目的探讨microRNA-1469(miR-1469)在肝细胞癌(HCC)中的表达及其对肝癌细胞生物学行为的影响。方法选取2010年1月至2012年12月于西安交通大学第一附属医院手术切除的HCC组织和对应的癌旁组织(距肿瘤边缘>2 cm)标本各76例,采用实时荧...目的探讨microRNA-1469(miR-1469)在肝细胞癌(HCC)中的表达及其对肝癌细胞生物学行为的影响。方法选取2010年1月至2012年12月于西安交通大学第一附属医院手术切除的HCC组织和对应的癌旁组织(距肿瘤边缘>2 cm)标本各76例,采用实时荧光定量聚合酶链反应检测HCC组织及癌旁组织中miR-1469的表达水平,分析miR-1469表达与HCC临床病理特征的关系,Kaplan-Meier生存曲线分析miR-1469表达与HCC患者3 a总体生存率的关系。采用实时荧光定量聚合酶链反应检测HCC细胞系HepG2、Hep3B、SMMC-7721、Huh7、Bel-7402及正常肝细胞系LO2中miR-1469的表达水平;培养Hep3B和SMMC-7721细胞,当细胞融合度达到70%时,分别应用miR-1469 mimics和相应的阴性对照脂质体转染Hep3B细胞,应用miR-1469 inhibitors和相应的阴性对照脂质体转染SMMC-7721细胞。转染48 h后,采用实时荧光定量聚合酶链反应检测各组细胞中miR-1469的表达水平,采用四甲基偶氮唑盐(MTT)法和平板克隆形成实验观察miR-1469对Hep3B和SMMC-7721细胞增殖及克隆形成能力的影响,流式细胞术检测miR-1469对Hep3B和SMMC-7721细胞周期的影响,Transwell实验观察miR-1469对Hep3B和SMMC-7721细胞侵袭及迁移能力的影响,Western blot法检测Hep3B和SMMC-7721细胞中NDRG1蛋白表达。结果miR-1469在HCC组织和癌旁组织中的相对表达量分别为0.71±0.03、5.49±0.04,HCC组织中miR-1469相对表达量显著低于癌旁组织( P <0.05)。miR-1469表达与肿瘤直径、肿瘤数目、TNM分期、组织分化程度及微血管侵犯相关( P <0.05),而与患者的年龄、性别、血清甲胎蛋白水平、静脉侵犯、Edmondson病理分级、肿瘤部位无相关性( P >0.05)。Kaplan-Meier生存分析显示,miR-1469高表达HCC患者的3 a总体生存率显著高于低表达者( P <0.05)。miR-1469在LO2、HepG2、Hep3B、SMMC-7721、Huh7、Bel-7402细胞中的相对表达量分别为1.01±0.02、0.45±0.01、0.05±0.01、0.61±0.02、0.14±0.01、0.29±0.02;miR-1469在HepG2、Hep3B、SMMC-7721、Huh7、Bel-7402细胞中的相对表达量显著低于LO2细胞( P <0.05);其中Hep3B细胞中miR-1469表达量最低,SMMC-7721细胞中miR-1469表达量最高。miR-1469 mimics组和miR-1469 control mimics组Hep3B细胞中miR-1469相对表达量分别为4.47±0.15、0.05±0.01,miR-1469 mimics组Hep3B细胞中miR-1469相对表达量显著高于miR-1469 control mimics组( P <0.05)。miR-1469 inhibitors组和miR-1469 control inhibitors组SMMC-7721细胞中miR-1469相对表达量分别为0.20±0.11、1.00±0.00,miR-1469 inhibitors组SMMC-7721细胞中miR-1469相对表达量显著低于miR-1469 control inhibitors组( P <0.05)。MTT结果显示,培养24、48、72 h时,miR-1469 mimics组Hep3B细胞增殖能力显著低于miR-1469 control mimics组,miR-1469 inhibitors组SMMC-7721细胞增殖能力显著高于miR-1469 control inhibitors组( P <0.05)。平板克隆实验结果显示,miR-1469 mimics组和miR-1469 control mimics组Hep3B细胞的克隆数分别为17.00±1.73、65.67±2.33,miR-1469 mimics组Hep3B细胞的克隆形成能力显著低于miR-1469 control mimics组( P <0.05);miR-1469 inhibitors组和miR-1469 control inhibitors组SMMC-7721细胞的克隆数分别为93.00±2.08、27.67±1.45,miR-1469 inhibitors组SMMC-7721细胞的克隆形成能力显著高于miR-1469 control inhibitors组( P <0.05)。流式细胞术结果显示,与miR-1469 control mimics组比较,miR-1469 mimics组G 1期Hep3B细胞显著增多,S期Hep3B细胞显著减少( P <0.05),但2组G 2期细胞比例比较差异无统计学意义( P >0.05)。与miR-1469 control inhibitors组比较,miR-1469 inhibitors组G 1期SMMC-7721细胞显著减少,S期SMMC-7721细胞显著增多( P <0.05);但2组G 2期SMMC-7721细胞比例比较差异无统计学意义( P >0.05)。Transwell实验结果显示,miR-1469 mimics组Hep3B细胞迁移数和侵袭数分别为59.00±2.08、29.00±2.08,miR-1469 control mimics组Hep3B细胞迁移数和侵袭数分别为35.00±1.53、20.33±1.45;miR-1469 mimics组Hep3B细胞的迁移和侵袭能力显著高于miR-1469 control mimics组( P <0.05)。miR-1469 inhibitors组SMMC-7721细胞迁移数和侵袭数分别为26.00±1.16、17.33±0.88,miR-1469 control inhibitors组SMMC-7721细胞迁移数和侵袭数分别为56.33±3.18、44.67±1.45;miR-1469 inhibitors组SMMC-7721细胞的迁移和侵袭能力显著低于miR-1469 control inhibitors组( P <0.05)。Western blot结果显示,miR-1469 mimics组和miR-1469 control mimics组Hep3B细胞中NDRG1蛋白相对表达量分别为0.23±0.04、1.00±0.00,miR-1469 mimics组Hep3B细胞中NDRG1蛋白相对表达量显著低于miR-1469 control mimics组( P <0.05);miR-1469 inhibitors组和miR-1469 control inhibitors组SMMC-7721细胞中NDRG1蛋白相对表达量分别为3.90±0.17、1.00±0.00,miR-1469 inhibitors组SMMC-7721细胞中NDRG1蛋白相对表达量显著高于miR-1469 control inhibitors组( P <0.05)。结论 MiR-1469在HCC中表达下调,且其表达与肿瘤数目、肿瘤直径、TNM分期、组织分化程度、微血管侵犯及患者预后密切相关;miR-1469可能通过靶向结合NDRG1并抑制其表达而抑制HCC细胞的增殖、周期、侵袭和迁移。展开更多
BACKGROUND Hepatocellular carcinoma(HCC)has been a pervasive malignancy throughout the world with elevated mortality.Efficient therapeutic targets are beneficial to treat and predict the disease.Currently,the exact mo...BACKGROUND Hepatocellular carcinoma(HCC)has been a pervasive malignancy throughout the world with elevated mortality.Efficient therapeutic targets are beneficial to treat and predict the disease.Currently,the exact molecular mechanisms leading to the progression of HCC are still unclear.Research has shown that the microRNA-142-3p level decreases in HCC,whereas bioinformatics analysis of the cancer genome atlas database shows the ASH1L expression increased among liver tumor tissues.In this paper,we will explore the effects and mechanisms of microRNA-142-3p and ASH1L affect the prognosis of HCC patients and HCC cell bioactivity,and the association between them.AIM To investigate the effects and mechanisms of microRNA-142-3p and ASH1L on the HCC cell bioactivity and prognosis of HCC patients.METHODS In this study,we grouped HCC patients according to their immunohistochemistry results of ASH1L with pathological tissues,and retrospectively analyzed the prognosis of HCC patients.Furthermore,explored the roles and mechanisms of microRNA-142-3p and ASH1L by cellular and animal experiments,which involved the following experimental methods:Immunohistochemical staining,western blot,quantitative real-time-polymerase chain reaction,flow cytometric analysis,tumor xenografts in nude mice,etc.The statistical methods involved in this study contained t-test,one-way analysis of variance,theχ^(2)test,the Kaplan-Meier approach and the log-rank test.RESULTS In this study,we found that HCC patients with high expression of ASH1L possess a more recurrence rate as well as a decreased overall survival rate.ASH1L promotes the tumorigenicity of HCC and microRNA-142-3p exhibits reduced expression in HCC tissues and interacts with ASH1L through targeting the ASH1L 3′untranslated region.Furthermore,microRNA-142-3p promotes apoptosis and inhibits proliferation,invasion,and migration of HCC cell lines in vitro via ASH1L.For the exploration mechanism,we found ASH1L may promote an immunosuppressive microenvironment in HCC and ASH1L affects the expression of the cell junction protein zonula occludens-1,which is potentially relevant to the immune system.CONCLUSION Loss function of microRNA-142-3p induces cancer progression and immune evasion through upregulation of ASH1L in HCC.Both microRNA-142-3p and ASH1L can feature as new biomarker for HCC in the future.展开更多
BACKGROUND Shifting from the inflammatory to the proliferative phase represents a pivotal step during managing diabetic foot ulcers(DFUs);however,existing medical interventions remain insufficient.MicroRNAs(miRs)highl...BACKGROUND Shifting from the inflammatory to the proliferative phase represents a pivotal step during managing diabetic foot ulcers(DFUs);however,existing medical interventions remain insufficient.MicroRNAs(miRs)highlight notable capacity for accelerating the repair process of DFUs.Previous research has demonstrated which miR-122-5p regulates matrix metalloproteinases under diabetic conditions,thereby influencing extracellular matrix dynamics.AIM To investigate the impact of miR-122-5p on the transition from the inflammatory to the proliferative stage in DFU.METHODS Analysis for miR-122-5p expression in skin tissues from diabetic ulcer patients and mice was analyzed using quantitative real-time polymerase chain reaction(qRT-PCR).A diabetic wound healing model induced by streptozotocin was used,with mice receiving intradermal injections of adeno-associated virus-DJ encoding empty vector or miR-122.Skin tissues were retrieved at 3,7,and 14 days after injury for gene expression analysis,histology,immunohistochemistry,and network studies.The study explored miR-122-5p’s role in macrophage-fibroblast interactions and its effect on transitioning from inflammation to proliferation in DFU healing.RESULTS High-throughput sequencing revealed miR-122-5p as crucial for DFU healing.qRT-PCR showed significant upregulation of miR-122-5p within diabetic skin among DFU individuals and mice.Western blot,along with immunohistochemical and enzyme-linked immunosorbent assay,demonstrating the upregulation of inflammatory mediators(hypoxia inducible factor-1α,matrix metalloproteinase 9,tumor necrosis factor-α)and reduced fibrosis markers(fibronectin 1,α-smooth muscle actin)by targeting vascular endothelial growth factor.Fluorescence in situ hybridization indicated its expression localized to epidermal keratinocytes and fibroblasts in diabetic mice.Immunofluorescence revealed enhanced increased presence of M1 macrophages and reduced M2 polarization,highlighting its role in inflammation.MiR-122-5p elevated inflammatory cytokine levels while suppressing fibrotic activity from fibroblasts exposed to macrophage-derived media,highlighting its pivotal role in regulating DFU healing.CONCLUSION MiR-122-5p impedes cutaneous healing of diabetic mice via enhancing inflammation and inhibiting fibrosis,offering insights into miR roles in human skin wound repair.展开更多
文摘目的探讨microRNA-1469(miR-1469)在肝细胞癌(HCC)中的表达及其对肝癌细胞生物学行为的影响。方法选取2010年1月至2012年12月于西安交通大学第一附属医院手术切除的HCC组织和对应的癌旁组织(距肿瘤边缘>2 cm)标本各76例,采用实时荧光定量聚合酶链反应检测HCC组织及癌旁组织中miR-1469的表达水平,分析miR-1469表达与HCC临床病理特征的关系,Kaplan-Meier生存曲线分析miR-1469表达与HCC患者3 a总体生存率的关系。采用实时荧光定量聚合酶链反应检测HCC细胞系HepG2、Hep3B、SMMC-7721、Huh7、Bel-7402及正常肝细胞系LO2中miR-1469的表达水平;培养Hep3B和SMMC-7721细胞,当细胞融合度达到70%时,分别应用miR-1469 mimics和相应的阴性对照脂质体转染Hep3B细胞,应用miR-1469 inhibitors和相应的阴性对照脂质体转染SMMC-7721细胞。转染48 h后,采用实时荧光定量聚合酶链反应检测各组细胞中miR-1469的表达水平,采用四甲基偶氮唑盐(MTT)法和平板克隆形成实验观察miR-1469对Hep3B和SMMC-7721细胞增殖及克隆形成能力的影响,流式细胞术检测miR-1469对Hep3B和SMMC-7721细胞周期的影响,Transwell实验观察miR-1469对Hep3B和SMMC-7721细胞侵袭及迁移能力的影响,Western blot法检测Hep3B和SMMC-7721细胞中NDRG1蛋白表达。结果miR-1469在HCC组织和癌旁组织中的相对表达量分别为0.71±0.03、5.49±0.04,HCC组织中miR-1469相对表达量显著低于癌旁组织( P <0.05)。miR-1469表达与肿瘤直径、肿瘤数目、TNM分期、组织分化程度及微血管侵犯相关( P <0.05),而与患者的年龄、性别、血清甲胎蛋白水平、静脉侵犯、Edmondson病理分级、肿瘤部位无相关性( P >0.05)。Kaplan-Meier生存分析显示,miR-1469高表达HCC患者的3 a总体生存率显著高于低表达者( P <0.05)。miR-1469在LO2、HepG2、Hep3B、SMMC-7721、Huh7、Bel-7402细胞中的相对表达量分别为1.01±0.02、0.45±0.01、0.05±0.01、0.61±0.02、0.14±0.01、0.29±0.02;miR-1469在HepG2、Hep3B、SMMC-7721、Huh7、Bel-7402细胞中的相对表达量显著低于LO2细胞( P <0.05);其中Hep3B细胞中miR-1469表达量最低,SMMC-7721细胞中miR-1469表达量最高。miR-1469 mimics组和miR-1469 control mimics组Hep3B细胞中miR-1469相对表达量分别为4.47±0.15、0.05±0.01,miR-1469 mimics组Hep3B细胞中miR-1469相对表达量显著高于miR-1469 control mimics组( P <0.05)。miR-1469 inhibitors组和miR-1469 control inhibitors组SMMC-7721细胞中miR-1469相对表达量分别为0.20±0.11、1.00±0.00,miR-1469 inhibitors组SMMC-7721细胞中miR-1469相对表达量显著低于miR-1469 control inhibitors组( P <0.05)。MTT结果显示,培养24、48、72 h时,miR-1469 mimics组Hep3B细胞增殖能力显著低于miR-1469 control mimics组,miR-1469 inhibitors组SMMC-7721细胞增殖能力显著高于miR-1469 control inhibitors组( P <0.05)。平板克隆实验结果显示,miR-1469 mimics组和miR-1469 control mimics组Hep3B细胞的克隆数分别为17.00±1.73、65.67±2.33,miR-1469 mimics组Hep3B细胞的克隆形成能力显著低于miR-1469 control mimics组( P <0.05);miR-1469 inhibitors组和miR-1469 control inhibitors组SMMC-7721细胞的克隆数分别为93.00±2.08、27.67±1.45,miR-1469 inhibitors组SMMC-7721细胞的克隆形成能力显著高于miR-1469 control inhibitors组( P <0.05)。流式细胞术结果显示,与miR-1469 control mimics组比较,miR-1469 mimics组G 1期Hep3B细胞显著增多,S期Hep3B细胞显著减少( P <0.05),但2组G 2期细胞比例比较差异无统计学意义( P >0.05)。与miR-1469 control inhibitors组比较,miR-1469 inhibitors组G 1期SMMC-7721细胞显著减少,S期SMMC-7721细胞显著增多( P <0.05);但2组G 2期SMMC-7721细胞比例比较差异无统计学意义( P >0.05)。Transwell实验结果显示,miR-1469 mimics组Hep3B细胞迁移数和侵袭数分别为59.00±2.08、29.00±2.08,miR-1469 control mimics组Hep3B细胞迁移数和侵袭数分别为35.00±1.53、20.33±1.45;miR-1469 mimics组Hep3B细胞的迁移和侵袭能力显著高于miR-1469 control mimics组( P <0.05)。miR-1469 inhibitors组SMMC-7721细胞迁移数和侵袭数分别为26.00±1.16、17.33±0.88,miR-1469 control inhibitors组SMMC-7721细胞迁移数和侵袭数分别为56.33±3.18、44.67±1.45;miR-1469 inhibitors组SMMC-7721细胞的迁移和侵袭能力显著低于miR-1469 control inhibitors组( P <0.05)。Western blot结果显示,miR-1469 mimics组和miR-1469 control mimics组Hep3B细胞中NDRG1蛋白相对表达量分别为0.23±0.04、1.00±0.00,miR-1469 mimics组Hep3B细胞中NDRG1蛋白相对表达量显著低于miR-1469 control mimics组( P <0.05);miR-1469 inhibitors组和miR-1469 control inhibitors组SMMC-7721细胞中NDRG1蛋白相对表达量分别为3.90±0.17、1.00±0.00,miR-1469 inhibitors组SMMC-7721细胞中NDRG1蛋白相对表达量显著高于miR-1469 control inhibitors组( P <0.05)。结论 MiR-1469在HCC中表达下调,且其表达与肿瘤数目、肿瘤直径、TNM分期、组织分化程度、微血管侵犯及患者预后密切相关;miR-1469可能通过靶向结合NDRG1并抑制其表达而抑制HCC细胞的增殖、周期、侵袭和迁移。
基金Supported by the Haihe Laboratory of Cell Ecosystem Innovation Fund,No.22HHXBJC00001the Key Discipline Special Project of Tianjin Municipal Health Commission,No.TJWJ2022XK016.
文摘BACKGROUND Hepatocellular carcinoma(HCC)has been a pervasive malignancy throughout the world with elevated mortality.Efficient therapeutic targets are beneficial to treat and predict the disease.Currently,the exact molecular mechanisms leading to the progression of HCC are still unclear.Research has shown that the microRNA-142-3p level decreases in HCC,whereas bioinformatics analysis of the cancer genome atlas database shows the ASH1L expression increased among liver tumor tissues.In this paper,we will explore the effects and mechanisms of microRNA-142-3p and ASH1L affect the prognosis of HCC patients and HCC cell bioactivity,and the association between them.AIM To investigate the effects and mechanisms of microRNA-142-3p and ASH1L on the HCC cell bioactivity and prognosis of HCC patients.METHODS In this study,we grouped HCC patients according to their immunohistochemistry results of ASH1L with pathological tissues,and retrospectively analyzed the prognosis of HCC patients.Furthermore,explored the roles and mechanisms of microRNA-142-3p and ASH1L by cellular and animal experiments,which involved the following experimental methods:Immunohistochemical staining,western blot,quantitative real-time-polymerase chain reaction,flow cytometric analysis,tumor xenografts in nude mice,etc.The statistical methods involved in this study contained t-test,one-way analysis of variance,theχ^(2)test,the Kaplan-Meier approach and the log-rank test.RESULTS In this study,we found that HCC patients with high expression of ASH1L possess a more recurrence rate as well as a decreased overall survival rate.ASH1L promotes the tumorigenicity of HCC and microRNA-142-3p exhibits reduced expression in HCC tissues and interacts with ASH1L through targeting the ASH1L 3′untranslated region.Furthermore,microRNA-142-3p promotes apoptosis and inhibits proliferation,invasion,and migration of HCC cell lines in vitro via ASH1L.For the exploration mechanism,we found ASH1L may promote an immunosuppressive microenvironment in HCC and ASH1L affects the expression of the cell junction protein zonula occludens-1,which is potentially relevant to the immune system.CONCLUSION Loss function of microRNA-142-3p induces cancer progression and immune evasion through upregulation of ASH1L in HCC.Both microRNA-142-3p and ASH1L can feature as new biomarker for HCC in the future.
基金Supported by the National Natural Science Foundation of China,No.82274528.
文摘BACKGROUND Shifting from the inflammatory to the proliferative phase represents a pivotal step during managing diabetic foot ulcers(DFUs);however,existing medical interventions remain insufficient.MicroRNAs(miRs)highlight notable capacity for accelerating the repair process of DFUs.Previous research has demonstrated which miR-122-5p regulates matrix metalloproteinases under diabetic conditions,thereby influencing extracellular matrix dynamics.AIM To investigate the impact of miR-122-5p on the transition from the inflammatory to the proliferative stage in DFU.METHODS Analysis for miR-122-5p expression in skin tissues from diabetic ulcer patients and mice was analyzed using quantitative real-time polymerase chain reaction(qRT-PCR).A diabetic wound healing model induced by streptozotocin was used,with mice receiving intradermal injections of adeno-associated virus-DJ encoding empty vector or miR-122.Skin tissues were retrieved at 3,7,and 14 days after injury for gene expression analysis,histology,immunohistochemistry,and network studies.The study explored miR-122-5p’s role in macrophage-fibroblast interactions and its effect on transitioning from inflammation to proliferation in DFU healing.RESULTS High-throughput sequencing revealed miR-122-5p as crucial for DFU healing.qRT-PCR showed significant upregulation of miR-122-5p within diabetic skin among DFU individuals and mice.Western blot,along with immunohistochemical and enzyme-linked immunosorbent assay,demonstrating the upregulation of inflammatory mediators(hypoxia inducible factor-1α,matrix metalloproteinase 9,tumor necrosis factor-α)and reduced fibrosis markers(fibronectin 1,α-smooth muscle actin)by targeting vascular endothelial growth factor.Fluorescence in situ hybridization indicated its expression localized to epidermal keratinocytes and fibroblasts in diabetic mice.Immunofluorescence revealed enhanced increased presence of M1 macrophages and reduced M2 polarization,highlighting its role in inflammation.MiR-122-5p elevated inflammatory cytokine levels while suppressing fibrotic activity from fibroblasts exposed to macrophage-derived media,highlighting its pivotal role in regulating DFU healing.CONCLUSION MiR-122-5p impedes cutaneous healing of diabetic mice via enhancing inflammation and inhibiting fibrosis,offering insights into miR roles in human skin wound repair.
