Histone Methyltransferase G9a And H3K9Dimethylation Inhibit The Self-renew Of Glioma Cancer Stem Cells

Cancer stem cells in tumor tissues are a small group of self-replicating andmulti-directional differentiation potential of special cells, replicating itself is the maincharacteristic of stem cells. Epigenetic regulation of self-replicating maintenance plays animportant role for stem cells, histone modification seems to be an important obstacle betweenstem cells and differentiated cells. In mammalian cells of H3K9methylation is one of themain modify tag cause inhibition of gene expression, G9a methyltransferase (histone lysine Nend) is the single catalytic histone H3K9methylation and methylation of two major enzymes,H3K9methylation (especially H3K9methylation) in maintaining self-replicating aspects therole of cancer stem cell remains unclear. This study mainly source of glioma cancer stem cellsas the research target, the system inquiry G9a and H3K9me2modification in the effect andmechanism of copies of themselves.This study first9cases of paraffin wax preservation of patients with gliomashistopathologic H3K9me2expression in the organization of chemical research, found thatmost gliomas (8cases) the organization of the tumor cells were tested positie for H3K9me2expression, the positive cells rate is40%; Have1case detection for H3K9me2expressnegative. Second we have9cases of pathological tissue samples of CD133(cancer stem cellmarker) expression in also has carried on the immunohistochemical detection, found that mostorganizations (8cases) expression of CD133negative, Only1case expression is positive,positive cells rate is3.82%; The positive rate of patients was H3K9me2express negativecases. This phenomenon shows that H3K9me2modification in self-replicating regulating tumor stem cells may be an important part of the switch. Third, in order to further test whethertumor stem cells is H3K9me2modification degree is low, we are out of clinical fresh gliomaspecimens of immunofluorescence double dye and westernblot detection, found H3K9me2cells and CD133exist a lot of can’t dye; And westernblot detection showed a similar trend ofboth the expression of mutual antagonism, namely H3K9me2high expression of specimenshows low CD133expression, H3K9me2lower expression of specimens is high expression ofCD133.Fourth, we in the training system of glioma stem cells into a ball, to the G9a andH3K9me2to replicate and marks the expression of CD133/Sox2stem cells were studiedsystematically. We use G9a inhibitors bix01294to suppress G9a enzyme function andH3K9me2, and use of G9a expression plasmid transfection method increases the expressionof G9a, from two aspects of function loss and gain. The results found that specific inhibitorsof G9a BIX01294can promote into a ball the self-replicating culture under the condition ofglioma cells, and stem cell molecular Sox2and the expression of CD133increases; Whereasoverexpression of G9a inhibits glioma cells self-replicating, promoted the expression ofH3K9me2, at the same time reduce the expression of CD133.Fifth, to further validate G9a’s impact on the number of CD133expression and tumorstem cells, we use flow cytometry (FC) to detect the number of CD133positive cells. Theresults found that bix01294number of CD133positive cells in the group processingproportion (31%) was obviously higher than normal control group (51%).Sixth, to test whether H3K9me2modifiers do occur in the promoter region of CD133and Sox2, we conducted chromatin (ChIP), immune coprecipitation experiment withH3K9me2specific antibody precipitation was modified DNA fragments, and then use theCD133and Sox2promoter region specific primers for quantitative PCR detection. Found thatbix01294after processing, CD133and Sox2promoter of PCR amplification significantlyreduced, Sox2amplification enhancer were reduced, suggests that this area H3K9me2inhibit lower.Through a series of experiments above, the conclusion is, G9a and H3K9me2maythrough direct inhibition of CD133and Sox2promoter region, self-replicating of gliomacancer stem cells and CD133, Sox2expression inhibition.