The Biological Effect And Mechanism Of Paired Genes Box 2(PAX2) In Esophageal Squamous Carcinoma Cells

Part one :The expression of PAX2 in esophageal squamous carcinoma(ESCC)tissues and its clinical significance Objective:Esophageal squamous cell carcinoma(ESCC) is one of the deadliest malignancies worldwide. To study the expression of paired genes box 2(PAX2) in esophageal squamous cell carcinoma tissue. Methods:Detected 15 cases of normal esophageal tissue samples by immunohistochemical staining method, next to 52 cases of esophageal squamous cell carcinoma and 120 cases of esophageal squamous cell carcinoma(ESCC) protein expression levels of PAX2 and observe patients with esophageal squamous cell carcinoma age, gender, tumor differentiation, lymph node metastasis, TNM stage and lymphatic invasion relationship. Results:PAX2 expression was significantly increased in tumor tissues and that its expression correlated with the ESCC stage(P = 0.001), lymph node metastasis(p N, P = 0.019) and lymphatic invasion(P = 0.005) in 120 ESCC tissue specimens. Conclusion: PAX2 expression in esophageal squamous cell carcinoma was significantly higher than normal esophageal tissue adjacent tissues and corresponding cancer, and with TNM stage and lymph node metastasis and lymphatic invasion or correlated.Part two :Functional characterization of PAX2 in esophageal squamous carcinoma cells Objective:To investigate the function of paired genes box 2(PAX2) in esophageal squamous cell carcinoma cell lines. Methods:ESCC derived TE-1 and Eca-109 cell lines were used. Cell viability was measured by MTT assay. Proliferation and clongenic formation was determined using colony formation wassay. Cell invasion was measured using Transwell chamber-based assay. Radiosensitivity of ESCC cells was determined by clongenic assay after irradiation of various doses. Protein expression was analyzed by Western blot using respective antibody. Results:PAX2 overexpression resulted in markedly reduced cell proliferation but increased metastasis capacity in ESCC TE-1 and Eca-109 cells. Knockdown of PAX2 expression with a short hairpin RNA confirmed a role in the promotion of metastasis in ESCC cells. PAX2 induced MMP-2 and MMP-9 expression, which are metastatic markers of cancer cells. Moreover, silencing of PAX2 resulted in increased radiosensitivity in both TE-1 and Eca-109 cells. Conclusion: PAX2 promotes the metastasis and radiosensitivity but inhibits the proliferation of ESCC cells.Part three :The mechanism of PAX2 in transcriptional regulation in esophegeal cancer cells Objective:To investigate the transcriptional network of PAX2 and identifiy new downstream effectors of PAX2. Methods:TE-1 cells were transfected with pc DNA3.1 or PAX2 overexpression vector. m RNA profiling was measured by microarray analysis. The upstream promoter of IL-5 was cloned into the p GL3- Basic vector and luciferase assay was measured by dual luciferase assay kit. RT-PCR was used to confirm m RNA expression level. Chromatin immunoprecipitation(Ch IP) was used to detect the binding between PAX2 and IL-5 promoter. Results:m RNA microarray screening revealed that PAX2 overexpression affected multiple genes that function in multiple pathways. Interleukin-5(IL-5), which was induced by PAX2 and has been shown to promote tumor metastasis, was further studied in greater detail. Two PAX2 binding sites were identified in the IL-5 promoter, and PAX2 was observed to stimulate IL-5 promoter activity and IL-5 expression in esophageal cancer cells. Chromatin immunoprecipitation(Ch IP) confirmed the direct binding of PAX2 in the IL-5 promoter. The expression of PAX2 m RNA significantly correlated with that of IL-5 in normal esophageal and ESCC tissues. Conclusion: We demonstrated the overexpression of PAX2 in esophageal carcinoma and identified IL-5 as PAX2’s effector for metastasis.