Lysine-specific Demethylase 2B(KDM2B) Silencing Induces Apoptosis Of Ovarian Cancer Cells By Affected The Expression Of Harakiri In Vitro And In Vivo

Objective:Using STRING database to predict the relationship between Harakiri(HRK)and lysine specific demethylase 2B(KDM2B)to investigate whether KDM2 B silencing regulates the expression of pro-apoptotic gene HRK in ovarian cancer.To investigate the effect of KDM2 B silencing on the biological behavior of epithelial ovarian cancer A2780 and SKOV3 cells and the relationship between silencing KDM2 B and the expression of pro-apoptotic gene HRK,and whether KDM2 B can induce apoptosis of ovarian cancer cells by regulating HRK gene in vitro and in vivo.To explore the possible mechanism of KDM2 B inducing apoptosis of epithelial ovarian cancer cells,it provides a new epigenetic regulatory target for the research and treatment of ovarian cancer.Methods:Protein-protein interaction network(PPI)analysis of HRK and KDM2 B using STRING database;The KDM2 B silenced was constructed and packaged and infected with ovarian cancer cells A2780 and SKOV3(sh KDM2B)to construct the silenced KDM2 B cell model.Then the stable cell lines expressing only Puromycin resistance were screened out.72 hours after transfection,the m RNA and protein expression levels of KDM2 B and HRK genes were detected by real-time fluorescence quantitative PCR(q RT-PCR)and Western Blotting respectively.The expression of H3K27 trimethyltransferase(H3K27Me3)after knockdown KDM2 B was detected by Western Blotting method and cellular immunofluorescence assay.MTT assay and Wound Healing assay were used to detect the effect of KDM2 B knockdown on the proliferation and migration of epithelial ovarian cancer cells A2780 and SKOV3;Meanwhile,flow cytometry and AO/EB double fluorescence detection kit were used to detect the apoptosis of ovarian cancer cells A2780 and SKOV3 after silencing KDM2B;The cell model of silenced HRK was constructed.Stable cell lines expressing only Puromycin resistance were screened out.Then Western Blotting and q RT-PCR were used to determine the protein and m RNA expression levels after HRK infected,and the expression level of HRK antagonistic apoptotic gene Bcl-2 was detected by Western Blotting.Western Blotting was used to detect the expression of apoptosis marker caspase3 in ovarian cancer A2780 and SKOV3 cells transfected sh HRK and cotransfected sh KDM2B/HRK.Flow cytometry was used to detect the apoptosis rate of ovarian cancer cells A2780 and SKOV3 after knockdown of KDM2 B and HRK.SKOV3 cells,a stable transfected cell line with logarithmic growth and only Puromycin resistance,were inoculated subcutaneously into BALBC/c nude mice in the experimental group(sh KDM2 B group)and the control group(sh NC group)respectively.Tumor growth was observed,and curve was plotted after tumor body presented,q RT-PCR,Western Blotting and immunohistochemistry(IHC)were used to detect the expression of KDM2 B and HRK in subcutaneous transplantation tumors of sh KDM2 B and sh NC groups.Results:1.(PPI)analysis of protein-protein interaction network in STRING database shows that there may be a protein relationship between HRK and KDM2 B.2.Successfully constructed lentivirus expression vector with the KDM2 B knockdown,and the cell line can reach subsequent experimental requirements.Real-time quantitative PCR and Western Blotting results showed that:compared with blank control group and negative control group,knockdown KDM2 B significantly inhibited the expression levels of KDM2 B m RNA and protein in ovarian cancer cells A2780 and SKOV3(p<0.05).Moreover,silencing KDM2 B can increase the m RNA and protein levels of HRK gene in ovarian cancer cells A2780 and SKOV3(p<0.05).Western Blotting and cell immunofluorescence experiments showed that knockout of KDM2 B inhibited the expression of H3K27me3 in ovarian cancer cells A2780 and SKOV3(p<0.05).The results of MTT assay and Wound healing assay showed that knockdown KDM2 B in sh KDM2 B group could significantly inhibit the proliferation and migration of ovarian cancer cells A2780 and SKOV3 compared with sh NC and blank control group(p<0.05);The results of AO/EB double fluorescence detection kit showed that in ovarian cancer cells A2780 and SKOV3,the number of apoptosis or dead cells in sh KDM2 B group was significantly increased than that in sh NC and blank control group(p<0.05).At the same time,the results of flow cytometry showed that the proportion of apoptotic cells in sh KDM2 B group was significantly higher than that in sh NC and blank control group,which was consistent with that of AO/EB double fluorescence detection kit(p < 0.05).It is suggested that silent KDM2 B can inhibit the expression of H3K27Me3 in ovarian cancer cells A2780 and SKOV3,and regulate the proliferation,migration and apoptosis of ovarian cancer cells A2780 and SKOV3.3.HRK silenced cell model was successfully constructed,and the selected stable cell lines can reach the subsequent experimental requirements.The results of Western Blotting showed that the expression of anti-apoptosis gene Bc L-2 of knockout HRK was significantly increased in ovarian cancer cells A2780 and SKOV3(p<0.05);In ovarian cancer cells A2780 and SKOV3,compared with silencing KDM2 B,the expression of apoptosis marker caspase3 in ovarian cancer cells A2780 and SKOV3 co-transfected with KDM2B/HRK was partially inhibited(p<0.05).In short,our results indicated that KDM2 B inhibited the expression of HRK and HRK mediated apoptosis induced by KDM2 B silences.4.The subcutaneous transplanted tumor model of nude mice was successfully established.After four weeks,the tumor volume of sh KDM2 B group was smaller than that of sh NC group(p<0.05),and the results of real-time fluorescence quantitative PCR,Western Blotting and IHC showed that the m RNA and protein levels of HRK increased after knockout KDM2B(p<0.05).It is also proved that knockdown KDM2 B increases the basic level of tumor cell apoptosis by increasing the expression of HRK and inhibiting the tumor growth in vivo.Conclusions:Taken together,our data suggest that KDM2 B knockdown reduces the level of H3K27me3,thus inhibiting the proliferation and migration of ovarian cancer cells.These results were related to the increased expression of the pro-apoptotic HRK gene,suggesting that KDM2 B inhibited the apoptosis of epithelial ovarian cancer cells by promoting H3K27me3 to inhibit HRK expression.