Inhibit LSD1 Affect Notch3 Signaling Pathways In Esophageal Squamous Carcinoma

Aim 1.To investigate the expression of LSD1 in esophageal squamous cell cacinoma cell lines and the regulatory relationship between LSD1 and Notch3 signaling pathway.2.To explore the effects of new synthetic LSD1 inhibitor on the proliferation of esophageal squamous cell cacinoma cells and the possible molecular mechanism.Methods 1. The protein expressions of LSD1 and the factors in Notch pathway were detected in five esophageal squamous cell carcinoma cell lines with different degrees of differentiation by Western blots.2. The m RNA expressions of LSD1 in esophageal squamous carcinoma cell lines were detected by Real-Time PCR.3. The effects of the new synthetic LSD1 inhibitor on proliferation in ECa109 and TE-1 cells were detected by CCK-8.4. The protein expressions of LSD1, Notch3 and CSL were detected in ESCC cells which were treatmented with different times or different concentrations of LSD1 inhibitor by Western blots.5. The histone expressions were detected in ECa109 cells which were treatmented with different concentrations of LSD1 inhibitor by Western blots.6. The histone and the protein expressions of Notch pathway were detected in ECa109 cells which were treatmented with the inhibitor of Notch pathway GSI or GSI in combination with LSD1 inhibitor by Western blots.7. The protein expressions of Notch3 and Bcl-2 were detected in Eca109 cells which was transfected with LSD1-sh RNA by Western blots.Results 1. The protein expression of LSD1 and Notch signaling pathway in the esophageal carcinoma cell lines do exist differences. The protein of LSD1 and Notch3 that present positive correlation have high expression in ECa109 cell.2. The m RNA expression of LSD1 in the esophageal carcinoma cell lines also do exist differences.3. LSD1 inhibitor inhibited the cell viability of esophageal squamous cell carcinoma cells.4. The new synthetic LSD1 inhibitor down-regulated the protein expression of Notch3 which affected the function of LSD1 by increasing the expression of the histone H3K9me2 on a dose-dependent manner.5. GSI inhibited the protein expression of Notch3, LSD1 and Bcl-2.6. The LSD1 inhibitor combination with GSI obviously inhibited the expression of Notch3 and LSD1 and the histone H3K4me2.7. The expression of Notch3 and Bcl-2 were down-regulated after Eca109 cells were transfected with LSD1-sh RNA.Conclusions 1. LSD1 overexpressed in esophageal squamous cancer cells,but its expression has no correlation with cell differentiation.2. The new synthetic LSD1 inhibitor can inhibit the proliferation of esophageal squamous cell carcinoma cells.3. Notch3 signaling pathway can be regulated by LSD1: The expressions of Notch3 were down-regulated by the suppression of LSD1 in the situation of the activation or deactivation of Notch3 signaling pathway. When Notch3 pathway is activated, LSD1 may make it possible by promoting the demethylation of H3K9me2, on the other side, achieving through stimulating the demethylation of H3K4me2.