Regulation Of Histone H3K9Methylation On The Selfrenew Of Glioma Cancer Stem Cells

Objective: Cancer stem cells are a small subpopulation of multipotential cells in thecancer tissue which can selfrenew. Histone3methylation is one of the main regulatorymechanisms of selfrenew and tumorigenecity of the cancer stem cells. Several lab’s worksuggest that the histone3lysine9may contribute to the transition of cancer stem cells fromthe resting state to the selfrenew state and the transition of cancer cells to cancer stem cells.But little is know of the precise mechanism of these events. We are trying to explore that howthe histone3lysine9methylation contributes to the selfrenew.We are also interested in howthe histone3lysine9crosstalk with the other cancer stem cell related molecules such asCD133and Sox2.Methods: Three inhibitors were used in this study, the5-AZ (5-azacytidine), thedemethylating agent; the Bix01294, the inhibitor of G9a-HMT (Histone H3lysine9histonemethyltransferase); and Sodium butyrate, the histone deacetylase inhibitor. NeurosphereClone formation assay was used to test the selfrenew of the cancer stem cells. Soft agaroseclone culture method was used to test the tumorigenecity of the cancer stem cells. The gliomacell line U87and U251cells were cultured in the serum free sphere formation medium to letthe cancer spheres form， with or without the above inhibitors. Real-TimePCR, RT-PCR andWestern blot were used to test the mRNA and protein expression of the cancer stem cellmarker CD133,Sox2etc. ChIP assay was used to confirm the direct inhibition of H3K9me2methylation on the promoter region of CD133and Sox2. Immunohistochemical comparativestudy was also performed between the H3K9me2and CD133expression levels on clinical glioma pathology specimens.Results：(1) The glioma cell line U87and U251cells were cultured in the serum freesphere formation medium to let the cancer spheres form. After cultured for8-10days, both theBix01294and Sodium butyrate treated cells formed more spheres, and the Bix01294plusSodium butyrate group formed the most. But the5-AC treatment caused cell death and fewsphere formation.(2) Real-TimePCR and Western blot results shown the similar active effectson the CD133expression. The Bix01294and Sodium butyrate treatment results theup-regulation of CD133expression.(3)For the soft agarose clone formation experiment, theBix01294and Sodium butyrate treatment also results an enhancement of the cloneformation.ChIP assay suggested that the promoter region of Sox2and CD133were inhibitedby H3K9me2methylation.(4).Immunohistochemical research found that CD133andH3K9me2expressed reversely (did not expressed in the same) in glioma specimens.Conclusions: Histone H3K9methylation inhibits the expression of Sox2and CD133.The inhibitors of G9a (Bix01294) promote the selfrenew of glioma stem cells through theremoving the H3K9methylation suppression on the Sox2.