Expression Of HER4 In Esophageal Carcinoma And Effects Of Expression Silencing Of HER4 By RNA Interference On Growth And Invasion Of Esophageal Carcinoma Cells

Part I Expression of HER4 in Esophageal Carcinoma and Its Relationshipwith Metastasis-related ProteinsObjective: To investigate the expression of HER4 in human esophageal carcinoma tissues, and its relationship with metastasis-related proteins—MMP-9, VEGF, E-cadherin and CD44v6.Methods: Immunohistochemical Envision technique was applied to detect the expressions of HER4, VEGF, E-cadherin, and CD44v6 in 45 specimens of esophageal carcinoma tissues.Results: The positive rate of HER4, CD44v6, VEGF, MMP-9 and E-cadherin expression was 73.3%, 68.9%, 62.2%, 42.2%, 22.4% respectively.; the expression of HER4 was correlated with TNM stage and lymph node metastasis(P<0.01), but was not correlated with histological grade (P>0.05); the expressions of CD44v6 and VEGF was also correlated with TNM stage and lymph node metastasis(P<0.05), but was not correlated with histological grade either(P>0.05); the expression of MMP-9 was correlated with invasion depth, TNM stage and lymph node metastasis(P<0.05); the expression of E-cadherin was reversely correlated with lymph node metastasis(P<0.05). The expression of HER4 in esophageal carcinoma tissues was related with the expressions of MMP-9, CD44v6, VEGF.Conclusion : The expression of HER4, MMP-9, CD44v6 and VEGF was correlated with TNM stage and lymph node metastasis of the carcinoma; further more, the expression of MMP-9 was also related with invasion depth; the over-expression of HER4 in esophageal carcinoma tissues was correlated with the expressions of MMP-9, CD44v6 and VEGF. Part II Vector Construction and Silencing Effect of HER4-Targeted SmallInterfering RNAObjective: To construct HER4-targeted small interfering RNA and its expression vector, then to observe its silencing effect in human esophageal carcinoma cell line Eca-109.Methods: Two complementary oligo DNA strands targeting HER4 gene were designed and synthesized according to the principles of designing siRNA. After annealing, oligo DNAs were inserted into pSUPER/Neo/GFP vector, then enzyme digestion analysis and DNA sequencing were performed. After transfecting it into human esophageal carcinoma cell line, we detected the level of expression of HER4 gene by real-time quantitative PCR and Western Blot assay to pick out the most effective siRNA and vector.Results: The enzyme digestion analysis and DNA sequencing showed that HER4-targeted small interfering RNA and its expression vector were constructed successfully. The results of real-time quantitative PCR and Western Blot showed that NO.2 siRNA and vector was the most effective one to silence HER4 gene, which was indicated by the lowest level of mRNA and protein of HER4 after it was transfected into Eca-109 cell line.Conclusion: HER4-targeted small interfering RNA and its expression vector were constructed successfully, and could depress the expression of HER4 gene in Eca-109 cell line, which laid the foundation for the following experiment. Part IE The Impact of HER4 -Targeted siRNA on the Expression of MMP-9, VEGF and CD44v6 In Vitro ExperimentObjective: To observe the impact of HER4-targeted siRNA on the expressions of MMP-9, VEGF and CD44v6 proteins in esophageal carcinoma cell line Eca-109, which are believed to be metastasis-related proteins.Methods: Eca-109 cells were divided into 4 groups: blank control group (nothing added), blank vector group (blank vectors added), non-specific interference group (non-specific vectors added) and specific interference group (the most effective vectors added). 24, 48, 72h after transfection, the expressions of MMP-9, VEGF and CD44v6 were detected by Western Blot assay.Results: 24,48, 72h after transfection, the expressions of MMP-9 and VEGF were suppressed remarkably in the specific interference group, compared with the blank control group, and the difference was significant statistically. Significant difference was not found between the blank vector group and the blank control group. The same result was shown between the non-specific interference group and the blank control group. 48h after transfection, the lowest level of the expression of both proteins was observed, indicating the greatest suppressive effect, with the suppressive ratio 56.8% and 32.3% respectively. Otherwise, significant change of the expression of CD44v6 was not observed in the specific interference group after transfection.Conclusion: The results of the in-vitro experiment indicated HER4-targeted siRNA and its vector had the ability of suppressing the expression of MMP-9 and VEGF in Eca-109 cell line, which implied that HER4 gene is related with the expression of MMP-9 and VEGF in esophageal carcinoma. HER4 might play a role in the modulating process of the two proteins and then affect the invasive and metastatic ability of esophageal carcinoma. Part IV The Impact of HER4 Gene Targeted siRNA on the Growth andInvasive Ability of Eca-109 Cell LineObjective: To observe the impact of HER4-targeted siRNA on the growth, viability, apoptosis and invasive ability of Eca-109 cell line, explore and discuss possible mechanisms of it.Methods: Eca-109 cells were divided into 3 groups: blank control group (nothing added), non-specific interference group (non-specific vector added) and specific interference group (the most effective vector added). After transfection, cell viability was determined by MTT assay, cell apoptosis was assessed by nuclear staining with Hoechst 33258 observed through fluorescence microscope, the invasion ability of Eca-109 cells was detected by Transwell chemoinvasion assay.Results:①Compared with the other two groups, the cell viability of specificinterference group was suppressed 48h after transfection, and the level of viability was about 23 % lower 5 days later. It confirmed that the siRNA could silence HER4 gene effectively, and the difference was significant statistically between the specific interference group and the control groups (P<0.05).②Nuclear staining with Hoechst 33258 showed moderate cell apoptosis in the specific interference group while the other two groups showed seldom.③Transwell chemoinvasion assay showed that the count of cells that penetrated membrane of Transwell chamber in the specific interference group was much less than those of the other two groups, both of the comparations had significant difference (P<0.05) .Conclusion: HER4-targeted siRNA could inhibit the viability and proliferation of Eca-109 cells obviously, further more, it also had the ability of inducing cell apoptosis in Eca-109 cells and suppressing the invasive ability of the cells.