目的探讨环状RNA(CircRNA)-CDR1as靶向miR-7-5p/DNA聚合酶ε亚基4(POLE4)对非小细胞肺癌(NSCLC)细胞恶性生物学行为的影响。方法收集2023年1月-2024年6月于儋州市人民医院接受手术的30例NSCLC患者的肿瘤组织和癌旁组织,检测CircRNA-CDR...目的探讨环状RNA(CircRNA)-CDR1as靶向miR-7-5p/DNA聚合酶ε亚基4(POLE4)对非小细胞肺癌(NSCLC)细胞恶性生物学行为的影响。方法收集2023年1月-2024年6月于儋州市人民医院接受手术的30例NSCLC患者的肿瘤组织和癌旁组织,检测CircRNA-CDR1as、miR-7-5p、POLE4在肿瘤组织、癌旁组织及NSCLC细胞(A549、PC9、NCI-H1299、NCI-H1650)、正常肺上皮细胞(BEAS-2B)中的表达水平;将A549细胞分为对照组、sh-CircRNA-CDR1as组、阴性对照(sh-NC)组、sh-CircRNA-CDR1as+miR-7-5p抑制剂(in-miR-7-5p)组、sh-CircRNACDR1as+miR抑制剂(in-miR-NC)组,细胞计数试剂盒-8(CCK-8)检测细胞增殖能力,Transwell实验检测细胞迁移、侵袭能力,流式细胞术检测细胞凋亡,Western blot检测基质金属蛋白酶-9(MMP-9)、程序性死亡配体-1(PD-L1)、抗BCL2相关x蛋白(BAX)、POLE4、增殖细胞核抗原(PCNA)蛋白表达;实时荧光定量PCR检测CircRNA-CDR1as、miR-7-5p、POLE4基因表达;并验证miR-7-5p与CircRNA-CDR1as或POLE4靶向关系。结果肿瘤组织中CircRNACDR1as、POLE4表达水平较癌旁组织显著升高,miR-7-5p表达水平较癌旁组织显著降低,差异均有统计学意义(t=16.877、21.546、16.383,P均<0.05);A549、PC9、NCI-H1299、NCI-H1650细胞中CircRNA-CDR1as、POLE4表达水平较BEAS-2B细胞显著升高,miR-7-5p表达水平较BEAS-2B细胞显著降低,差异均有统计学意义(F=32.878、33.836、64.688,P均<0.05)。与对照组、sh-NC组比较,sh-CircRNA-CDR1as组细胞凋亡、BAX表达、miR-7-5p表达显著增加,细胞24和48 h OD450值、PCNA表达、侵袭、迁移及MMP-9、PD-L1表达、CircRNA-CDR1as、POLE4蛋白及mRNA表达显著降低,差异均有统计学意义(P均<0.05);与sh-CircRNA-CDR1as+in-miR-NC组比较,sh-CircRNA-CDR1as+in-miR-7-5p组细胞凋亡、BAX表达、miR-7-5p表达显著降低,细胞24和48 h OD450值、PCNA表达、侵袭、迁移及MMP-9、PD-L1表达、POLE4蛋白及mRNA表达显著增加,差异均有统计学意义(P均<0.05);各组细胞凋亡、BAX、miR-7-5p表达、细胞24和48 h OD450值、PCNA表达、侵袭、迁移、MMP-9、PD-L1、CircRNA-CDR1as、POLE4蛋白及mRNA表达比较差异均有统计学意义(F=149.200、95.529、33.807、29.678、28.905、71.011、43.873、50.978、59.597、46.174、103.372、54.760、45.096,P均<0.05)。CircRNA-CDR1as-WT、POLE4-WT分别与miR-7-5p或miR-NC共转染,两组间荧光素酶活性差异均有统计学意义(P均<0.05)。结论干扰CircRNA-CDR1as靶向miR-7-5p/POLE4可对NSCLC细胞恶性生物学行为发挥抑制作用。展开更多
Background:Drug resistance is the main factor contributing to cancer recurrence and poor prognosis.Exploration of drug resistance-related mechanisms and effective therapeutic targets are the aim of molecular targeted ...Background:Drug resistance is the main factor contributing to cancer recurrence and poor prognosis.Exploration of drug resistance-related mechanisms and effective therapeutic targets are the aim of molecular targeted therapy.In our study,the role of long non-coding RNA(lncRNA)AFAP1-AS1 in gemcitabine resistance and related mechanisms were explored in cervical cancer cells.Methods:Gemcitabine-resistant cervical cancer cell lines HT-3-Gem and SW756-Gem were constructed using the gemcitabine concentration gradient method.The overall survival rates and recurrence-free survival rates were evaluated by Kaplan-Meier analysis.The interaction was verified through a Dual-luciferase reporter gene assay and a Biotinylated RNA pull-down assay.Cell proliferation ability was assessed through methyl-thiazolyl-tetrazolium(MTT),soft agar,and colony formation experiments.Cell cycle and apoptosis were detected byflow cytometry.Results:Up-regulation of AFAP1-AS1 in cervical cancer predicted a poor prognosis.Besides,patients in the gemcitabine-resistance group had higher levels of AFAP1-AS1 than the gemcitabine-sensitive group.AFAP1-AS1 promoted tumor growth and induced gemcitabine tolerance of cervical cancer cells.In addition,AFAP1-AS1 mediated epidermal growth factor receptor(EGFR)expression by serving as a molecular sponge for microRNA-7a-5p(miR-7-5p).This present study also proved that the knockdown of EGFR or overexpression of miR-7a-5p abolished the accelerative role of AFAP1-AS1 overexpression in cancer progression and gemcitabine tolerance.Conclusions:In general,the AFAP1-AS1/miR-7-5p/EGFR axis was tightly related to the progression and gemcitabine tolerance of cervical cancer,providing potential targets for the management of cervical cancer.展开更多
文摘目的探讨环状RNA(CircRNA)-CDR1as靶向miR-7-5p/DNA聚合酶ε亚基4(POLE4)对非小细胞肺癌(NSCLC)细胞恶性生物学行为的影响。方法收集2023年1月-2024年6月于儋州市人民医院接受手术的30例NSCLC患者的肿瘤组织和癌旁组织,检测CircRNA-CDR1as、miR-7-5p、POLE4在肿瘤组织、癌旁组织及NSCLC细胞(A549、PC9、NCI-H1299、NCI-H1650)、正常肺上皮细胞(BEAS-2B)中的表达水平;将A549细胞分为对照组、sh-CircRNA-CDR1as组、阴性对照(sh-NC)组、sh-CircRNA-CDR1as+miR-7-5p抑制剂(in-miR-7-5p)组、sh-CircRNACDR1as+miR抑制剂(in-miR-NC)组,细胞计数试剂盒-8(CCK-8)检测细胞增殖能力,Transwell实验检测细胞迁移、侵袭能力,流式细胞术检测细胞凋亡,Western blot检测基质金属蛋白酶-9(MMP-9)、程序性死亡配体-1(PD-L1)、抗BCL2相关x蛋白(BAX)、POLE4、增殖细胞核抗原(PCNA)蛋白表达;实时荧光定量PCR检测CircRNA-CDR1as、miR-7-5p、POLE4基因表达;并验证miR-7-5p与CircRNA-CDR1as或POLE4靶向关系。结果肿瘤组织中CircRNACDR1as、POLE4表达水平较癌旁组织显著升高,miR-7-5p表达水平较癌旁组织显著降低,差异均有统计学意义(t=16.877、21.546、16.383,P均<0.05);A549、PC9、NCI-H1299、NCI-H1650细胞中CircRNA-CDR1as、POLE4表达水平较BEAS-2B细胞显著升高,miR-7-5p表达水平较BEAS-2B细胞显著降低,差异均有统计学意义(F=32.878、33.836、64.688,P均<0.05)。与对照组、sh-NC组比较,sh-CircRNA-CDR1as组细胞凋亡、BAX表达、miR-7-5p表达显著增加,细胞24和48 h OD450值、PCNA表达、侵袭、迁移及MMP-9、PD-L1表达、CircRNA-CDR1as、POLE4蛋白及mRNA表达显著降低,差异均有统计学意义(P均<0.05);与sh-CircRNA-CDR1as+in-miR-NC组比较,sh-CircRNA-CDR1as+in-miR-7-5p组细胞凋亡、BAX表达、miR-7-5p表达显著降低,细胞24和48 h OD450值、PCNA表达、侵袭、迁移及MMP-9、PD-L1表达、POLE4蛋白及mRNA表达显著增加,差异均有统计学意义(P均<0.05);各组细胞凋亡、BAX、miR-7-5p表达、细胞24和48 h OD450值、PCNA表达、侵袭、迁移、MMP-9、PD-L1、CircRNA-CDR1as、POLE4蛋白及mRNA表达比较差异均有统计学意义(F=149.200、95.529、33.807、29.678、28.905、71.011、43.873、50.978、59.597、46.174、103.372、54.760、45.096,P均<0.05)。CircRNA-CDR1as-WT、POLE4-WT分别与miR-7-5p或miR-NC共转染,两组间荧光素酶活性差异均有统计学意义(P均<0.05)。结论干扰CircRNA-CDR1as靶向miR-7-5p/POLE4可对NSCLC细胞恶性生物学行为发挥抑制作用。
文摘Background:Drug resistance is the main factor contributing to cancer recurrence and poor prognosis.Exploration of drug resistance-related mechanisms and effective therapeutic targets are the aim of molecular targeted therapy.In our study,the role of long non-coding RNA(lncRNA)AFAP1-AS1 in gemcitabine resistance and related mechanisms were explored in cervical cancer cells.Methods:Gemcitabine-resistant cervical cancer cell lines HT-3-Gem and SW756-Gem were constructed using the gemcitabine concentration gradient method.The overall survival rates and recurrence-free survival rates were evaluated by Kaplan-Meier analysis.The interaction was verified through a Dual-luciferase reporter gene assay and a Biotinylated RNA pull-down assay.Cell proliferation ability was assessed through methyl-thiazolyl-tetrazolium(MTT),soft agar,and colony formation experiments.Cell cycle and apoptosis were detected byflow cytometry.Results:Up-regulation of AFAP1-AS1 in cervical cancer predicted a poor prognosis.Besides,patients in the gemcitabine-resistance group had higher levels of AFAP1-AS1 than the gemcitabine-sensitive group.AFAP1-AS1 promoted tumor growth and induced gemcitabine tolerance of cervical cancer cells.In addition,AFAP1-AS1 mediated epidermal growth factor receptor(EGFR)expression by serving as a molecular sponge for microRNA-7a-5p(miR-7-5p).This present study also proved that the knockdown of EGFR or overexpression of miR-7a-5p abolished the accelerative role of AFAP1-AS1 overexpression in cancer progression and gemcitabine tolerance.Conclusions:In general,the AFAP1-AS1/miR-7-5p/EGFR axis was tightly related to the progression and gemcitabine tolerance of cervical cancer,providing potential targets for the management of cervical cancer.
