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Objective To explore the effect of circular RNA(circRNA)transcription factor 25(TCF25)-targeting microRNA-128b(miR-128b)on the proliferation and apoptosis of MCF-7 human breast cancer cells.Methods MCF-7 cells were cultured until the loga-rithmic growth stage.Cells were randomly divided into the blank(untransfected),si-NC(transfected si-NC),si-circRNA TCF25(transfected si-circRNA TCF25),pcDNA-circRNA TCF25(transfected pcDNA-circRNA TCF25),miR-NC(transfected miR-NC),miR-128b mimic(transfected miR-128b mimic),miR-128b inhibitor(transfected miR-128b inhibitor),and pcDNA-circRNA TCF25+miR-128b mimic(transfected with pcDNA-circRNA TCF25 and miR-128b mimic)groups.Each group included six multiple pores.Forty-eight hours after transfection,the expression of circRNA TCF25 and miR-128b in each group was determined using real-time reverse transcription-quanti-tative polymerase chain reaction(RT-qPCR).An RNase R enzyme digestion assay was used to identify circular RNA.Subcellular locali-zation of circRNA TCF25 was determined through cytoplasmic-nuclear separation.Cell proliferative activity was measured using the 2,5-diphenyl-2H-tetrazolium bromide(MTT)assay.Apoptosis was detected using flow cytometry.RT-qPCR was performed to determine the mRNA expression levels of phosphatase and tensin homolog(PTEN),proliferating nuclear antigen 67(Ki-67),andcaspase-3.Western blotting was performed to measure PTEN,Ki-67,caspase-3,and cleaved caspase-3 protein expression.The dual-luciferase reporter(DLR)assay was performed to analyze the relationship between circRNA TCF25 and miR-128b.Results Compared to the control group,the relative expression of circRNA TCF25 did not exhibit significant changes after RNase R enzyme treatment(P＞0.05),whereas that of linear TCF25 decreased after RNase R enzyme treatment(P＜0.05).The relative expression of circRNA TCF25 in the cytoplasm was higher than that in the nucleus(P＜0.05).Compared with the blank and si-NC groups,the cell proliferation activity of the si-circRNA TCF25 group decreased;the apoptosis rate increased;Ki-67mRNA and protein expression decreased;and PTEN,caspase-3,and cleaved caspase-3mRNA and protein expression increased.In addition,cell proliferation activity increased and apoptosis rate decreased in the pcDNA-circRNA TCF25 group.Ki-67 mRNA and protein expression were increased,and PTEN,caspase-3,and cleaved caspase-3 mRNA and protein expression decreased,with statistical significance(all P＜0.05).Compared with the blank and miR-NC groups,the cell proliferation activity of the miR-128b mimic group decreased;the apoptosis rate increased;Ki-67mRNA and protein expression decreased;and PTEN,caspase-3,and cleaved caspase-3 mRNA and protein expression were increased,whereas the cell proliferation activity increased and apoptosis rate decreased in the miR-128b inhibitor group.Ki-67mRNA and protein expression were increased,and PTEN,caspase-3,and cleavedcaspase-3mRNA and protein expression decreased,with statistical significance(all P＜0.05).Compared with the pcDNA circRNA TCF25 group,the cell proliferation activity of the pcDNA circRNA TCF25+miR-128b mimic group decreased;the apoptosis rate increased;Ki-67mRNA and protein expression decreased;and PTEN,caspase-3,and cleavedcaspase-3mRNA and protein expression increased,with statistical significance(all P＜0.05).The DLR assay results confirmed that circRNA TCF25 targets miR-128b.Conclusion CircRNAs may play a key role in promoting the proliferation of MCF-7 human breast cancer cells and inhibiting their apoptosis by targeting miR-128b expression;promoting Ki-67 expression;and inhibiting PTEN,caspase-3,and cleaved caspase-3 expression.
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Objective To investigate the expressions of ATPase family AAA domain-containing protein 2 (ATAD2) and β-catenin,and to analyze their correlations with clinicopathological features and prognostic significance in patients with hepatocellular carcinoma (HCC).Methods The HCC tissues of 40 patients were tested by real-time PCR to study the expressions of ATAD2 and β-catenin.Real-time PCR and Western blot were performed to detect the proteins and mRNA levels of ATAD2,APC,β-catenin and wnt signaling pathway downstream.The HCC tissues of 80 patients and 20 peritumoral tissues were tested by immunohistochemistry (IHC).The cumulative survival-rate was analyzed by the Kaplan-Meier method,and univariate and multivariate were analyzed by the Cox proportional hazards model.Results ISH:The positive rates of ATAD2 and β-catenin were 65.0％ and 55.0％,respectively.These rates were significantly higher than those in the peritumoral tissues (30.0％ and 25.0％,respectively).The ATAD2 expression was related to tumor size (P ＜ 0.05),metastasis (P ＜ 0.05),serum AFP level (P ＜ 0.05) and TNM stag ing (P ＜ 0.05).The β-catenin expression was only significantly related to metastasis (P ＜ 0.05).Correlation analysis showed that the ATAD2 expression was positively related to the β-catenin expression (Pearson =0.578,P ＜ 0.01,R2 =0.3607,Spearman =0.495).This positive relationship was also found in the remaining 4 cell lines except the SK-hep1.Depleting ATAD2 up-regulated APC and down-regulated β-catenin protein and mRNA expression.Univariate and multivariate analyses showed that ATAD2 and β-catenin expressions,tumor size,metastasis,serum AFP,and TNM staging were poor prognostic factors for HCC,and ATAD2 and β-catenin expressions,metastasis,serum.AFP were independent prognostic factors.Patients whose ATAD2 and β-catenin were both positive had worse survival than those with only one positive expression or both negative expressions (P ＜ 0.05).Depleting ATAD2 down-regulated survivin,cyclinD1,c-myc,MMP7,Vimentin in wnt signaling pathway and EMT related proteins.Conclusions ATAD2 and β-catenin expressions were positively related in patients with HCC.Abnormal expressions between ATAD2 and β-catenin might participate in the wnt signaling pathway.