ABSTRACT
Objective To construct the Hut78 cell line with EZH2 gene knocked into by CRISPR/Cas9 system. Methods The EZH2 expression vector pMD-18T-EZH2 with homologous arm and the sgRNA expression vector pSpCas9 (BB)-2A-Puro-sgRNA, which could cut the double stranded genomic DNA, were constructed, and the two vectors were co-transfected into Hut78 cells. Then the expression of EZH2 mRNA was detected by qPCR, and the expressions of EZH2 and H3K27me3 proteins were detected by Western blot assay. Results The pMD-18T-EZH2 and pSpCas9(BB)-2A-Puro-sgRNA recombinant vectors were confirmed by DNA sequencing. When Hut78 cells were transfected with the two recombinant plasmid, qPCR results showed that the expression of EZH2 mRNA was significantly increased, and Western blot analysis showed that the expressions of EZH2 and H3K27me3 proteins were significantly increased. Conclusion EZH2 gene is successfully knocked into Hut78 cells by CRISPR/Cas9 system.
ABSTRACT
Objective To investigate the effects of siRNA silencing enhancer of zeste homolo 2 (EZH2) on invasion and migration of cervical cancer cells and the molecular mechanisms.Methods The small interfering RNAs (siRNA) targeting EZH2 were transiently transfected into C33A cell line by lipofectamin2000.The effects of EZH2 on cell invasion and migration were detected by wound-healing assay, Transwell assay and soft agar colony assay.The expression of MMP2 was detected by Western blot.Results Downregulation of EZH2 expression by siRNA in C33A cell line significantly inhibited cell invasion and migration in vitro.Meanwhile, siRNA-mediated depletion of EZH2 reduced the expression of MMP2.Conclusion Knocking down EZH2 expression by siRNA could surpress invasion and migration of human cervical cancer cells, which might be related to downregulating MMP2 expression.
ABSTRACT
Objective To investigate the inhibitory effects of silencing expression ofEZH2 gene on the cell proliferation of human QBC939 cells and its mechanisms. Methods The targeting siRNA was designed and trans-fected into QBC939cells. The expressions of EZH2 mRNA and protein were detected by real-time qPCR and west-ern blotting,respectively. The ability of cellproliferationwas analyzed by MTT assay and plate clone formation assay. Cell apoptosis and cycle percentage weremeasured by flow cytometry. Cell senescence was assessed byβ-galactosi-dase dyeing.The expressions of H3K27me3,P14ARF,P16INK4a,P53,P21 and E2F1 proteinwere determined by West-ern blotting.Results Compared with the control group ,the expressions of mRNAand protein were significantly elevat-ed in experimental group. The ability of cellproliferation in the experiment group was significantly down regulated , which could also cause a rise of G1/S phase ,but not a marked variation of apoptosis rate. Silencing EZH2 would induce a obvious senescence phenotype in QBC939 cells. EZH2-siRNA transferredcould also down-regulate the expressions of H3K27me3 and E2F1 protein,while up-regulating the expressions of P14ARF,P16INK4a,P53 and P21 protein in QBC939 cells.Conclusions Silencing EZH2 could induce a significant inhibition on cell proliferation of QBC939 cells,the mechanism of which may be associated with the senescence pathway regulation.
ABSTRACT
Objective: To study the expressions of enhancer of zeste homolog 2 (EZH2) and deleted in liver cancer-1 (DLC1) in breast cancer tissues and cell lines, and to explore their relationship. Methods: The expressions of EZH2 and DLC1 proteins in 120 cases of breast cancer tissues and 63 cases of para-cancerous tissues were detected by immunohistochemistry. The expression levels of EZH2 and DLC1 mRNAs and proteins in 20 cases of breast cancer tissues, 20 cases of the corresponding para-cancerous tissues, breast cancer MCF-7 and MDA-MB-231 cells and the normal mammary epithelial HBL100 cells were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The specific siRNA targeting EZH2 gene was transfected into MDA-MB-231 cells by liposome, then the expression levels of EZH2 and DLC1 mRNAs and proteins in MDA-MB-231 cells were detected again by realtime fluorescent quantitative PCR and Western blotting, respectively. Results: The positive expression rate of EZH2 in breast cancer tissues was significantly higher than that in corresponding para-cancerous tissues (P = 0.000). The positive expression rate of DLC1 in breast cancer tissues was significantly lower than that in corresponding para-cancerous tissues (P = 0.008). The expression rates of EZH2 and DLC1 were significantly correlated with tumor size and lymph node metastasis (both P < 0.05). The expression levels of EZH2 mRNA and protein in breast cancer tissues and breast cancer MCF7 and MDA-MB-231 cells were signifiicantly higher than those in corresponding para-cancerous tissues and normal breast epithelial HBL100 cells (all P < 0.01), but the expression levels of DLC1 mRNA and protein were lower than those in corresponding para-cancerous tissues and normal breast epithelial HBL100 cells (all P < 0.01). After EZH2 gene-silencing, the expression levels of DLC1 mRNA and protein in MDA-MB-231 cells were increased (both P < 0.05). Conclusion: There is a negative correlation between the expressions of EZH2 and DLC1 in breast cancer, and the inhibition of EZH2 expression can restore the expression of DLC1; which suggests that EZH2 maybe promote the occurrence and development of tumor by inhibiting the expression of DLC1.