目的:研究钾双孔域通道亚家族K成员1(potassium two pore domain channel subfamily K member 1,KCNK1)在甲状腺乳头状癌(papillary thyroid cancer,PTC)中的表达、与患者临床病理参数的关系及促进PTC细胞转移能力的作用机制。方法:收...目的:研究钾双孔域通道亚家族K成员1(potassium two pore domain channel subfamily K member 1,KCNK1)在甲状腺乳头状癌(papillary thyroid cancer,PTC)中的表达、与患者临床病理参数的关系及促进PTC细胞转移能力的作用机制。方法:收集我院2019年1月至2020年12月从PTC患者切除的癌组织及癌旁组织85例。采用免疫组织化学(IHC)检测PTC癌和癌旁组织中KCNK1的表达,并分析其表达水平与患者临床病理参数及预后的关系。Western-blot检测KCNK1在PTC各细胞系(KTC-1,IHH-4,TPC-1)中的表达,选择KCNK1高表达的PTC细胞系TPC-1分为shNC组、shKCNK1-1组和shKCNK1-2组,Western-blot检测各组细胞中KCNK1的表达;Transwell实验和划痕实验检测各组细胞转移能力;Western-blot检测各组细胞中E-cadherin和N-cadherin蛋白表达;shNC组和shKCNK1-1组差异表达基因采用全转录组测序技术分析,并进行KEGG信号通路富集;Western-blot和功能实验检测KCNK1对PI3K/AKT信号通路的调控作用。结果:与癌旁组织相比,PTC组织中KCNK1阳性率增加(50.59%VS 21.18%;χ^(2)=15.98,P=0.000),KCNK1阳性率在淋巴结转移、癌浸润周围组织和TNM分期晚期的PTC患者中表达增加(P<0.05),KCNK1阳性表达的PTC患者预后较差(复发转移率34.88%VS 14.29%;χ^(2)=4.842,P=0.028)。与人正常的甲状腺滤泡上皮细胞系Nthyori3-1相比,KCNK1在PTC细胞系中表达增加,在TPC-1细胞中的表达最高(P<0.05)。TPC-1细胞转染KCNK1敲减慢病毒,与shNC组相比,shKCNK1-1组和shKCNK1-2组细胞中KCNK1蛋白表达降低(P<0.05),shKCNK1-1组和shKCNK1-2组细胞转移能力降低(P<0.05),KEGG信号通路富集分析显示shNC组和shKCNK1-1组差异表达基因富集在PI3K/AKT信号通路,与shNC组相比,shKCNK1-1组细胞中pPI3K,pAKT蛋白表达降低(P<0.05)。AKT激动剂SC79激活shKCNK1-1组细胞PI3K/AKT信号通路活性,可以减弱敲减KCNK1对PTC细胞转移能力的抑制作用(P<0.05)。结论:KCNK1在PTC中表达增加,与临床病理参数及不良预后相关,敲减KCNK1抑制PI3K/AKT信号通路抑制PTC细胞转移能力。展开更多
Objective To investigate the effects of electroacupuncture(EA)on the expression and phosphorylation of insulin signal transduction-related proteins in the hypothalamus of insulin resistance(IR)rats.Methods There were ...Objective To investigate the effects of electroacupuncture(EA)on the expression and phosphorylation of insulin signal transduction-related proteins in the hypothalamus of insulin resistance(IR)rats.Methods There were totally seventy-five Wistar rats.Ten rats were randomly assigned to the Normal group(N).The remaining 65 rats were fed a high-fat diet,fifty successfully modeled rats were randomly divided into the Model(M),EA,Sham EA(S+EA),l-leucine(L),and l-leucine+EA(L+EA)groups,with 10 rats in each group.EA was applied at acupoints“Guanyuan(CV 4)”,“Zhongwan(CV 12)”,“Zusanli(ST 36)”,and“Fenglong(ST 40)”,with each session lasting 10 min,three times per week for 8 weeks.The S+EA group received needle insertion to a depth of≤2 mm without electrical stimulation,with the same treatment duration and same acupoint selection.Body weight,fasting blood glucose(FBG),and insulin sensitivity(glucose infusion rate,GIR)were measured.Western blot analysis was used to assess insulin receptor substrate-1(IRS-1),(Protein kinase B)Akt,glycogen synthase kinase-3β(GSK-3β),mechanistic target of rapamycin complex 1(mTORC1),and Ribosomal S6 kinase 1(S6K1),along with their phosphorylated forms.PCR was used to evaluate mRNA expression of IRS-1,Akt,and GSK-3β.Immunofluorescence was used to detect hypothalamic Akt localization.Results(1)Compared to the N group,the M group exhibited increased body weight,FBG,and phosphorylation of GSK-3β,mTORC1,and S6K1,with decreased GIR,IRS-1,Akt phosphorylation,and mRNA expression(P<0.05,P<0.01).(2)Compared to the M group,the EA and S+EA groups showed reduced body weight,FBG,GSK-3β,mTORC1,and S6K1 phosphorylation,with increased GIR,IRS-1,Akt phosphorylation,and mRNA expression(P<0.05,P<0.01).(3)Compared to the EA group,the S+EA group had higher body weight,GSK-3βphosphorylation,and mRNA expression,with reduced p-IRS-1 and p-Akt expression(P<0.05);the L and L+EA groups showed increased GSK-3β,mTORC1,and S6K1 phosphorylation,with decreased GIR,IRS-1,and Akt mRNA expression(P<0.05).(4)Compared to the L+EA group,the L group exhibited higher GSK-3β,mTORC1,and S6K1 phosphorylation,with lower GIR,Akt mRNA,and p-Akt expression(P<0.05,P<0.01).Conclusion EA positively influences body weight,glucose-lipid metabolism,and insulin sensitivity in IR rats,with regulatory effects on central insulin signal transduction-related proteins,potentially linked to its suppression of hypothalamic mTORC1/S6K1 pathway activity.展开更多
文摘目的:研究钾双孔域通道亚家族K成员1(potassium two pore domain channel subfamily K member 1,KCNK1)在甲状腺乳头状癌(papillary thyroid cancer,PTC)中的表达、与患者临床病理参数的关系及促进PTC细胞转移能力的作用机制。方法:收集我院2019年1月至2020年12月从PTC患者切除的癌组织及癌旁组织85例。采用免疫组织化学(IHC)检测PTC癌和癌旁组织中KCNK1的表达,并分析其表达水平与患者临床病理参数及预后的关系。Western-blot检测KCNK1在PTC各细胞系(KTC-1,IHH-4,TPC-1)中的表达,选择KCNK1高表达的PTC细胞系TPC-1分为shNC组、shKCNK1-1组和shKCNK1-2组,Western-blot检测各组细胞中KCNK1的表达;Transwell实验和划痕实验检测各组细胞转移能力;Western-blot检测各组细胞中E-cadherin和N-cadherin蛋白表达;shNC组和shKCNK1-1组差异表达基因采用全转录组测序技术分析,并进行KEGG信号通路富集;Western-blot和功能实验检测KCNK1对PI3K/AKT信号通路的调控作用。结果:与癌旁组织相比,PTC组织中KCNK1阳性率增加(50.59%VS 21.18%;χ^(2)=15.98,P=0.000),KCNK1阳性率在淋巴结转移、癌浸润周围组织和TNM分期晚期的PTC患者中表达增加(P<0.05),KCNK1阳性表达的PTC患者预后较差(复发转移率34.88%VS 14.29%;χ^(2)=4.842,P=0.028)。与人正常的甲状腺滤泡上皮细胞系Nthyori3-1相比,KCNK1在PTC细胞系中表达增加,在TPC-1细胞中的表达最高(P<0.05)。TPC-1细胞转染KCNK1敲减慢病毒,与shNC组相比,shKCNK1-1组和shKCNK1-2组细胞中KCNK1蛋白表达降低(P<0.05),shKCNK1-1组和shKCNK1-2组细胞转移能力降低(P<0.05),KEGG信号通路富集分析显示shNC组和shKCNK1-1组差异表达基因富集在PI3K/AKT信号通路,与shNC组相比,shKCNK1-1组细胞中pPI3K,pAKT蛋白表达降低(P<0.05)。AKT激动剂SC79激活shKCNK1-1组细胞PI3K/AKT信号通路活性,可以减弱敲减KCNK1对PTC细胞转移能力的抑制作用(P<0.05)。结论:KCNK1在PTC中表达增加,与临床病理参数及不良预后相关,敲减KCNK1抑制PI3K/AKT信号通路抑制PTC细胞转移能力。
基金Supported by the Hubei Natural Science Foundation Joint Fund Project:2023AFD139,2023AFD140the "Shizhen Young Talent" Training Program of the College of Acupuncture and Orthopedics,Hubei University of Chinese Medicinethe National Famous Traditional Chinese Medicine Expert Inheritance Studio Construction Project。
文摘Objective To investigate the effects of electroacupuncture(EA)on the expression and phosphorylation of insulin signal transduction-related proteins in the hypothalamus of insulin resistance(IR)rats.Methods There were totally seventy-five Wistar rats.Ten rats were randomly assigned to the Normal group(N).The remaining 65 rats were fed a high-fat diet,fifty successfully modeled rats were randomly divided into the Model(M),EA,Sham EA(S+EA),l-leucine(L),and l-leucine+EA(L+EA)groups,with 10 rats in each group.EA was applied at acupoints“Guanyuan(CV 4)”,“Zhongwan(CV 12)”,“Zusanli(ST 36)”,and“Fenglong(ST 40)”,with each session lasting 10 min,three times per week for 8 weeks.The S+EA group received needle insertion to a depth of≤2 mm without electrical stimulation,with the same treatment duration and same acupoint selection.Body weight,fasting blood glucose(FBG),and insulin sensitivity(glucose infusion rate,GIR)were measured.Western blot analysis was used to assess insulin receptor substrate-1(IRS-1),(Protein kinase B)Akt,glycogen synthase kinase-3β(GSK-3β),mechanistic target of rapamycin complex 1(mTORC1),and Ribosomal S6 kinase 1(S6K1),along with their phosphorylated forms.PCR was used to evaluate mRNA expression of IRS-1,Akt,and GSK-3β.Immunofluorescence was used to detect hypothalamic Akt localization.Results(1)Compared to the N group,the M group exhibited increased body weight,FBG,and phosphorylation of GSK-3β,mTORC1,and S6K1,with decreased GIR,IRS-1,Akt phosphorylation,and mRNA expression(P<0.05,P<0.01).(2)Compared to the M group,the EA and S+EA groups showed reduced body weight,FBG,GSK-3β,mTORC1,and S6K1 phosphorylation,with increased GIR,IRS-1,Akt phosphorylation,and mRNA expression(P<0.05,P<0.01).(3)Compared to the EA group,the S+EA group had higher body weight,GSK-3βphosphorylation,and mRNA expression,with reduced p-IRS-1 and p-Akt expression(P<0.05);the L and L+EA groups showed increased GSK-3β,mTORC1,and S6K1 phosphorylation,with decreased GIR,IRS-1,and Akt mRNA expression(P<0.05).(4)Compared to the L+EA group,the L group exhibited higher GSK-3β,mTORC1,and S6K1 phosphorylation,with lower GIR,Akt mRNA,and p-Akt expression(P<0.05,P<0.01).Conclusion EA positively influences body weight,glucose-lipid metabolism,and insulin sensitivity in IR rats,with regulatory effects on central insulin signal transduction-related proteins,potentially linked to its suppression of hypothalamic mTORC1/S6K1 pathway activity.
