目的:探讨miR-20a-3p基因干扰对人牙髓干细胞(hDPSCs)成牙本质分化的影响及信号通路调控机制。方法:对hDPSCs转染miR-20a-3p-mimic和miR-20a-3p-inhibitor来上调或下调其表达,然后对hDPSCs进行成牙本质分化诱导培养。通过茜素红染色观...目的:探讨miR-20a-3p基因干扰对人牙髓干细胞(hDPSCs)成牙本质分化的影响及信号通路调控机制。方法:对hDPSCs转染miR-20a-3p-mimic和miR-20a-3p-inhibitor来上调或下调其表达,然后对hDPSCs进行成牙本质分化诱导培养。通过茜素红染色观察细胞钙基质的矿化程度并检测ALP活性。通过双荧光素酶报告基因测定实验验证miR-20a-3p与mothers against decapentaplegic homolog(SMAD)特异性E3泛素蛋白连接酶1(SMURF1)的靶向调节关系。通过RT-qPCR检测hDPSCs中miR-20a-3p以及SMURF1、Runt相关转录因子2(RUNX2)、骨钙蛋白(OCN)和牙本质涎磷蛋白(DSPP)mRNA水平。结果:与Control组相比,miR-20a-3p-mimic组的茜素红相对染色强度升高了67.09%,miR-20a-3p-inhibitor组降低了46.13%(P<0.05)。与Control组相比,miR-20a-3p-mimic组的相对ALP活性升高了52.89%,miR-20a-3p-inhibitor组降低了53.32%(P<0.05)。与Control组相比,miR-20a-3p-mimic组的RUNX2、OCN和DSPP mRNA相对表达量分别升高了2.19倍、1.86倍和2.35倍,miR-20a-3p-inhibitor组分别降低了63.26%、58.84%和68.12%(P<0.05)。双荧光素酶报告基因测定显示miR-20a-3p靶向抑制SMURF1(P<0.05)。过表达SMURF1抑制了miR-20a-3p对成牙本质分化的影响(P<0.05)。结论:miR-20a-3p通过靶向抑制SMURF1及其下游基因促进hDPSCs的成牙本质分化。展开更多
为了探讨hsa-miR-125a-3p调控肺癌细胞侵袭的作用机制,分别检测了转染正义和反义hsa-miR-125a-3p的人肺癌A549细胞中Rho A mRNA和蛋白水平表达情况,结果显示,两种方式处理的细胞中Rho A mRNA均无明显变化,但Rho A蛋白浓度分别下降和增加...为了探讨hsa-miR-125a-3p调控肺癌细胞侵袭的作用机制,分别检测了转染正义和反义hsa-miR-125a-3p的人肺癌A549细胞中Rho A mRNA和蛋白水平表达情况,结果显示,两种方式处理的细胞中Rho A mRNA均无明显变化,但Rho A蛋白浓度分别下降和增加;再用包含Rho A 3’-未翻译区(3’-UTR)荧光素酶报告基因与hsa-miR-125a-3p mimics共转染发现,当hsa-miR-125a-3p异常表达时,报告基因活性降低。进一步的实验显示,hsa-miR-125a-3p下调可诱导A549细胞侵袭;Rho抑制剂CT04阻断Rho A后,转染反义或正义hsa-miR-125a-3p的A549细胞侵袭率均无变化;转染正义的hsa-miR-125a-3p的A549细胞中活化Rho A(Rho A-GTP)、Rho A和肌动蛋白丝聚集表达均降低;相反,当转染反义的hsa-miR-125a-3p时,Rho A-GTP、Rho A和肌动蛋白丝聚集表达均增强。上述研究表明,hsa-miR-125a-3p可能通过Rho A-肌动蛋白途径影响肺癌细胞系A549的侵袭能力。展开更多
目的:探讨hsa-miR-103a-3p影响食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)细胞对奥沙利铂(oxaliplatin,OXA)耐药的机制。方法:采用RT-qPCR验证测序结果中hsa-miR-103a-3p在不同ESCC细胞系中的表达;转染hsa-miR-103a-3p m...目的:探讨hsa-miR-103a-3p影响食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)细胞对奥沙利铂(oxaliplatin,OXA)耐药的机制。方法:采用RT-qPCR验证测序结果中hsa-miR-103a-3p在不同ESCC细胞系中的表达;转染hsa-miR-103a-3p mimics或inhibitor,验证hsa-miR-103a-3p是否可以逆转ESCC细胞系的耐药表型。应用双萤光素酶报告基因实验和RT-qPCR验证hsa-miR-103a-3p与RASSF8(Ras association domain family member 8)的靶向调控关系;挽救实验验证改变RASSF8的表达是否可以逆转由hsa-miR-103a-3p引起的耐药表型。PCR-array实验检测hsa-miR-103a-3p参与ESCC的耐药机制;Kaplan-Meier生存分析预测hsa-miR-103a-3p高表达与患者生存时间的关系。结果:hsa-miR-103a-3p在OXA敏感ESCC细胞系中高表达(P<0.05);hsa-miR-103a-3p的表达影响ESCC细胞对OXA的敏感性(P<0.05);与对照组相比,过表达hsa-miR-103a-3p能够降低RASSF83’端非翻译区萤光素酶报告基因质粒的活性(P<0.05);挽救实验结果显示,改变RASSF8的表达可有效挽救由hsa-miR-103a-3p引起的耐药表型(P<0.05)。PCR-array实验结果显示,RASSF8高表达能够促进XPA(xeroderma pigmentosum complementation group A)和XPC(xeroderma pigmentosum complementation group C)表达。配对t检验分析结果显示,hsamiR-103a-3p在肿瘤组织中高表达;Kaplan-Meier生存分析显示,与hsa-miR-103a-3p低表达的患者相比,hsa-miR-103a-3p高表达的患者生存时间显著缩短(P<0.05)。结论:hsa-miR-103a-3p低表达通过靶向上调RASSF8的表达促进ESCC细胞对OXA耐药。展开更多
BACKGROUND The incidence of inflammatory bowel disease,a chronic intestinal inflammatory disorder that includes Crohn’s disease(CD)and ulcerative colitis,is rising.Circular RNAs are considered valuable diagnostic bio...BACKGROUND The incidence of inflammatory bowel disease,a chronic intestinal inflammatory disorder that includes Crohn’s disease(CD)and ulcerative colitis,is rising.Circular RNAs are considered valuable diagnostic biomarkers for CD.Current evidence supports the views that epithelial-mesenchymal transition(EMT)plays an important role in CD pathogenesis,and that hsa-miR-130a-3p can inhibit transforming growth factor-β1(TGF-β1)-induced EMT.Our previous study revealed that hsa_circRNA_102610 was upregulated in CD patients.Moreover,we predicted an interaction between hsa_circRNA_102610 and hsa-miR-130a-3p.Thus,we hypothesized that hsa_circRNA_102610 may play roles in the proliferation and EMT of intestinal epithelial cells by sponging hsa-miR-130a-3p to participate in the pathogenesis of CD.AIM To explore the mechanism of hsa_circRNA_102610 in the pathogenesis of CD.METHODS The relative expression levels of hsa_circRNA_102610 and hsa-miR-130a-3p in patients were detected by quantitative reverse transcription-polymerase chain reaction.The proliferation of human intestinal epithelial cells(HIECs)and normal-derived colon mucosa cell line 460(NCM460)cells was detected by cell counting kit-8,5-ethynyl-2’-deoxyuridine staining and cell cycle assays following overexpression or downregulation of hsa_circRNA_102610.Cell proliferation assays were performed as described above in a rescue experiment with hsa-miR-130a-3p mimics.The interaction of hsa_circRNA_102610 and hsa-miR-130a-3p was verified by fluorescence in situ hybridization and dual luciferase reporter assays.The relative expression levels of CyclinD1,mothers against decapentaplegic homolog 4(SMAD4),E-cadherin,N-cadherin and Vimentin were detected by western blotting following hsa_circRNA_102610 overexpression,TGF-β1-induced EMT or hsa-miR-130a-3p mimic transfection(in rescue experiments).RESULTS Upregulation of hsa_circRNA_102610 was determined to be positively correlated with elevated fecal calprotectin levels in CD(r=0.359,P=0.007)by Pearson correlation analysis.Hsa_circRNA_102610 promoted the proliferation of HIECs and NCM460 cells,while hsa-miR-130a-3p reversed the cell proliferationpromoting effects of hsa_circRNA_102610.Fluorescence in situ hybridization and dual luciferase reporter assays showed that hsa_circRNA_102610 directly bound hsa-miR-130a-3p in NCM460 and 293T cells.An inverse correlation between downregulation of hsa-miR-130a-3p and upregulation of hsa_circRNA_102610 in CD patients was observed(r=-0.290,P=0.024)by Pearson correlation analysis.Moreover,overexpression of hsa_circRNA_102610 promoted SMAD4 and CyclinD1 protein expression validated by western-blotting.Furthermore,overexpression of hsa_circRNA_102610 promoted TGF-β1 induced EMT in HIECs and NCM460 cells via targeting of hsa-miR-130a-3p,with increased expression of Vimentin and N-cadherin and decreased expression of E-cadherin.CONCLUSION Hsa_circRNA_102610 upregulation in CD patients could promote the proliferation and EMT of intestinal epithelial cells via sponging of hsa-miR-130a-3p.展开更多
文摘目的:探讨miR-20a-3p基因干扰对人牙髓干细胞(hDPSCs)成牙本质分化的影响及信号通路调控机制。方法:对hDPSCs转染miR-20a-3p-mimic和miR-20a-3p-inhibitor来上调或下调其表达,然后对hDPSCs进行成牙本质分化诱导培养。通过茜素红染色观察细胞钙基质的矿化程度并检测ALP活性。通过双荧光素酶报告基因测定实验验证miR-20a-3p与mothers against decapentaplegic homolog(SMAD)特异性E3泛素蛋白连接酶1(SMURF1)的靶向调节关系。通过RT-qPCR检测hDPSCs中miR-20a-3p以及SMURF1、Runt相关转录因子2(RUNX2)、骨钙蛋白(OCN)和牙本质涎磷蛋白(DSPP)mRNA水平。结果:与Control组相比,miR-20a-3p-mimic组的茜素红相对染色强度升高了67.09%,miR-20a-3p-inhibitor组降低了46.13%(P<0.05)。与Control组相比,miR-20a-3p-mimic组的相对ALP活性升高了52.89%,miR-20a-3p-inhibitor组降低了53.32%(P<0.05)。与Control组相比,miR-20a-3p-mimic组的RUNX2、OCN和DSPP mRNA相对表达量分别升高了2.19倍、1.86倍和2.35倍,miR-20a-3p-inhibitor组分别降低了63.26%、58.84%和68.12%(P<0.05)。双荧光素酶报告基因测定显示miR-20a-3p靶向抑制SMURF1(P<0.05)。过表达SMURF1抑制了miR-20a-3p对成牙本质分化的影响(P<0.05)。结论:miR-20a-3p通过靶向抑制SMURF1及其下游基因促进hDPSCs的成牙本质分化。
文摘目的:探讨hsa-miR-103a-3p影响食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)细胞对奥沙利铂(oxaliplatin,OXA)耐药的机制。方法:采用RT-qPCR验证测序结果中hsa-miR-103a-3p在不同ESCC细胞系中的表达;转染hsa-miR-103a-3p mimics或inhibitor,验证hsa-miR-103a-3p是否可以逆转ESCC细胞系的耐药表型。应用双萤光素酶报告基因实验和RT-qPCR验证hsa-miR-103a-3p与RASSF8(Ras association domain family member 8)的靶向调控关系;挽救实验验证改变RASSF8的表达是否可以逆转由hsa-miR-103a-3p引起的耐药表型。PCR-array实验检测hsa-miR-103a-3p参与ESCC的耐药机制;Kaplan-Meier生存分析预测hsa-miR-103a-3p高表达与患者生存时间的关系。结果:hsa-miR-103a-3p在OXA敏感ESCC细胞系中高表达(P<0.05);hsa-miR-103a-3p的表达影响ESCC细胞对OXA的敏感性(P<0.05);与对照组相比,过表达hsa-miR-103a-3p能够降低RASSF83’端非翻译区萤光素酶报告基因质粒的活性(P<0.05);挽救实验结果显示,改变RASSF8的表达可有效挽救由hsa-miR-103a-3p引起的耐药表型(P<0.05)。PCR-array实验结果显示,RASSF8高表达能够促进XPA(xeroderma pigmentosum complementation group A)和XPC(xeroderma pigmentosum complementation group C)表达。配对t检验分析结果显示,hsamiR-103a-3p在肿瘤组织中高表达;Kaplan-Meier生存分析显示,与hsa-miR-103a-3p低表达的患者相比,hsa-miR-103a-3p高表达的患者生存时间显著缩短(P<0.05)。结论:hsa-miR-103a-3p低表达通过靶向上调RASSF8的表达促进ESCC细胞对OXA耐药。
基金Supported by the Suzhou Special Project of Diagnosis and Treatment for Key Clinical Disease,No.LCZX201715the Natural Science Foundation of Jiangsu Province,No.BK20161232the Science and Technology Development Fund of Nanjing Medical University,No.NMUB2018215.
文摘BACKGROUND The incidence of inflammatory bowel disease,a chronic intestinal inflammatory disorder that includes Crohn’s disease(CD)and ulcerative colitis,is rising.Circular RNAs are considered valuable diagnostic biomarkers for CD.Current evidence supports the views that epithelial-mesenchymal transition(EMT)plays an important role in CD pathogenesis,and that hsa-miR-130a-3p can inhibit transforming growth factor-β1(TGF-β1)-induced EMT.Our previous study revealed that hsa_circRNA_102610 was upregulated in CD patients.Moreover,we predicted an interaction between hsa_circRNA_102610 and hsa-miR-130a-3p.Thus,we hypothesized that hsa_circRNA_102610 may play roles in the proliferation and EMT of intestinal epithelial cells by sponging hsa-miR-130a-3p to participate in the pathogenesis of CD.AIM To explore the mechanism of hsa_circRNA_102610 in the pathogenesis of CD.METHODS The relative expression levels of hsa_circRNA_102610 and hsa-miR-130a-3p in patients were detected by quantitative reverse transcription-polymerase chain reaction.The proliferation of human intestinal epithelial cells(HIECs)and normal-derived colon mucosa cell line 460(NCM460)cells was detected by cell counting kit-8,5-ethynyl-2’-deoxyuridine staining and cell cycle assays following overexpression or downregulation of hsa_circRNA_102610.Cell proliferation assays were performed as described above in a rescue experiment with hsa-miR-130a-3p mimics.The interaction of hsa_circRNA_102610 and hsa-miR-130a-3p was verified by fluorescence in situ hybridization and dual luciferase reporter assays.The relative expression levels of CyclinD1,mothers against decapentaplegic homolog 4(SMAD4),E-cadherin,N-cadherin and Vimentin were detected by western blotting following hsa_circRNA_102610 overexpression,TGF-β1-induced EMT or hsa-miR-130a-3p mimic transfection(in rescue experiments).RESULTS Upregulation of hsa_circRNA_102610 was determined to be positively correlated with elevated fecal calprotectin levels in CD(r=0.359,P=0.007)by Pearson correlation analysis.Hsa_circRNA_102610 promoted the proliferation of HIECs and NCM460 cells,while hsa-miR-130a-3p reversed the cell proliferationpromoting effects of hsa_circRNA_102610.Fluorescence in situ hybridization and dual luciferase reporter assays showed that hsa_circRNA_102610 directly bound hsa-miR-130a-3p in NCM460 and 293T cells.An inverse correlation between downregulation of hsa-miR-130a-3p and upregulation of hsa_circRNA_102610 in CD patients was observed(r=-0.290,P=0.024)by Pearson correlation analysis.Moreover,overexpression of hsa_circRNA_102610 promoted SMAD4 and CyclinD1 protein expression validated by western-blotting.Furthermore,overexpression of hsa_circRNA_102610 promoted TGF-β1 induced EMT in HIECs and NCM460 cells via targeting of hsa-miR-130a-3p,with increased expression of Vimentin and N-cadherin and decreased expression of E-cadherin.CONCLUSION Hsa_circRNA_102610 upregulation in CD patients could promote the proliferation and EMT of intestinal epithelial cells via sponging of hsa-miR-130a-3p.
