AIM:To determine whether the microRNA-27b-3p(miR-27b-3p)/NF-E2-related factor 2(Nrf2)pathway plays a role in human retinal pigment epithelial(hRPE)cell response to high glucose,how miR-27b-3p and Nrf2 expression are r...AIM:To determine whether the microRNA-27b-3p(miR-27b-3p)/NF-E2-related factor 2(Nrf2)pathway plays a role in human retinal pigment epithelial(hRPE)cell response to high glucose,how miR-27b-3p and Nrf2 expression are regulated,and whether this pathway could be specifically targeted.METHODS:hRPE cells were cultured in normal glucose or high glucose for 1,3,or 6d before measuring cellular proliferation rates using cell counting kit-8 and reactive oxygen species(ROS)levels using a dihydroethidium kit.miR-27b-3p,Nrf2,NAD(P)H quinone oxidoreductase 1(NQO1)and heme oxygenase-1(HO-1)mRNA and protein levels were analyzed using reverse transcription quantitative polymerase chain reaction(RT-qPCR)and immunocytofluorescence(ICF),respectively.Western blot analyses were performed to determine nuclear and total Nrf2 protein levels.Nrf2,NQO1,and HO-1 expression levels by RT-qPCR,ICF,or Western blot were further tested after miR-27b-3p overexpression or inhibitor lentiviral transfection.Finally,the expression level of those target genes was analyzed after treating hRPE cells with pyridoxamine.RESULTS:Persistent exposure to high glucose gradually suppressed hRPE Nrf2,NQO1,and HO-1 mRNA and protein levels and increased miR-27b-3p mRNA levels.High glucose also promoted ROS release and inhibited cellular proliferation.Nrf2,NQO1,and HO-1 mRNA levels decreased after miR-27b-3p overexpression and,conversely,both mRNA and protein levels increased after expressing a miR-27b-3p inhibitor.After treating hRPE cells exposed to high glucose with pyridoxamine,ROS levels tended to decreased,proliferation rate increased,Nrf2,NQO1,and HO-1 mRNA and protein levels were upregulated,and miR-27b-3p mRNA levels were suppressed.CONCLUSION:Nrf2 is a downstream target of miR-27b-3p.Furthermore,the miR-27b-3p inhibitor pyridoxamine can alleviate high glucose injury by regulating the miR-27b-3p/Nrf2 axis.展开更多
目的探讨催产素(oxytocin,OXT)对卵巢癌细胞增殖与凋亡的影响及其作用机制。方法人卵巢癌细胞系SKOV3和A2780细胞来源于美国模式培养物保藏所(ATCC)。OXT和OXT受体(oxytocin receptor,OXTR)拮抗剂Atosiban刺激卵巢癌细胞,改变细胞中miR-...目的探讨催产素(oxytocin,OXT)对卵巢癌细胞增殖与凋亡的影响及其作用机制。方法人卵巢癌细胞系SKOV3和A2780细胞来源于美国模式培养物保藏所(ATCC)。OXT和OXT受体(oxytocin receptor,OXTR)拮抗剂Atosiban刺激卵巢癌细胞,改变细胞中miR-196b-3p的水平,使用MTT法、ELISA试验、Western blot、生物信息学工具及荧光素酶报告基因实验等研究以上处理对卵巢癌细胞增殖与凋亡的作用和机制。结果OXT刺激SKOV3细胞OXTR激活(F=28.842,P<0.05),增殖活性降低(F=12.988,P<0.05),Caspase3活性增加(F=26.676,P<0.05);Ki67、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)及p53凋亡刺激蛋白抑制剂(inhibitor of apoptosis-stimulating of p53 protein,iASPP)表达下调,p53凋亡刺激蛋白(apoptosis-stimulating of p53 protein,ASPP)1(ASPP1)及2(ASPP2)表达上调;但Atosiban部分逆转OXT的效果。OXT刺激卵巢癌细胞miR-196b-3p水平减少(A2780:F=76.406,P<0.05;SKOV3:F=45.874,P<0.05)。miR-196b-3p-mimic转染联合OXT刺激,导致细胞增殖活性增加(F=9.232,P<0.05),Caspase3活性降低(F=36.350,P<0.05),同时ASPP2表达降低(F=83.013,P<0.05)。而miR-196b-3p-inhibitor转染联合OXT刺激,ASPP2表达增加(F=83.013,P<0.05)。生物信息学工具预测联合荧光素酶报告基因实验确认ASPP2是miR-196b-3p的靶基因。结论OXT介导的OXTR激活通过调节miR-196b-3p/ASPP2信号通路调节卵巢癌细胞生长。展开更多
基金Supported by National Natural Science Foundation of China(No.2020J01652)the Training Project for Young and Middleaged Core Talents in Health System of Fujian Province(No.2016-ZQN-62).
文摘AIM:To determine whether the microRNA-27b-3p(miR-27b-3p)/NF-E2-related factor 2(Nrf2)pathway plays a role in human retinal pigment epithelial(hRPE)cell response to high glucose,how miR-27b-3p and Nrf2 expression are regulated,and whether this pathway could be specifically targeted.METHODS:hRPE cells were cultured in normal glucose or high glucose for 1,3,or 6d before measuring cellular proliferation rates using cell counting kit-8 and reactive oxygen species(ROS)levels using a dihydroethidium kit.miR-27b-3p,Nrf2,NAD(P)H quinone oxidoreductase 1(NQO1)and heme oxygenase-1(HO-1)mRNA and protein levels were analyzed using reverse transcription quantitative polymerase chain reaction(RT-qPCR)and immunocytofluorescence(ICF),respectively.Western blot analyses were performed to determine nuclear and total Nrf2 protein levels.Nrf2,NQO1,and HO-1 expression levels by RT-qPCR,ICF,or Western blot were further tested after miR-27b-3p overexpression or inhibitor lentiviral transfection.Finally,the expression level of those target genes was analyzed after treating hRPE cells with pyridoxamine.RESULTS:Persistent exposure to high glucose gradually suppressed hRPE Nrf2,NQO1,and HO-1 mRNA and protein levels and increased miR-27b-3p mRNA levels.High glucose also promoted ROS release and inhibited cellular proliferation.Nrf2,NQO1,and HO-1 mRNA levels decreased after miR-27b-3p overexpression and,conversely,both mRNA and protein levels increased after expressing a miR-27b-3p inhibitor.After treating hRPE cells exposed to high glucose with pyridoxamine,ROS levels tended to decreased,proliferation rate increased,Nrf2,NQO1,and HO-1 mRNA and protein levels were upregulated,and miR-27b-3p mRNA levels were suppressed.CONCLUSION:Nrf2 is a downstream target of miR-27b-3p.Furthermore,the miR-27b-3p inhibitor pyridoxamine can alleviate high glucose injury by regulating the miR-27b-3p/Nrf2 axis.
文摘目的探讨催产素(oxytocin,OXT)对卵巢癌细胞增殖与凋亡的影响及其作用机制。方法人卵巢癌细胞系SKOV3和A2780细胞来源于美国模式培养物保藏所(ATCC)。OXT和OXT受体(oxytocin receptor,OXTR)拮抗剂Atosiban刺激卵巢癌细胞,改变细胞中miR-196b-3p的水平,使用MTT法、ELISA试验、Western blot、生物信息学工具及荧光素酶报告基因实验等研究以上处理对卵巢癌细胞增殖与凋亡的作用和机制。结果OXT刺激SKOV3细胞OXTR激活(F=28.842,P<0.05),增殖活性降低(F=12.988,P<0.05),Caspase3活性增加(F=26.676,P<0.05);Ki67、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)及p53凋亡刺激蛋白抑制剂(inhibitor of apoptosis-stimulating of p53 protein,iASPP)表达下调,p53凋亡刺激蛋白(apoptosis-stimulating of p53 protein,ASPP)1(ASPP1)及2(ASPP2)表达上调;但Atosiban部分逆转OXT的效果。OXT刺激卵巢癌细胞miR-196b-3p水平减少(A2780:F=76.406,P<0.05;SKOV3:F=45.874,P<0.05)。miR-196b-3p-mimic转染联合OXT刺激,导致细胞增殖活性增加(F=9.232,P<0.05),Caspase3活性降低(F=36.350,P<0.05),同时ASPP2表达降低(F=83.013,P<0.05)。而miR-196b-3p-inhibitor转染联合OXT刺激,ASPP2表达增加(F=83.013,P<0.05)。生物信息学工具预测联合荧光素酶报告基因实验确认ASPP2是miR-196b-3p的靶基因。结论OXT介导的OXTR激活通过调节miR-196b-3p/ASPP2信号通路调节卵巢癌细胞生长。
