Expression And Significance Of SEMA3B In Esophageal Squamous Cell Carcinoma And Construction And Identification Of Recombinant Plasmid PcDNA3.1-SEMA3B

Background and ObjectiveEsophageal squamous cell carcinoma(ESCC), is one of the most frequent malignant tumors and ranked as the sixth leading cause of cancer death worldwide. ESCC is characterized by its remarkable geographic distribution, especially in Linzhou (formerly Linxian) and the nearby counties in Henan province of Northern China. Many factors,such as environment,smoking,habit of diet,contribute to the malignant progression and prognosis of ESCC.Like other types of solid tumors, the development of ESCC is also the accumulation of the abnormal expression of oncogenes and tumor suppressor genes (TSGs). Many genes associated with the development and progression of ESCC have been characterized,for example P53 and Rb. Comparative genomic hybridization and loss of heterozygosity(LOH) have characterized the common deletion regions at 3p in ESCC and several candidate TSGs within the region have been studied, such as SEMA3B,FHIT,RASSF1A, CACNA2D2,DLEC1,PLCδ1.Previous studies have characterized the down-expression of SEMA3B gene in lung cancer and breast cancer, which associated with the development and progression of lung cancer and breast cancer. SEMA3B effectively suppress cell growth through inducing apoptosis,decreasing foci formation and suppressing tumor formation in nude mice. RT-PCR and S-P immunohistochemistry were used to determine the expression of SEMA3B in 51 primary ESCC tumors and their paired nontumorous tissues in order to investigate its effect in ESCC. Successfully construct the eukaryotic expression recombinant plasim pcDNA3.1 (+)-SEMA3B and transfected the recombinant vector into KYSE30 cells, which will provide the foundation for further research.Materials and Method1. Fifity-one specimens resected by surgical intervention were obtained from the Cancer Hospital of Anyang City from March 2007 to December 2007(the same case included esophageal squamous cell carcinoma and their paired tumor-adjacent nontumorous tissues beyond 5 cm), which were identified as esophageal squamous cell carcinoma by pathobiology with sections stained by HE.None of these cases was treated by radiotherapy and chemotherapy.2. RT-PCR and S-P immunohistochemistry were used to determine the expression of SEMA3B in 51 primary ESCC tumors and their paired nontumorous tissues, separately on the level of mRNA or protein.3. The data was analyzed by SPSS 16.0 statistical package,throughχ2-test or Fish exact-test,whose value of test a is 0.05.4. Construct the eukaryotic expression recombinant plasim pcDNA3.1 (+)-SEMA3B, cell culture and transfected the recombinant vector into KYSE30 cells. 5. RT-PCR and western blot were used to detect the expression of pcDNA3.1 (+)-SEMA3B in transfected cell line, separately on the level of mRNA or protein.Result1. In 51 cases of esophageal squamous cell carcinoma, absent expression of SEMA3B mRNA was detected in 30 cases,and the frequency of down-expression is 58.8%. Expression of SEMA3B mRNA was observed in all 51 tested nontumorous tissues.The result showed that there was a significant difference in expressions of SEMA3B mRNA between esophageal squamous cell carcinoma and their paired nontumorous tissues(P<0.05).2. In 51 cases of esophageal squamous cell carcinoma, absent expression of SEMA3B protein was detected in 28 cases. and the frequency of down-expression is 54.9%. Expression of SEMA3B protein was observed in all 51 tested nontumorous tissues. The result showed that there was a significant difference in expressions of SEMA3B protein between esophageal squamous cell carcinoma and their paired nontumorous tissues(P<0.05).3. In 51 cases of esophageal squamous cell carcinoma,the frequecy of SEMA3B protein down-expression in the group with lymphatic metastasis is 79.8%(15/19), which was higher than in the group of no lymphatic metastasis40.6%(13/32), and the difference had statistical significance(P<0.05). The frequecy of SEMA3B protein down-expression in the group with invision all layers is85.7%(24/28), which was higher than in the group of non-invision all layers 17.4%(4/23), and the difference had statistical significance(P<0.05). According to the standard of TNM staging, the frequecy of SEMA3B protein down-expression in the group ofⅢstage was 92.9%(13/14), which was higher than in the group ofⅠstage0%(0/13) andⅡstage46.9%(15/32), and the difference had statistical significance (P<0.05).4. In 51 cases of esophageal squamous cell carcinoma, expression of SEMA3B protein had no statistical significance with gender,age,cell differentiation and general classification (P>0.05).5. Successfully construct the eukaryotic expression recombinant plasim pcDNA3.1 (+) -SEMA3B, and DNA sequence result showed the fragment was identical with human SEMA3B cDNA in GenBank.6. RT-PCR showed expression of SEMA3B mRNA in transfected group was higher than control.7. Western blot showed expression of SEMA3B protein in transfected group was higher than control. Conclusion1. Down-expression of SEMA3B was observed in ESCC. The abnormal expression of SEMA3B significantly correlated with tumor invision, lymph node metastasis, and clinicopathological staging. It is indicated that the abnormal down-regulation expressin of SEMA3B gene may play an significant role in the malignant progression and prognosis of ESCC.2. Successful cloning of SEMA3B gene,construction of pcDNA3.1 (+)-SEMA3B and lasting expression in KYSE30 cell line after transfecting pcDNA3.1 (+)-SEMA3B may provide the foundation for further research.