Identify The Mechanism Of EZH2 Gene Promote The Growth Of Bladder Cancer

Objective Explore the mechanism of EZH2 promote the growth of bladder cancer.Methods T24 and 5637 human bladder cancer cell lines were treated in transient transfection knock down of EZH2 gene, high expression or inhibition of miRNA-143. Cell viability assay was used to study the effect of EZH2 and miRNA-143 on T24 and 5637 human bladder cancer cell lines. Colony-formation assay was used to examine bladder cancer cell growth with siEZH2, high expression or inhibition of miRNA-143. Real-time fluorescence quantitative nucleic acid amplification detection system (Q-PCR) was used to detect changes in RNA levels of treated bladder cancer cells. To verify the miRNA-143 is a target of EZH2 gene, chromatin immunoprecipitation (CHIP) was used to detect the binding situation of EZH2 protein and miRNA-143. .Extrcting the protein of T24 and 5637 cell treated by transfection knock down of EZH2 gene, high expression or inhibition of miRNA-143, western blot assay was used to determine the protein level of CyclinD1、CD44、Notch-1、Sox-2、EZH2 and its target protein H3K27me3.Results In Vitro, T24 and 5637 bladder cancer cells were treated with siEZH2 and cell viability was assayed at 24,48 and 72 hours using cell viability assays. Treatment with siEZH2 resulted in growth was inhibition. Clonogenic assay results in inhibition in clonogenicity formation. Western blot analysis indicated that treatment of T24 and 5637 bladder cancer cell with siEZH2 resulted in a decreased expression of CD44,Notch-1,Sox-2,EZH2 and its target protein H3K27me3, the expression of cell cycle protein CyclinD1 also decreased.In Vitro, T24 and 5637 bladder cancer cells were treated with siEZH2 and QPCR Shows with knock down of EZH2 in T24 and 5637 bladder cancer cells, the expression of miRNA-143 and miRNA-145 was increased. Our previous studies have confirmed herbal extracts honokiol (HNK) can suppress the growth of bladder cancer T24 and 5637 by reducing EZH2, bladder cancer cell T24 and 5637 were treatment with HNK of 24 hours, and detection of the expression of miRNA-143 through Q-PCR, The results showed a significant increase in expression of miRNA-143 compared with the control group. Then we use CHIP experiments to detect how EZH2 do in miRNA-143, the experiment showed that EZH2 through binding to the promoter region to interfere with the expression of miRNA-143.In Vitro, T24 and 5637 bladder cancer cell lines were treated in transient transfection high expression of miRNA-143 (mimic miR-143). Cell viability showed that compared to the control group high expression of miRNA-143 can inhibit the growth of T24 and 5637. Clonogenic assay results in inhibition in clonogenicity formation of high expression of miRNA-143. Western blot analysis indicated that treatment of T24 and 5637 bladder cancer cell with high expression of miRNA-143 resulted in a decreased expression of cancer stem cell marker protein CD44,Sox-2, the expression of cell cycle protein CyclinD1 also decreased.In Vitro, T24 and 5637 bladder cancer cell lines were treated in transient transfection with siEZH2 or inhibition of miRNA-143. Cell viability showed that compared to the control group transfection with siEZH2 group inhibit the growth of T24 and 5637, but Simultaneously knocking down EZH2 and inhibition of micRNA-143 can stimulate the growth of T24 and 5637. Clonogenic assay results were smilar to Cell viability. Western blot analysis indicated that compare to knocking down EZH2 group alone, joint with inhibition of miRNA-143 resulted in a high expression of cancer stem cell marker protein CD44,Sox-2, the expression of cell cycle protein CyclinD1 also increased. Prompts EZH2 induced proliferation changes can be reversed by its downstream micRNA-143.Conclusion EZH2 can directly inhibit miRNA-143 and promote the growth of bladder cancer cells.