siRNA inhibits the expression of VDR, the cell grows rapidly.3. The colony formation assay showed that cells with VDR overexpression were unable to form colony, however, if the expression of VDR was inhibited, cell clone was developed.4. When SiRNA plasmid were transfected into gastric carcinoma cell, its invasion increased significantly, on the contrary, cells with VDR overexpression lost the invasive activity.5. Gastric cancer cell lines (N87) was treated with 1,25 (OH) 2D3 for 0,2,5,10,30, 60 hours respectively. Western blot indicated that VDR expression levels in gastric cancer cells treated with 1,25 (OH) 2D3 showed a time dependent increased expression; and with the increase of the VDR expression, the expression of P-catenin decreased gradually, but the expression of E-cadherin showed a time dependent increase (P< 0.05).6. The gastric cancer cell line N87 was transfected with wild-type and mutant VDR gene plasmids, and was treated with 1,25 (OH) 2D3 for 0,2,5,10,30,60 hours respectively. The expression of β-catenin and c-myc protein were decreased, especially after a long time (10h) treatment, but the expression of E-cadherin in the cytoplasm increased over time. Compared with the wild-type VDR gene cells, the decline of β-catenin expression was significantly enhanced after transfected with mutant VDR gene plasmids (p<0.05).7. The expression of β-catenin and the cell proliferation was significantly decreased after increased the VDR expression, in contrast to enhanced by interference of VDR expression (P< 0.05).Conclusion1. The results of this study indicate that VDR plays an important role in the malignant phenotype of gastric cancer cells, such as proliferation, invasion and clone formation.2. Compared with the cells transfected with wild-type VDR gene plasmids, the decrease of β-catenin and c-myc protein expression after transfection of VDR Fok I mutation plasmids were significantly enhanced and rapid, which suggested that VDR Fok I mutation can play a role of cancer suppressor via Wnt/β-catenin signaling pathway. This is consistent with our previous study of the role of VDR polymorphisms played in gastric cancer patients.3. In gastric cancer cells stimulated by VD3, the active form of Vitamin D, the VDR expression increased, the β-catenin expression in nucleus reduced accordingly, and E-cadherin expression showed a corresponding increase, which indicates that, when the VDR is activated by its ligand, it can induce a series of reactions to prevent the nuclear import of P-catenin, thereby inhibiting the proliferation of gastric cancer cells, and regulating the invasion and migration of gastric cancer cells via affecting E-cadherin level.4. Increasing the expression of VDR in gastric cancer cells, the expression of P-catenin decreased, while the cell proliferation was significantly inhibited; on the contrary, the expression of P-catenin increased, and cell proliferation was enhanced with the decrease of VDR expression, which further confirmed the effect of VDR on the proliferation and apoptosis of gastric carcinoma cells by regulating the level of β-catenin. |
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