Differential Expression Of Tumor Suppressor Gene Methylation And Its Relationship With DNMT1 In Esophageal Squamous Cell Cancer Cell Lines

Objective:To applicate the targeting DNA methyltransferase 1(DNMT1) gene’s short hairpin RNA(shRNA) recombinant plasmid was used to transfect the esophageal squamous cell carcinoma cells, and the nude mice xenograft animal model was established to study the proliferation, metastasis and invasion of esophageal squamous cell carcinoma cells. Methods:1.The experimental group was given shRNA-DNMT1 sequences for transfection, the control group was only given negative control shRNA-NC sequence, we constructed lentiviral gene silencing DNMT1 and obtained stable cell lines infected with esophageal squamous cell carcinoma cells, established an animal xenograft model in nude mice. 2.Cell Counting Kit-8(CCK8) was used to evaluate cell growth of ESCC cells. 3.Colony formation assays were used to evaluate forming ability of colony formation. 4.Transwell assays were used to evaluate invasion and metastasis of tumor cells. 5.Detection apoptosis and cycle of tumor cells with flow cytometry. 6.Quantitative real-time PCR(Real-time PCR) was used to detect mRNA expression of suppressor gene P16, CDH13, RASSF1 A, APC, MGMT, ASC, DAPK and DNMT1 in esophageal squamous cell carcinoma cells and xenograft tumor. 7.The methylation specific PCR(MSP) was used to detect methylation status of suppressor gene RASSF1 A and DAPK in esophageal squamous cell carcinoma cells and xenograft tumortissue. 8.Western blot was used to detect the expression of tumor suppressor gene RASSF1 A and DAPK regulated proteins and DNMT1 protein in esophageal squamous cell carcinoma cells and xenograft tumor tissues. 9.Immunohistochemical was used to detect DAPK and RASSF1 A regulated proteins in xenograft tumor tissues. Results: 1.The recombinant lentivirus of silence gene DNMT1 was successfully constructed and transfected to esophageal squamous cell carcinoma cell(KYSE150、KYSE410、KYSE450)for obtaining stable cell lines. 2.Compared with the control group, the results of qRT-PCR showed that in esophageal squamous carcinoma cells and xenograft tumors, the expression of DNMT1 mRNA reduced significantly in the experimental group, while the mRNA expression of RASSF1 A and DAPK increased significantly(P<0.01). 3.Western blot showed that in the esophageal squamous cell carcinoma cells and xenograft tumors, the levels of DNMT1 protein were reduced significantly in experimental group, while the levels of protein expressed RASSF1 A and DAPK increased significantly(P<0.05). 4.CCK test showed that cell proliferation slowed significantly in the experimental group(P<0.05). 5.Colony formation assays showed the number of colonies of cells in the experimental group was significantly reduced(P<0.01). 6.Transwell Experiment showed that the ability of invasion and metastasis of esophageal squamous cell carcinoma decreased significantly in experimental group(P<0.01). 7. Flow cytometry results showed that: compared with the control group, the percentage of apoptotic cells increased in experimental group of esophageal tumor cells(P<0.01), focused on the early stages of adjustment; compared with the control group, the experimental group esophagus tumor cells showed the following changes: the percentage of cells increased during the early DNA synthesis stage(G0 / G1 stage)(P <0.001), the percentage of cells reduced during DNA synthesis stage(S stage)(P <0.001), the percentage of cells decrease during the late DNA synthesis stage(G2 / M stage)(P <0.001). 8.Immunohistochemistry showed that the expression of RASSF1 A and DAPK protein was increased significantly in the experimental group(P<0.05). 9.Methylation-specific PCR(MSP) showed the methylation of RASSF1 A and DAPK was inhibited in esophageal squamous cell carcinoma cells and xenograft tumors. Conclusion: Silencing DNMT1 inhibits proliferation, metastasis and invasion in ESCCby modulatingmethylationof RASSF1 A andDAPK.