Effect of circular RNA transcription factor 25-targeting microRNA-128b on the proliferation and apoptosis of MCF-7 human breast cancer cells / 中国医科大学学报

ABSTRACT

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.