Inhibition Effect And Epigenetic Regulation Mechanism Of Histone H3K27 Demethylase Inhibitor GSK-J4 On Proliferation,Invasion And Metastasis Of Cervical Cancer Cells

Background and objective:According to the latest global cancer burden data in 2020,cervical cancer ranks fourth in the incidence and mortality of all cancer type in women.In China,the morbidity and mortality of cervical cancer ranks first and second in the world respectively,which seriously threaten the lives and health of women.For advanced cervical cancer cases,the treatment options are limited,and the side effects are serious.It is urgent to explore new therapeutic targets and drugs.Studies have shown that histone H3K27 methylation modification is involved in the mediation of a variety of human tumor cell proliferation,invasion and metastasis and other malignant biological processes.In our previous studies,we found that GSK-J4（a novel specific inhibitor of histone H3K27me3/2 demethylase）can significantly inhibit the proliferation activity,clone formation ability,invasion and metastasis ability of HeLa and Siha cells,but the specific mechanism has not been further studied.On this basis,this study intends to further explore the regulation effect and molecular mechanism of H3K27me3/2demethylation on malignant biological behavior of cervical cancer cells through in vitro cell experiments.Methods:1.Experimental grouping:HeLa and Siha cells were randomly divided into experimental group（treatment with complete medium containing 8.0μM GSK-J4）and control group（treatment with complete medium containing equal volume of solvent DMSO）.2.Flow cytometry was used to detect the effects of GSK-J4 on cell cycle changes and apoptosis of HeLa and Siha cells.3.Gene expressions of cell proliferation,apoptosis,metastasis,EMT and inflammation related genes（Ki-67,Cyclin B1,CDK1,P16INK4A,Bax,Bcl-2,CDH1,CDH2,VIM,ZEB1,ZEB2 and IL-6）m RNA were detected by q RT-PCR.4.Protein levels of JMJD3,UTX,H3K27me1,H3K27me3,Cyclin B1,CDK1,Bax,Bcl-2,Caspase-7,Caspase-3,MMP-1,MMP-2,MMP-9,E-cadherin,N-cadherin and Vimentin were detected by Western Blot.5.Confocal laser technology was used to detects the expression level of Ki-67.6.Chromatin immunoprecipitation assay combined with real-time fluorescence quantitative PCR（Ch IP-q PCR）was used to analysis the enrichment levels of histone H3K27me3 in the promoter regions of target genes MMP-1,MMP-2,MMP-9,Ki-67,P16INK4A,Cyclin B1,ZEB2,Bax,Bcl-2 and IL-6.Results:1.GSK-J4 induce cervical cancer cell cycle arrest:Flow cytometry assay results showed that after GSK-J4 treatment,the percentage of cells in G₂/M phase was significantly increased（p<0.05）,while the percentage of cells in G₀/G₁ phase was significantly reduced（p<0.05）,and there was no significant change in S phase,indicating that GSK-J4 can induce HeLa and Siha cells cycle arrest in G2/M phase.Meanwhile,q RT-PCR results showed that the cell cycle control checkpoints m RNA levels of Ki-67,Cyclin B1,CDK1,and P16INK4A were significantly inhibited after GSK-J4 treatment（p<0.01）.The protein expression levels of Cyclin B1,CDK1 and Ki-67 were further detected by Western Blot and cell immunofluorescence assay.Compared with the control group,the protein expression levels of Cyclin B1,CDK1and Ki-67 in the experimental group were significantly decreased（p<0.05）.2.GSK-J4 promoted cervical cancer cell apoptosis:Flow cytometry was used to detect apoptosis after treatment with GSK-J4.The results showed that GSK-J4 had a strong inhibitory effect on both HeLa and Siha cells,and cell apoptosis was significantly increased（p<0.01）.The expression levels of apoptotic genes Bax and Bcl-2 m RNA were detected by q RT-PCR.After GSK-J4 treatment,the expression level of Bcl-2 m RNA was significantly decreased（p<0.01）,and the expression level of Bax m RNA in HeLa cells was significantly increased（p<0.01）,while the expression level of Bax m RNA in Siha cells was also up-regulated,but the difference was not statistically significant（p>0.05）.The expression of mitochondrial apoptotic pathway related proteins Bax,Bcl-2,Caspase-7 and Caspase-3 were detected by Western Blot,and the results showed that GSK-J4 significantly decreased the expression of Bcl-2（p<0.01）,while increased the expression of Bax,Caspase-7 and the ratio of Cleaved Caspase-3/Pro-Caspase-3（p<0.05）.3.GSK-J4 can inhibit the expression of JMJD3 and UTX protein and simultaneously up-regulate the Global H3K27me3 protein levels:The protein levels of JMJD3,UTX,H3K27me1 and H3K27me3 in the cells were detected by Western Blot.The results showed that the protein levels of JMJD3 and H3K27me1 in HeLa cells were significantly decreased after GSK-J4 treatment（p<0.01）,while the protein levels of UTX were not significantly change（p>0.05）.In Siha cells,JMJD3,UTX and H3K27me1 protein levels were significantly reduced in the experimental group compared with the control group（p<0.05）.concurrently,the levels of H3K27me3 in both HeLa and Siha experimental groups were significantly increased（p<0.05）.4.GSK-J4 inhibited the invasion and metastasis of tumor cells by decreasing the expression of matrix metalloproteinases and reversing the process of EMT in cervical cancer cells:q RT-RCR results showed that GSK-J4 could significantly reduce the m RNA expression of CDH2,VIM,ZEB1 and ZEB2 and increase the expression of CDH1 m RNA（p<0.05）.Western Blot results showed that the expression levels of MMP-1,MMP-9,N-cadherin and Vimentin were significantly decreased after GSK-J4treatment（p<0.05）,while the protein level of E-cadherin protein was significantly increased（p<0.05）.At the same time,GSK-J4 significantly inhibited the expression of MMP-2 protein in Siha cells（p<0.001）.and there was a decrease in the expression of MMP-2 protein in HeLa cells,but there was no statistical difference（p>0.05）.5.Related gene promoter regions H3K27me3 enrichment level analysis:Ch IP-q PCR was used to detect the H3K27me3 enrichment level of related gene promoter regions.the results showed that MMP-1,MMP-9,Ki-67,Cyclin B1,ZEB2,Bcl-2 gene promoter regions,the levels of H3K27me3 were significantly higher than that of control group（p<0.05）,while the MMP-2,P16INK4A and Bax gene promoter region H3K27me3 enrichment level has no obvious change（p>0.05）.6.GSK-J4 inhibits the expression of inflammatory cytokine IL-6:q RT-PCR was used to detect the level of IL-6 m RNA,and the results showed that the level of IL-6m RNA was significantly reduced after GSK-J4 treatment（p<0.01）.Meanwhile,Ch IP-q PCR detected that GSK-J4 could significantly up-regulate the enrichment level of H3K27me3 in the IL-6 promoter region of HeLa cells（p<0.05）,while in Siha cells,the H3K27me3 enrichment level of IL-6 promoter region was also up-regulated,but there was no statistical difference（p>0.05）.Conclusion:GSK-J4 regulates gene expression by up-regulating the H3K27me3 enrichment level in the promoter regions of HeLa and Siha cells,in which related to proliferation,anti-apoptosis,cell cycle and epithelial-mesenchymal transition.Induces cell cycle arrest and apoptosis,and inhibits cell proliferation in vitro.Meanwhile,the cell invasion and metastasis were inhibited by down-regulating the expression of matrix metalloproteinases and reversing the process of EMT.