AIM: To explore the role and potential mechanism of miR-30 b regulation of autophagy in hepatic ischemiareperfusion injury(IRI).METHODS: An animal model of hepatic IRI was generated in C57BL/6 mice. For in vitro studi...AIM: To explore the role and potential mechanism of miR-30 b regulation of autophagy in hepatic ischemiareperfusion injury(IRI).METHODS: An animal model of hepatic IRI was generated in C57BL/6 mice. For in vitro studies, AML12 cells were immersed in mineral oil for 1 h and then cultured in complete Dulbecco's Modified Eagle's Medium(DMEM)/F12 to simulate IRI. Mice and cells were transfected with miR-30 b agomir/mimics or antagomir/inhibitor to examine the effect of miR-30 b on autophagy to promote hepatic IRI. The expression of miR-30 b was measured by real-time polymerase chain reaction. Apoptotic cells were detected by terminal uridine nickend labeling(TUNEL) staining, and cell viability was detected by methylthiazole tetrazolium assay. The expression of light chain 3, autophagy-related gene(Atg)12, Atg5, P62, and caspase-3 were detected by western blotting analysis.RESULTS: miR-30 b levels were significantly downregulated after hepatic IRI, and the numbers of autophagosomes were increased in response to IRI both in vivo and in vitro. These findings demonstrate that low levels of miR-30 b could promote hepatic IRI. Furthermore, we found that miR-30 b interacted with Atg12-Atg5 conjugate by binding to Atg12. Overexpression of miR-30 b diminished Atg12 and Atg12-Atg5 conjugate levels, which promoted autophagy in response to IR. In contrast, downregulation of miR-30 b was associated with increased Atg12-Atg5 conjugate levels and increased autophagy.CONCLUSION: miR-30 b inhibited autophagy to alleviate hepatic ischemia-reperfusion injury via decreasing the Atg12-Atg5 conjugate.展开更多
AIM To investigate autophagy-related genes, particularly ATG12, in apoptosis and cell cycle in hepatitis B virus(HBV)-associated hepatocellular carcinoma(HCC) and non-HBV-HCC cell lines.METHODS The expression of autop...AIM To investigate autophagy-related genes, particularly ATG12, in apoptosis and cell cycle in hepatitis B virus(HBV)-associated hepatocellular carcinoma(HCC) and non-HBV-HCC cell lines.METHODS The expression of autophagy-related genes in HBVassociated hepatocellular carcinoma and non-HBV-HCC cell lines and human liver tissues was examined by quantitative real-time reverse transcriptase-polymerase chain reaction(q RT-PCR) and western blotting. The silencing of target genes was used to examine the function of various genes in apoptosis and cell cycle progression. RESULTS The expression of autophagy related genes ATG5, ATG12, ATG9 A and ATG4 B expression was analyzed in Hep G2.2.15 cells and compared with Hep G2 and THLE cells. We found that ATG5 and ATG12 m RNA expression was significantly increased in Hep G2.2.15 cells compared to HepG 2 cells(P < 0.005). Moreover, ATG5-ATG12 protein levels were increased in tumor liver tissues compared to adjacent non-tumor tissues mainly from HCC patients with HBV infection. We also analyzed the function of ATG12 in cell apoptosis and cell cycle progression. The percentage of apoptotic cells increased by 11.4% in ATG12-silenced Hep G2.2.15 cells(P < 0.005) but did not change in ATG12-silenced HepG 2 cells under starvation with Earle's balanced salt solution. However, the combination blockade of Notch signaling and ATG12 decreased the apoptotic rate of HepG 2.2.15 cells from 55.6% to 50.4%(P < 0.05). CONCLUSION ATG12 is important for HBV-associated apoptosis and a potential drug target for HBV-HCC. Combination inhibition of ATG12/Notch signaling had no additional effect on HepG 2.2.15 apoptosis.展开更多
自噬是一种保守的细胞内降解过程,在多种生物体内证实自噬有重要的生物学意义。但是自噬在牦牛这一高原特色物种体内的研究未见报道。因此为探索正常生理条件下自噬相关基因在牦牛生殖过程中的表达,本研究分别采集牦牛不同繁殖周期(卵...自噬是一种保守的细胞内降解过程,在多种生物体内证实自噬有重要的生物学意义。但是自噬在牦牛这一高原特色物种体内的研究未见报道。因此为探索正常生理条件下自噬相关基因在牦牛生殖过程中的表达,本研究分别采集牦牛不同繁殖周期(卵泡期、黄体期及妊娠期)的主要生殖器官(输卵管、卵巢、子宫),克隆牦牛自噬相关基因5(Autophagy related 5,Atg5)基因并进行生物信息学分析;利用qRT-PCR检测Atg5 基因在组织中的相对表达量;并采用蛋白质免疫印迹(Western-blot, WB)检测Atg5和Atg5-Atg12(Autophagy related 12,自噬相关基因12)复合体在不同组织中的表达水平;免疫组织化学方法分析Atg5在各生殖器官中的分布特征。结果显示,成功克隆牦牛Atg5基因在进化过程中高度保守,编码的蛋白质为可溶性的非跨膜蛋白;qRT-PCR和WB检测结果显示Atg5和Atg5-Atg12复合体在牦牛输卵管、卵巢和子宫中均有表达。其中卵泡期卵巢Atg5-Atg12表达显著高于黄体期和妊娠期,卵泡期Atg5的表达却显著低于黄体期和妊娠期;黄体期输卵管Atg5-Atg12表达量显著高于卵泡期和妊娠期,妊娠期输卵管中Atg5的表达量显著高于卵泡期和黄体期;卵泡期和妊娠期子宫中Atg5-Atg12的表达量显著高于黄体期,而黄体期子宫中的Atg5的表达量又显著高于妊娠期和卵泡期,在蛋白水平上Atg5和Atg5-Atg12的表达呈负相关。免疫组织化学结果显示Atg5在输卵管黏膜上皮,卵巢卵泡膜、颗粒层、生殖上皮、黄体细胞,子宫内膜和子宫腺体均有表达。研究结果表明自噬在牦牛体内与其他物种相似具有保守性,通过检测Atg5-Atg12复合体在牦牛生殖器官中的表达,推测自噬可能参与牦牛生殖生理过程的调控。该研究结果对自噬在其他大型哺乳动物以及高寒低氧环境中动物的研究具有借鉴意义,有助于自噬参与其他动物生殖生理作用机制的研究。展开更多
As an important horticultural plant,the orchid is widely distributed in its natural habitat and faces various environmental stresses,among which nutrient recycling and stress resistance are of great concern.During the...As an important horticultural plant,the orchid is widely distributed in its natural habitat and faces various environmental stresses,among which nutrient recycling and stress resistance are of great concern.During these processes,autophagy is an essential pathway,which is a conserved self-eating process that degrades macromolecular components and recycles cell materials or nutrients during developmental processes or under stress conditions.Two ubiquitin-like systems(UBLs)play a major role in the initiation of autophagy and are associated with two key proteins:ATG8 and ATG12.In this study,we identified and refined the UBL-related genes in orchids and performed phylogenetic reconstruction together with other plant species.We found that the orchid had unique domains in UBL-related genes,indicating potential functional diversification in the ATG8 system in plants.Transcriptome and protein tertiary structure prediction indicated that conserved domains that are vital for the canonical function of ATG12 are incomplete in orchids,in which a novel mechanism of autophagy may have evolved.展开更多
基金Supported by National High Technology Research and Development Program(863)of China,No.2012AA021001National Natural Science Foundation of China,No.81270554+1 种基金Special Fund for Health Research in the Public Interest of China,No.201302009National Key Specialty Construction of Clinical Projects,No.201354409
文摘AIM: To explore the role and potential mechanism of miR-30 b regulation of autophagy in hepatic ischemiareperfusion injury(IRI).METHODS: An animal model of hepatic IRI was generated in C57BL/6 mice. For in vitro studies, AML12 cells were immersed in mineral oil for 1 h and then cultured in complete Dulbecco's Modified Eagle's Medium(DMEM)/F12 to simulate IRI. Mice and cells were transfected with miR-30 b agomir/mimics or antagomir/inhibitor to examine the effect of miR-30 b on autophagy to promote hepatic IRI. The expression of miR-30 b was measured by real-time polymerase chain reaction. Apoptotic cells were detected by terminal uridine nickend labeling(TUNEL) staining, and cell viability was detected by methylthiazole tetrazolium assay. The expression of light chain 3, autophagy-related gene(Atg)12, Atg5, P62, and caspase-3 were detected by western blotting analysis.RESULTS: miR-30 b levels were significantly downregulated after hepatic IRI, and the numbers of autophagosomes were increased in response to IRI both in vivo and in vitro. These findings demonstrate that low levels of miR-30 b could promote hepatic IRI. Furthermore, we found that miR-30 b interacted with Atg12-Atg5 conjugate by binding to Atg12. Overexpression of miR-30 b diminished Atg12 and Atg12-Atg5 conjugate levels, which promoted autophagy in response to IR. In contrast, downregulation of miR-30 b was associated with increased Atg12-Atg5 conjugate levels and increased autophagy.CONCLUSION: miR-30 b inhibited autophagy to alleviate hepatic ischemia-reperfusion injury via decreasing the Atg12-Atg5 conjugate.
基金Supported by National Research Council of Thailand 2013 and the Ratchadaphiseksomphot Matching Fund from the Faculty of Medicine,Chulalongkorn UniversityInternational Research Integration,Chula Research Scholar,Ratchadaphiseksomphot Endowment FundCenter of Excellence in Immunology and Immune-mediated Diseases and the Rachadapisaek Sompote Post-Doctoral Fund,Chulalongkorn University
文摘AIM To investigate autophagy-related genes, particularly ATG12, in apoptosis and cell cycle in hepatitis B virus(HBV)-associated hepatocellular carcinoma(HCC) and non-HBV-HCC cell lines.METHODS The expression of autophagy-related genes in HBVassociated hepatocellular carcinoma and non-HBV-HCC cell lines and human liver tissues was examined by quantitative real-time reverse transcriptase-polymerase chain reaction(q RT-PCR) and western blotting. The silencing of target genes was used to examine the function of various genes in apoptosis and cell cycle progression. RESULTS The expression of autophagy related genes ATG5, ATG12, ATG9 A and ATG4 B expression was analyzed in Hep G2.2.15 cells and compared with Hep G2 and THLE cells. We found that ATG5 and ATG12 m RNA expression was significantly increased in Hep G2.2.15 cells compared to HepG 2 cells(P < 0.005). Moreover, ATG5-ATG12 protein levels were increased in tumor liver tissues compared to adjacent non-tumor tissues mainly from HCC patients with HBV infection. We also analyzed the function of ATG12 in cell apoptosis and cell cycle progression. The percentage of apoptotic cells increased by 11.4% in ATG12-silenced Hep G2.2.15 cells(P < 0.005) but did not change in ATG12-silenced HepG 2 cells under starvation with Earle's balanced salt solution. However, the combination blockade of Notch signaling and ATG12 decreased the apoptotic rate of HepG 2.2.15 cells from 55.6% to 50.4%(P < 0.05). CONCLUSION ATG12 is important for HBV-associated apoptosis and a potential drug target for HBV-HCC. Combination inhibition of ATG12/Notch signaling had no additional effect on HepG 2.2.15 apoptosis.
文摘自噬是一种保守的细胞内降解过程,在多种生物体内证实自噬有重要的生物学意义。但是自噬在牦牛这一高原特色物种体内的研究未见报道。因此为探索正常生理条件下自噬相关基因在牦牛生殖过程中的表达,本研究分别采集牦牛不同繁殖周期(卵泡期、黄体期及妊娠期)的主要生殖器官(输卵管、卵巢、子宫),克隆牦牛自噬相关基因5(Autophagy related 5,Atg5)基因并进行生物信息学分析;利用qRT-PCR检测Atg5 基因在组织中的相对表达量;并采用蛋白质免疫印迹(Western-blot, WB)检测Atg5和Atg5-Atg12(Autophagy related 12,自噬相关基因12)复合体在不同组织中的表达水平;免疫组织化学方法分析Atg5在各生殖器官中的分布特征。结果显示,成功克隆牦牛Atg5基因在进化过程中高度保守,编码的蛋白质为可溶性的非跨膜蛋白;qRT-PCR和WB检测结果显示Atg5和Atg5-Atg12复合体在牦牛输卵管、卵巢和子宫中均有表达。其中卵泡期卵巢Atg5-Atg12表达显著高于黄体期和妊娠期,卵泡期Atg5的表达却显著低于黄体期和妊娠期;黄体期输卵管Atg5-Atg12表达量显著高于卵泡期和妊娠期,妊娠期输卵管中Atg5的表达量显著高于卵泡期和黄体期;卵泡期和妊娠期子宫中Atg5-Atg12的表达量显著高于黄体期,而黄体期子宫中的Atg5的表达量又显著高于妊娠期和卵泡期,在蛋白水平上Atg5和Atg5-Atg12的表达呈负相关。免疫组织化学结果显示Atg5在输卵管黏膜上皮,卵巢卵泡膜、颗粒层、生殖上皮、黄体细胞,子宫内膜和子宫腺体均有表达。研究结果表明自噬在牦牛体内与其他物种相似具有保守性,通过检测Atg5-Atg12复合体在牦牛生殖器官中的表达,推测自噬可能参与牦牛生殖生理过程的调控。该研究结果对自噬在其他大型哺乳动物以及高寒低氧环境中动物的研究具有借鉴意义,有助于自噬参与其他动物生殖生理作用机制的研究。
文摘As an important horticultural plant,the orchid is widely distributed in its natural habitat and faces various environmental stresses,among which nutrient recycling and stress resistance are of great concern.During these processes,autophagy is an essential pathway,which is a conserved self-eating process that degrades macromolecular components and recycles cell materials or nutrients during developmental processes or under stress conditions.Two ubiquitin-like systems(UBLs)play a major role in the initiation of autophagy and are associated with two key proteins:ATG8 and ATG12.In this study,we identified and refined the UBL-related genes in orchids and performed phylogenetic reconstruction together with other plant species.We found that the orchid had unique domains in UBL-related genes,indicating potential functional diversification in the ATG8 system in plants.Transcriptome and protein tertiary structure prediction indicated that conserved domains that are vital for the canonical function of ATG12 are incomplete in orchids,in which a novel mechanism of autophagy may have evolved.
