目的探讨脂多糖(LPS)作用于人牙髓细胞后,B细胞淋巴瘤-2(Bc1-2)蛋白、Bcl-2相关X蛋白(Bax)在正常人牙髓和炎症牙髓中的表达,探讨其在牙髓炎症过程中的作用机制。方法在体外用不同质量浓度LPS刺激人牙髓细胞后,采用免疫细胞化学染色方法...目的探讨脂多糖(LPS)作用于人牙髓细胞后,B细胞淋巴瘤-2(Bc1-2)蛋白、Bcl-2相关X蛋白(Bax)在正常人牙髓和炎症牙髓中的表达,探讨其在牙髓炎症过程中的作用机制。方法在体外用不同质量浓度LPS刺激人牙髓细胞后,采用免疫细胞化学染色方法在不同的时间点检测Bcl-2、Bax的表达,用图像分析系统Simple PCI version 5.1分析。结果正常人牙髓细胞中Bcl-2、Bax表达量不高,而在牙髓炎症过程中两者均增强,但是随着LPS质量浓度的增加,Bcl-2的表达量开始下降,而Bax的表达则持续增强。结论内毒素能使人牙髓细胞膜上的Bcl-2、Bax表达增强,但Bax的表达强于Bcl-2,内毒素可能通过两者介导的信号传导途径引起细胞凋亡。展开更多
Objective:Oxaliplatin(OXA)and 5-fluorouracil(5-FU)are 2 commonly used chemotherapeutic agents for colorectal cancer(CRC).MicroRNAs(miRNAs,miRs)play crucial roles in the development of chemoresistance in various cancer...Objective:Oxaliplatin(OXA)and 5-fluorouracil(5-FU)are 2 commonly used chemotherapeutic agents for colorectal cancer(CRC).MicroRNAs(miRNAs,miRs)play crucial roles in the development of chemoresistance in various cancers.However,the role and mechanism of miR-224-5p in regulating CRC chemoresistance remain unclear.This study aims to investigate the function of miR-224-5p in chemoresistant CRC cells and the underlying mechanisms.Methods:CRC datasets GSE28702 and GSE69657 were downloaded from the Gene Expression Omnibus(GEO)database.Differentially expressed miRNAs between drug sensitive and resistant groups(OXA or 5-FU)were analyzed,and miR-224-5p was identified as the target miRNA.Chemoresistant cell lines HCT15-OXR,HCT15-5-FU,SW480-OXR,and SW480-5-FU were established.Transient transfections were performed using miR-224-5p mimics,inhibitors,and their respective negative controls(control mimic,control inhibitor)in these cell lines.Cells were treated with different concentrations of OXA or 5-FU post-transfection,and the half-maximal inhibitory concentration(IC_(50))was determined using the cell counting kit-8(CCK-8)assay.Cell proliferation was assessed by CCK-8 and colony formation assays.The expression levels of miR-224-5p,LC3,and P62 were measured by real-time polymerase chain reaction(real-time PCR)and/or Western blotting.Autophagic flux was assessed using a tandem fluorescent-tagged LC3 reporter assay.TargetScan 8.0,miRTarBase,miRPathDB,and HADb were used to predict B-cell lymphoma-2(Bcl-2)as a potential miR-244-5p target,which was further validated by dual luciferase reporter assays.Results:Chemoresistant CRC cells exhibited down-regulated miR-224-5p expression,whereas up-regulation of miR-224-5p enhanced chemotherapy sensitivity.Exposure to OXA or 5-FU significantly increased autophagic activity in chemoresistant CRC cells,which was reversed by miR-224-5p overexpression.Dual-luciferase assays verified Bcl-2 as a direct target of miR-224-5p.Conclusion:MiR-224-5p regulates chemoresistance in CRC by modulating autophagy through direct targeting of Bcl-2.展开更多
文摘目的探讨脂多糖(LPS)作用于人牙髓细胞后,B细胞淋巴瘤-2(Bc1-2)蛋白、Bcl-2相关X蛋白(Bax)在正常人牙髓和炎症牙髓中的表达,探讨其在牙髓炎症过程中的作用机制。方法在体外用不同质量浓度LPS刺激人牙髓细胞后,采用免疫细胞化学染色方法在不同的时间点检测Bcl-2、Bax的表达,用图像分析系统Simple PCI version 5.1分析。结果正常人牙髓细胞中Bcl-2、Bax表达量不高,而在牙髓炎症过程中两者均增强,但是随着LPS质量浓度的增加,Bcl-2的表达量开始下降,而Bax的表达则持续增强。结论内毒素能使人牙髓细胞膜上的Bcl-2、Bax表达增强,但Bax的表达强于Bcl-2,内毒素可能通过两者介导的信号传导途径引起细胞凋亡。
基金supported by the National Natural Science Foundation(82072729)Wuhan Municipal Health Commission Medical Research Project(WX19Q30),China。
文摘Objective:Oxaliplatin(OXA)and 5-fluorouracil(5-FU)are 2 commonly used chemotherapeutic agents for colorectal cancer(CRC).MicroRNAs(miRNAs,miRs)play crucial roles in the development of chemoresistance in various cancers.However,the role and mechanism of miR-224-5p in regulating CRC chemoresistance remain unclear.This study aims to investigate the function of miR-224-5p in chemoresistant CRC cells and the underlying mechanisms.Methods:CRC datasets GSE28702 and GSE69657 were downloaded from the Gene Expression Omnibus(GEO)database.Differentially expressed miRNAs between drug sensitive and resistant groups(OXA or 5-FU)were analyzed,and miR-224-5p was identified as the target miRNA.Chemoresistant cell lines HCT15-OXR,HCT15-5-FU,SW480-OXR,and SW480-5-FU were established.Transient transfections were performed using miR-224-5p mimics,inhibitors,and their respective negative controls(control mimic,control inhibitor)in these cell lines.Cells were treated with different concentrations of OXA or 5-FU post-transfection,and the half-maximal inhibitory concentration(IC_(50))was determined using the cell counting kit-8(CCK-8)assay.Cell proliferation was assessed by CCK-8 and colony formation assays.The expression levels of miR-224-5p,LC3,and P62 were measured by real-time polymerase chain reaction(real-time PCR)and/or Western blotting.Autophagic flux was assessed using a tandem fluorescent-tagged LC3 reporter assay.TargetScan 8.0,miRTarBase,miRPathDB,and HADb were used to predict B-cell lymphoma-2(Bcl-2)as a potential miR-244-5p target,which was further validated by dual luciferase reporter assays.Results:Chemoresistant CRC cells exhibited down-regulated miR-224-5p expression,whereas up-regulation of miR-224-5p enhanced chemotherapy sensitivity.Exposure to OXA or 5-FU significantly increased autophagic activity in chemoresistant CRC cells,which was reversed by miR-224-5p overexpression.Dual-luciferase assays verified Bcl-2 as a direct target of miR-224-5p.Conclusion:MiR-224-5p regulates chemoresistance in CRC by modulating autophagy through direct targeting of Bcl-2.
