目的探讨食管鳞癌组织中microRNA let-7a-3甲基化状态与血浆中类胰岛素样生长因子2(Insulin like growth factor 2,IGF-Ⅱ)表达的相关性。方法采用甲基化特异性PCR法(Methylation specific PCR,qMSP)检测83例食管癌及相对应的癌旁正常...目的探讨食管鳞癌组织中microRNA let-7a-3甲基化状态与血浆中类胰岛素样生长因子2(Insulin like growth factor 2,IGF-Ⅱ)表达的相关性。方法采用甲基化特异性PCR法(Methylation specific PCR,qMSP)检测83例食管癌及相对应的癌旁正常组织中let-7a-3甲基化状态,采用酶联免疫吸附试验(Enzyme linked immunosorbent assay,ELISA)检测血浆中IGF-Ⅱ的表达水平。结果83例食管鳞癌患者癌组织中的microRNA let-7a-3甲基化程度显著高于癌旁正常组织(P<0.001)。83例食管鳞癌患者血浆中IGF-Ⅱ的表达水平与let-7a-3基因的甲基化程度总体上呈正相关,具有统计学意义(r=0.600,P<0.001)。结论microRNA let-7a-3可能通过对下游分子的甲基化调控参与食管鳞癌的发生发展,这对了解食管鳞癌形成的机制具有重要意义,可为食管鳞癌的诊断和预后提供依据。展开更多
[目的]研究微小RNA let-7a(microRNA let-7a,miR-let-7a)对脂磷壁酸(Lipoteichoic acid,LTA)诱导的炎性奶牛乳腺上皮细胞(MAC-T)的增殖和凋亡作用。[方法]通过实时荧光定量PCR(Quantitative real time polymerase chain reaction,qRT-P...[目的]研究微小RNA let-7a(microRNA let-7a,miR-let-7a)对脂磷壁酸(Lipoteichoic acid,LTA)诱导的炎性奶牛乳腺上皮细胞(MAC-T)的增殖和凋亡作用。[方法]通过实时荧光定量PCR(Quantitative real time polymerase chain reaction,qRT-PCR)、实时细胞分析仪和流式细胞术检测过表达和抑制表达miR-let-7a在炎性MAC-T中的影响。[结果]qRT-PCR检测结果显示,转染miR-let-7a模拟物会使炎性MAC-T细胞中miR-let-7a的表达量极显著上调(P<0.001),转染miR-let-7a抑制剂会使细胞中miR-let-7a的表达量极显著下降(P<0.001);RTCA结果显示,转染miR-let-7a模拟物能明显促进MAC-T细胞增殖,转染miR-let-7a抑制物会使MAC-T细胞的增殖受到抑制;流式细胞术结果显示,转染miR-let-7a模拟物的MAC-T细胞的凋亡率显著降低(P<0.01);而转染miR-let-7a抑制剂则与之相反,MAC-T细胞的凋亡率显著升高(P<0.01)。即过表达miR-let-7a可以促进MAC-T细胞增殖,并降低细胞的凋亡率。而抑制表达miR-let-7a的作用与之相反,抑制表达miR-let-7a使MAC-T细胞的增殖下降,细胞的凋亡率增大。[结论]miR-let-7a是一种抑制炎症调节因子,其过表达可以缓解奶牛乳腺炎,为阐明奶牛乳腺炎的分子调控机制奠定基础。展开更多
BACKGROUND Primary sclerosing cholangitis(PSC)is characterized by chronic inflammation and it predisposes to cholangiocarcinoma due to lack of effective treatment options.Recombinant adeno-associated virus(rAAV)provid...BACKGROUND Primary sclerosing cholangitis(PSC)is characterized by chronic inflammation and it predisposes to cholangiocarcinoma due to lack of effective treatment options.Recombinant adeno-associated virus(rAAV)provides a promising platform for gene therapy on such kinds of diseases.A microRNA(miRNA)let-7a has been reported to be associated with the progress of PSC but the potential therapeutic implication of inhibition of let-7a on PSC has not been evaluated.AIM To investigate the therapeutic effects of inhibition of a miRNA let-7a transferred by recombinant adeno-associated virus 8(rAAV8)on a xenobiotic-induced mouse model of sclerosing cholangitis.METHODS A xenobiotic-induced mouse model of sclerosing cholangitis was induced by 0.1% 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine(DDC)feeding for 2 wk or 6 wk.A single dose of rAAV8-mediated anti-let-7a-5p sponges or scramble control was injected in vivo into mice onset of DDC feeding.Upon sacrifice,the liver and the serum were collected from each mouse.The hepatobiliary injuries,hepatic inflammation and fibrosis were evaluated.The targets of let-7a-5p and downstream molecule NF-κB were detected using Western blot.RESULTS rAAV8-mediated anti-let-7a-5p sponges can depress the expression of let-7a-5p in mice after DDC feeding for 2 wk or 6 wk.The reduced expression of let-7a-5p can alleviate hepato-biliary injuries indicated by serum markers,and prevent the proliferation of cholangiocytes and biliary fibrosis.Furthermore,inhibition of let-7a mediated by rAAV8 can increase the expression of potential target molecules such as suppressor of cytokine signaling 1 and Dectin1,which consequently inhibit of NF-κB-mediated hepatic inflammation.CONCLUSION Our study demonstrates that a rAAV8 vector designed for liver-specific inhibition of let-7a-5p can potently ameliorate symptoms in a xenobiotic-induced mouse model of sclerosing cholangitis,which provides a possible clinical translation of PSC of human.展开更多
Neurotrophic factors,particularly nerve growth factor,enhance neuronal regeneration.However,the in vivo applications of nerve growth factor are largely limited by its intrinsic disadvantages,such as its short biologic...Neurotrophic factors,particularly nerve growth factor,enhance neuronal regeneration.However,the in vivo applications of nerve growth factor are largely limited by its intrinsic disadvantages,such as its short biological half-life,its contribution to pain response,and its inability to cross the blood-brain barrier.Considering that let-7(human miRNA)targets and regulates nerve growth factor,and that let-7 is a core regulator in peripheral nerve regeneration,we evaluated the possibilities of let-7 application in nerve repair.In this study,anti-let-7a was identified as the most suitable let-7 family molecule by analyses of endogenous expression and regulatory relationship,and functional screening.Let-7a antagomir demonstrated biosafety based on the results of in vivo safety assessments and it entered into the main cell types of the sciatic nerve,including Schwann cells,fibroblasts and macrophages.Use of hydrogel effectively achieved controlled,localized,and sustained delivery of let-7a antagomir.Finally,let-7a antagomir was integrated into chitosan conduit to construct a chitosan-hydrogel scaffold tissue-engineered nerve graft,which promoted nerve regeneration and functional recovery in a rat model of sciatic nerve transection.Our study provides an experimental basis for potential in vivo application of let-7a.展开更多
文摘[目的]研究微小RNA let-7a(microRNA let-7a,miR-let-7a)对脂磷壁酸(Lipoteichoic acid,LTA)诱导的炎性奶牛乳腺上皮细胞(MAC-T)的增殖和凋亡作用。[方法]通过实时荧光定量PCR(Quantitative real time polymerase chain reaction,qRT-PCR)、实时细胞分析仪和流式细胞术检测过表达和抑制表达miR-let-7a在炎性MAC-T中的影响。[结果]qRT-PCR检测结果显示,转染miR-let-7a模拟物会使炎性MAC-T细胞中miR-let-7a的表达量极显著上调(P<0.001),转染miR-let-7a抑制剂会使细胞中miR-let-7a的表达量极显著下降(P<0.001);RTCA结果显示,转染miR-let-7a模拟物能明显促进MAC-T细胞增殖,转染miR-let-7a抑制物会使MAC-T细胞的增殖受到抑制;流式细胞术结果显示,转染miR-let-7a模拟物的MAC-T细胞的凋亡率显著降低(P<0.01);而转染miR-let-7a抑制剂则与之相反,MAC-T细胞的凋亡率显著升高(P<0.01)。即过表达miR-let-7a可以促进MAC-T细胞增殖,并降低细胞的凋亡率。而抑制表达miR-let-7a的作用与之相反,抑制表达miR-let-7a使MAC-T细胞的增殖下降,细胞的凋亡率增大。[结论]miR-let-7a是一种抑制炎症调节因子,其过表达可以缓解奶牛乳腺炎,为阐明奶牛乳腺炎的分子调控机制奠定基础。
基金Supported by the National Natural Science Foundation of China,No.82172297Natural Science Foundation of Jiangsu Province of China,No.BK20211346 and No.BK20201011+1 种基金Natural Science Foundation of Jiangsu Higher Education Institutions of China,No.22KJA310007Xuzhou Science and Technology Project,No.KC22055.
文摘BACKGROUND Primary sclerosing cholangitis(PSC)is characterized by chronic inflammation and it predisposes to cholangiocarcinoma due to lack of effective treatment options.Recombinant adeno-associated virus(rAAV)provides a promising platform for gene therapy on such kinds of diseases.A microRNA(miRNA)let-7a has been reported to be associated with the progress of PSC but the potential therapeutic implication of inhibition of let-7a on PSC has not been evaluated.AIM To investigate the therapeutic effects of inhibition of a miRNA let-7a transferred by recombinant adeno-associated virus 8(rAAV8)on a xenobiotic-induced mouse model of sclerosing cholangitis.METHODS A xenobiotic-induced mouse model of sclerosing cholangitis was induced by 0.1% 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine(DDC)feeding for 2 wk or 6 wk.A single dose of rAAV8-mediated anti-let-7a-5p sponges or scramble control was injected in vivo into mice onset of DDC feeding.Upon sacrifice,the liver and the serum were collected from each mouse.The hepatobiliary injuries,hepatic inflammation and fibrosis were evaluated.The targets of let-7a-5p and downstream molecule NF-κB were detected using Western blot.RESULTS rAAV8-mediated anti-let-7a-5p sponges can depress the expression of let-7a-5p in mice after DDC feeding for 2 wk or 6 wk.The reduced expression of let-7a-5p can alleviate hepato-biliary injuries indicated by serum markers,and prevent the proliferation of cholangiocytes and biliary fibrosis.Furthermore,inhibition of let-7a mediated by rAAV8 can increase the expression of potential target molecules such as suppressor of cytokine signaling 1 and Dectin1,which consequently inhibit of NF-κB-mediated hepatic inflammation.CONCLUSION Our study demonstrates that a rAAV8 vector designed for liver-specific inhibition of let-7a-5p can potently ameliorate symptoms in a xenobiotic-induced mouse model of sclerosing cholangitis,which provides a possible clinical translation of PSC of human.
基金supported by the National Natural Science Foundation of China,No.31970968(to SYL)the Collegiate Natural Science Foundation of Jiangsu Province,No.16KJA310005(to SYL)+1 种基金Priority Academic Program Development of Jiangsu Higher Education Institutions[PAPD]the Natural Science Foundation of Jiangsu Province,No.BK20200976(to XHW).
文摘Neurotrophic factors,particularly nerve growth factor,enhance neuronal regeneration.However,the in vivo applications of nerve growth factor are largely limited by its intrinsic disadvantages,such as its short biological half-life,its contribution to pain response,and its inability to cross the blood-brain barrier.Considering that let-7(human miRNA)targets and regulates nerve growth factor,and that let-7 is a core regulator in peripheral nerve regeneration,we evaluated the possibilities of let-7 application in nerve repair.In this study,anti-let-7a was identified as the most suitable let-7 family molecule by analyses of endogenous expression and regulatory relationship,and functional screening.Let-7a antagomir demonstrated biosafety based on the results of in vivo safety assessments and it entered into the main cell types of the sciatic nerve,including Schwann cells,fibroblasts and macrophages.Use of hydrogel effectively achieved controlled,localized,and sustained delivery of let-7a antagomir.Finally,let-7a antagomir was integrated into chitosan conduit to construct a chitosan-hydrogel scaffold tissue-engineered nerve graft,which promoted nerve regeneration and functional recovery in a rat model of sciatic nerve transection.Our study provides an experimental basis for potential in vivo application of let-7a.
