Effects Of Histone H3K27 Methylation Modification On The Proliferation, Migration And Invasive Ability Of Cervical Cancer Cells

Background and objective:Cervical cancer is the second most common female malignant tumor in China,which seriously threatens the life health and quality of life of women and shows a younger trend.Although early screening and preventive measures have effectively reduced the incidence and mortality of cervical cancer,for patients with advanced and metastatic cervical cancer,the prognosis is still poor after surgery,radiotherapy and chemotherapy,and the 5-year survival rate remains low.In recent years,the study of cancer epigenetics has become the frontier and focus of international scientific research,and it has been found that histone methylation modification as one of the important regulation mechanisms of epigenetics,histone H3K27 methylation imbalance is closely related to the occurrence,development,proliferation and metastasis of many tumors.GSK-J4 is a new specific inhibitor of histone H3K27me3 demethylase,because of its low concentration and reversible modification,it has been studied in many tumor diseases,such as breast cancer,prostate cancer,acute lymphoid leukemia,endometrial cancer and so on,but it has not been reported in cervical cancer.Therefore,in this study,human cervical cancer HeLa and SiHa cells were treated with GSK-J4 to explore the effects of specific blocking histone H3K27me3 demethylation on the proliferation,migration and invasive ability of human cervical cancer cells in vitro,and to analyze the possible molecular mechanism.Methods:1.Experimental grouping: human cervical cancer cells treated with histone H3K27me3 demethylase specific inhibitor(GSK-J4)were set as the experimental group,and human cervical cancer cells treated with equal volume of solvent dimethyl sulfoxide(DMSO)were set as the control group.2.Effect of GSK-J4 on the proliferation activity of human cervical cancer cells was detected by cell counting Kit-8.3.Effect of GSK-J4 on the clonogenesis of human cervical cancer cells was detected by clone formation experiment.4.Effect of GSK-J4 on the migration and invasion ability of human cervical cancer cells was detected by scratch test,Transwell cell migration test and tumor invasion assay.5.Effect of GSK-J4 on the level of histone H3K27me3 in human cervical cancer cells was detected by flow cytometry.6.Effect of GSK-J4 on expression levels of ki-67,CyclinD1 mRNA and matrix metalloproteinases MMP-1,MMP-2,MMP-9mRNA in human cervical cancer cells was determined by real-time fluorescence quantitative PCR.Results:1.The results of CCK-8 assay showed that after HeLa cells were treated with different concentrations(0.25,0.5,1.0,2.0,4.0)μM GSK-J4 for 48 hours and 72 hours,the cell proliferation activity was inhibited at the concentration of 0.5μM,and the differences were statistically significant compared with the control group(all p<0.05);After SiHa cells were treated with different concentrations(0.5,1.0,2.0,4.0,8.0)μM GSK-J4 for 48 hours and 72 hours,the proliferation activity was inhibited at the concentration of 4.0μM,and the differences were statistically significant compared with the control group(all p<0.05),the results showed that GSK-J4 could inhibit the proliferation of cervical cancer cells.2.The results of cell plate cloning formation experiment showed that after HeLa cells were treated with 0.5μM GSK-J4 for 72 hours,the number of cell clones in the experimental group(142.30±3.67)was less than that in the control group(173.30±5.36),and the difference was statistically significant(p<0.05);After SiHa cells were treated with 4.0μM GSK-J4 for 72 hours,the number of cell clones in the experimental group was(16.00±4.36),compared with that of the control group(33.67±6.03),the number of cell clones decreased,and the difference was statistically significant(p<0.05),the results show that GSK-J4 could inhibit the ability of cervical cancer cells to form clones.3.The results of scratch test showed that after HeLa cells were treated with 0.5μM GSK-J4 for 48 hours,the scratch healing rate of the experimental group was 31.80±1.33%,which was lower than that of the control group(39.27±0.91%),the difference was statistically significant(p<0.05);After treated with 4.0μM GSK-J4 for 48 hours,the scratch healing rate of SiHa cells in the experimental group was 46.99±0.69%,which was significantly lower than that of the control group(81.49±0.14%),the difference was statistically significant(p<0.05),the results showed that GSK-J4 could effectively inhibit the migration of cervical cancer cells.4.The results of Transwell cell migration assay showed that after HeLa cells were treated with 0.5μM GSK-J4 for 48 hours,the number of perforated cells in the experimental group was(18.00±0.93),which was less than that in the control group(34.43±2.72),the difference was statistically significant(p<0.05);After SiHa cells were treated with 4.0μM GSK-J4 for 48 hours,the number of perforated cells in the experimental group was(54.57±1.55),which was significantly less than that in the control group(71.53±0.47),the difference was statistically significant(p<0.05).The results of Transwell cell invasion assay showed that after HeLa cells were treated with 0.5μM GSK-J4 for 48 hours,the number of perforated cells in the experimental group was(11.17±0.35),which was less than that in the control group(19.00±0.70),the difference was statistically significant(p<0.05);After SiHa cells were treated with 4.0μM GSK-J4 for 48 hours,the number of perforated cells in the experimental group was(29.77±0.83),compared with that of the control group(50.63±2.40),the number of perforated cells was reduced,and the difference was statistically significant(p<0.05),the results showed that GSK-J4 could effectively inhibit the migration and invasion of cervical cancer cells.5.The results of flow cytometry showed that after HeLa cells and SiHa cells were treated with 0.5μM and 4.0μM GSK-J4 for 72 hours,the relative fluorescence intensity of histone H3K27me3 in the experimental group was 1.63±0.12 and 1.10±0.03,respectively,and the differences were statistically significant compared with that of control group 1(both p<0.05),the results showed that GSK-J4 could up-regulate the level of histone H3K27me3 in cervical cancer cells.6.The results of real-time fluorescence quantitative PCR showed that after HeLa cells were treated with 0.5μM GSK-J4 for 48 hours,the expression levels of MMP-1mRNA and MMP-9 mRNA in the experimental group were down-regulated,and the differences were statistically significant compared with the control group(both p<0.05),after 72 hours of treatment with the same concentration,the expression levels of Ki-67 mRNA and CyclinD1 mRNA in the experimental group were down-regulated,and the differences were statistically significant compared with the control group(both p<0.05);After SiHa cells were treated with 4.0μM GSK-J4 for 48 hours,the expression levels of MMP-2 mRNA and MMP-9 mRNA in the experimental group were down-regulated,and the differences were statistically significant compared with the control group(both P<0.05),after 72 hours of treatment with the same concentration,the expression levels of Ki-67 mRNA and CyclinD1 mRNA in the experimental group were down-regulated,and the differences were statistically significant compared with the control group(both P<0.05).Discussion:In this study,human cervical cancer HeLa and SiHa cells were treated with low concentration of small molecular inhibitor GSK-J4,the results of in vitro experiments showed that up-regulation of intracellular histone H3K27me3 level could inhibit the proliferation,migration and invasion of cervical cancer cells,the mechanism may be related to the up-regulation of H3K27me3 level,resulting in transcriptional inhibition of Ki-67,CyclinD1 and MMP-1,-2,-9 genes.The preliminary results showed that the methylation level of histone H3K27 was related to the proliferation and metastasis of cervical cancer.