The Study On Expression Of DcR3 In ESCC Tumor Tissue And Its Mechanism

Background: Esophageal cancer is one of the eight most popular malignant tumors in human. Among 300,000 new diagnosed cases every year in the world, seventy percent were in China and the disease incidence still increases recently. One of the main problems of esophageal cancer is that long-term survival is very low, the totally 2-year survival rate is 35%-42% and only 15%-24% for 5-year survival. Esophageal cancer can be characterized into ESCC (esophageal squamous cell carcinoma) and esophageal adenocarcinoma, while ninty percent disease cases are ESCC. So it is very urgent to study the pathogenesis of ESCC and develop new target for diagnosis and treatment.As we known, the balance between the immunological surveillance of host and the immune escape of tumor is tightly regulated during the tumorigenesis and tumor development. Inducing the tumor-specific CTL response and/or the apotosis of tumor cells are two important methods to eliminate tumor cells. LIGHT and FasL are two members of TNF super family (TNFRSF). They are the most common pathway for inducing the apotosis in vivo, and play essential role in immune regulation and the tumor immune monitoring. DcR3 is a new member of TNFRSF, which can competitively bind to Fas, LIGHT and TL1A and then inhibite the apotosis of tumor cells induced by these three molecular. In the other hand, during the immune response, DcR3 can inhibit the proliferation of T cells and the differentiation and maturation of dendritic cells (DCs), especially the proliferation of CD4+ T cells in MLR. Besides, DcR3 can also inhibit the chemotaxis of T cells and reduce the interact between T cells and antigen processing cells.Previous studies showed that DcR3 mRNA expression level was very low in many normal human tissues, such as stomach, spinal cord, lymph node, lung, spleen, colon tissue and most hematopoietic cells, but contrarily, in many kinds of malignant tumor, including gastrointestinal cancer, breast cancer, carcinoma of colon, lung cancer, spongiocytoma and lymphoma, DcR3 mRNA expression level was very high. It was reported that overexpression of DcR3 was an important mechanism for tumor cells escaping from immune surveillance and destruction. In many different kinds of cancer, the expression level of DcR3 is clearly related with the progress of carcinogenesis, cancer metastasis, the resistance to chemotherapy and the prognosis of tumor. So, DcR3, as a candidate cancer biomarker and target for cancer gene therapy, becomes a very hot point in cancer research. Studying the expression of DcR3 on the mRNA and protein level in ESCC cell strains and patient biopsies, exploring the relationship between DcR3 expression and the invasion, metastasis, recurrence and prognosis of tumor are very helpful to evaluate the possibility of using DcR3 as a molecular marker in ESCC's clinical diagnose and prognosis prediction. In the meantime, investigating the regulation mechanism behind DcR3 expression can help us develop a new gene therapy approach to treat ESCC. These facts suggested that studying the expression of DcR3 and how it is regulated are very important in the prevention and treatment of ESCC.Aims: To identify the expression of DcR3 gene in ESCC patient's samples and analyze its possible correlation between DcR3 expression and their clinicopathologic features. The polymorphic sites frequencies of DcR3 promoter and coding region in Chongqing normal population and ESCC patients would to be detected, in order to address the possible correlation between polymorphism of DcR3 promoter and ESCC risk, and screen the SNP sites associated with DcR3 high expression. For exploring whether the the methylation was involved in the regulation of DcR3 expression, the methylation difference in promoter region of DcR3 was to be tested. In addition, the effects of DcR3 RNAi on ESCC tumor metastasis, invasion and immune escape were to be detected, and the biological behavior and the mechanism of DcR3 in the progression of human ESCC cells were also to be investigated.Methods: 1. Semi-quantitative RT-PCR assay was utilized to detect the expression of DcR3 mRNA in 109 ESCC tumor tissues and corresponding normal tissues far away from tumor.2. Immunohistochemistry and semi-quantitative analysis were performed to test DcR3 protein expressions from 52 ESCC patients.3. DcR3 promotor and four polymorphic sites (-369G/T,-323A/C,-321C/T,147C/T) in coding region were selected, these polymorphic sites frequencies in Chongqing normal population and their frequencies in ESCC patients were studied with PCR sequencing to address the possible correlation between polymorphism of DcR3 promoter and ESCC risk.4. Based on their significant difference of DcR3 expression with each other, four pairs of sample from the tumor tissues and corresponding normal tissues far away from tumor were choosed, and their methylation status of the CpG in the promoter region of DcR3 were detected by bisulfite sequencing assay(BSA).5. A DcR3 targeted RNA interfering lentivirus vector based on a new miR30 like was constructed to inhibit the expression of DcR3 in esophageal squamous cell line KYSE150.6. The verified recombined vector was co-transfect with pMD2g and psAX2 into 293FT packing cell line to produce the virus.7. ESCC cell model of DcR3 RNAi was obtained by FACS.8. The DcR3 expression level of infected cells were evaluated by Real-time PCR and Western blotting.9. The growth curves of infected cells, controlled cells and wild cells were drawn.10. The effects of DcR3 RNAi collaborated with hLIGHT-Fc on invasion activity of infected ESCC cells were studied by Transwell loculus assay.11. The effects of DcR3 RNAi collaborated with hLIGHT-Fc on apoptosis sensitivity of infected ESCC cells were investigated by Tunel assay.Results: 1. The expression of DcR3 mRNA in 109 ESCC tumor tissues was 73.4% and 47.7% in the corresponding normal tissues far away from tumor. The expression of DcR3 mRNA was no close relation with gender, age, localization and tissue differential degree of patients, but strong related with tumor infiltration (p<0.05), regional lymph node metastasis (p<0.05) and clinical TNM stage (p<0.05).2. DcR3 protein expression was consistent with DcR3 mRNA expression. The overexpression rate of DcR3 was 28.8%, and there was a correlation between DcR3 overexpression and regional lymph node metastasis (p<0.05) and clinical TNM stage (p<0.05).3. Polymorphism was found only existe at 147C/T, instead of -369G/T,-323A/C or -321 C/T, on Chongqing Han population. The minor allel frequence (MAF) was 43.7%, which was a little bit higher than the whole Chinese Han population(39.0%) and Janpense population(40.0%). 147C/T polymorphism was a risk site of susceptibility to ESCC in Chongqing Han population, and the 147C had the dominant inheritance effect comparing with 147T. The risk of susceptibility to ESCC was significantly increased in CC or CT, comparing with TT (p<0.05). But no significant correlation between this site and the upregulation of DcR3 protein was found.4. There was no difference of the methylation status between the cancer tissues and matched normal tissues. All the CpG sites in the promoter region were unmethylated.5. The new miR30 like sequence based lentivirus interfering vector was successfully constructed and the virus-containing cell culture supernatants could infect KYSE150 cells efficiently.6. FACS sorting and Western Blotting showed that the pPRIME-DcR3.3i vector could inhibit the expression of DcR3 protein with the 82.6% inhibition rate.7. There was no significant suppressing effect of DcR3 RNAi on infected KYSE150 cells.8. The invasion activity could be inhibited by the joint action of DcR3 RNAi and hLIGHT-Fc.9. DcR3 RNAi obviously promoted the apoptosis of KYSE150 cell induced by hLIGHT-Fc.Conclusion: The mRNA and protein expression level of DcR3 in ESCC tumor tissues are higher than that in matched normal tissues. Increased expression of DcR3 is significant related with regional lymph node metastasis and clinical TNM stage. 147C/T polymorphism is a risk site of susceptibility to ESCC in Chongqing Han population, and the 147C has the dominant inheritance effect. The abnormal high expression of DcR3 in ESCC patients is no relationship with methylation status of the CpG in the promoter region of DcR3. DcR3 RNAi has no direct inhibition on growth of ESCC, but it can inhibit invasion effectly and induce apoptosis with the collaboration of hLIGHT-Fc. Above all, DcR3 is a potential ESCC clinical biomarker.