Effect Of EZH2 On Biological Characteristics Of HPV16 Positive Cervical Cancer Cell Lines

Objective:Enhancement of zeste homolog 2(EZH2),an important member of the Pc G family(poly-comb group)of epigenetic repressors,can modify the methylation of nucleosomal histone H3K27.Methylation of genes can inactivate or transcriptionally repress certain genes,while demethylation can induce gene activation or re-expression.Therefore,methylation level can affect the silencing of downstream oncogenes,overexpression of oncogenes and aggravate the defects in DNA repair mechanism,which eventually leads to tumorigenesis and plays a key role in the proliferation,differentiation and inhibition of apoptosis of cancer cells.In this experiment,we intend to use HPV16 positive cervical cancer SiHa cell line to construct an EZH2 low expression cell model to investigate the effect of EZH2 on the biological properties of SiHa cells,in order to further the understanding of cervical cancer at the molecular biology level,and to provide ideas for exploring the potential molecular mechanisms of malignant biological properties of cervical cancer,relevant therapeutic targets and predicting prognosis.Methods:In this experiment,EZH2 siRNA and negative control siRNA were transfected into SiHa cells,and the transfection rate of EZH2 was detected by qRT-PCR,and the functional level of EZH2 protein was determined by Western Blotting.The effect of knockdown of EZH2 on the invasive ability of SiHa cells was examined by transwell assay,and the effect of low expression of EZH2 on apoptosis was examined by flow cytometry.All statistical analyses were performed by SPSS 23.0 and Graphpad Prism 8.0 software,and the measurement data were expressed as mean ± standard deviation(±s),and the comparison between two groups was analyzed by independent sample t-test,and all experiments were repeated at least three times,and P<0.05 indicated that the differences were statistically significant.Results:1.SiHa cells were first transfected with siRNA,and the best siRNA for transfection efficiency was determined by qRT-PCR,and an EZH2 low-expressing cell model was constructed.qRT-PCR to detect the m RNA level of EZH2 indicated that: the expression of m RNA in the si-EZH2 group(0.29±0.02),compared with the si-NC group(1.02±0.11)compared to si-NC group(1.02±0.03),the expression level was significantly lower(P<0.001);2.The protein level of EZH2 was examined by Western Blotting,and the expression level of EZH2 protein in si-EZH2 group(0.39±0.06),compared to si-NC group(1.02±0.03),was significantly lower(P<0.001),verifying that EZH2 protein expression was inhibited,followed by cell function loss assay;3.EDU assay to detect cell proliferation rate(%)in each group: relative to si-NC group(35.72±2.61),cell proliferation ability was significantly reduced in si-EZH2 group(16.65±1.16)(P<0.001),and knockdown of EZH2 expression could effectively inhibit DNA synthesis in cervical cancer cells with The cell proliferation activity was diminished;4.Scratch assay detected cell migration rate(%)in each group: at the 12 th h,the cell migration ability was significantly reduced(P < 0.001)in the si-EZH2 group(16.59 ± 1.41)compared with the si-NC group(30.50 ± 0.27);at the 24 th h,the cell migration ability was significantly reduced(P < 0.001)in the si-EZH2 group(23.24 ± 0.72)compared with the si-NC group(35.61±1.98)compared to the si-NC group(P<0.01);at the 48 thh,cell migration ability was reduced in the si-EZH2 group(34.26±2.24)compared to the si-NC group(47.54±0.38)(P<0.01),and cell migration ability was significantly reduced after knockdown of EZH2;5.Transwell assay detected the effect of low Expression of EZH2 on the invasive ability of SiHa cells: compared with si-NC group(1119.00±48.09),the number of invasive cells in si-EZH2 group(742.33±109.39)was reduced(P<0.05),the number of cervical cancer cells invading downward was significantly reduced after EZH2 knockdown,and knockdown of EZH2 could effectively inhibit the distant metastasis of cells.6.The percentage of apoptosis(%)was analyzed by flow cytometry: relative to the si-NC group(2.79 ± 0.11),the apoptosis rate was significantly higher in the si-EZH2 group(4.39 ± 0.17)(P < 0.001),and knockdown of EZH2 could significantly increase the percentage of apoptosis.Conclusion:In this study,the expression level of cellular EZH2 was altered by siRNA transfection of HPV16-positive cervical cancer cells,which led to a series of cellular function deficiency experiments on EZH2.Preliminary studies revealed malignant oncogenic effects of EZH2 on the proliferation,migration and invasion of SiHa cells.We tentatively concluded that EZH2 can promote the development,progression and metastasis of cervical cancer,providing a potential target for the treatment of cervical cancer patients.