AIM:To investigate the regulative effect of miRNA-3383p(miR-338-3p) on cell growth in colorectal carcinoma(CRC).METHODS:The lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed.The re...AIM:To investigate the regulative effect of miRNA-3383p(miR-338-3p) on cell growth in colorectal carcinoma(CRC).METHODS:The lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed.The recombinant viral vector encoding the pre-miR338-3p or miR-338-3p-inhibitor and the two packaging plasmids psPAX2 and pMD2.G were cotransfected into human embryonic kidney 293T cells to package lentivirus.The supernatant containing the lentivirus particles was harvested to determine the viral titer,and this supernatant was then used to transduce CRCderived cell line,SW-620.Flow cytometry was utilized for sorting the green fluorescent protein(GFP) + cells to establish the SW-620 cell line stably expressing premiR-338-3p or miR-338-3p-inhibitor.Moreover,the expression of miR-338-3p was determined by real-time reverse transcriptase polymerase chain reaction,andWestern blotting was used to detect the expression of the smoothened(SMO,the possible target of miR-3383p) protein in SW-620 cells.Furthermore,the status of CRC cell proliferation and apoptosis were detected by 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay and flow cytometry,respectively.RESULTS:Restriction enzyme digestion and DNA sequencing demonstrated that the lentiviral vector pLVTHM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed successfully.GFP was expressed after the SW-620 cells were transduced by the lentivirus.Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p was significantly increased(relative expression 3.91 ± 0.51 vs 2.36 ± 0.44,P < 0.01).Furthermore,overexpression of miR-338-3p inhibited the expression of SMO protein in SW-620 cells,which showed obviously suppressed proliferation ability [cellular proliferation inhibition rate(CPIR) 61.9% ± 5.2% vs 41.6% ± 4.8%,P < 0.01].Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p-inhibitor was significantly decreased(relative expression 0.92 ± 0.29 vs 2.36 ± 0.44,P < 0.01).Moreover,the downregulated expression of miR-338-3p caused upregulated expression of the SMO protein in SW-620 cells,which showed significantly enhanced proliferation ability(CPIR 19.2% ± 3.8% vs 41.6% ± 4.8%,P < 0.01).However,anti-SMO-siRNA largely,but not completely,reversed the effects induced by blockage of miR-338-3p,suggesting that the regulative effect of miR-338-3p on CRC cell growth was indeed mediated by SMO.CONCLUSION:miR-338-3p could suppress CRC growth by inhibiting SMO protein expression.展开更多
目的探讨通过沉默Smoothened(Smo)基因抑制Hedgehog通路后,小鼠胚胎成纤维细胞NIH/3T3中mRNA表达谱的变化。方法提取Smo沉默和对照NIH/3T3细胞的总RNA,并进行测序。与对照组比较,分析差异表达的mRNA,通过BLAST2GO和京都基因与基因组百...目的探讨通过沉默Smoothened(Smo)基因抑制Hedgehog通路后,小鼠胚胎成纤维细胞NIH/3T3中mRNA表达谱的变化。方法提取Smo沉默和对照NIH/3T3细胞的总RNA,并进行测序。与对照组比较,分析差异表达的mRNA,通过BLAST2GO和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)分析和预测这些差异表达mRNA的相关功能。选取差异表达mRNA进行实时定量PCR验证。结果通过测序在Smo沉默的NIH/3T3细胞中发现有2289个mRNA差异表达,其中1646个mRNA在Smo沉默后表达下调,而643个mRNA表达上调。荧光实时定量PCR结果与测序结果一致。生物信息学分析显示,差异表达mRNA参与细胞过程、单有机体过程、生物学调节。差异表达mRNA富集在轴突导向(ko04360)、癌症通路(ko05200)、癌症中的microRNA(ko05206)等通路。利用GEPIA数据库分析CXCL12、IL-33、TGF-β2和FGF10及其受体在食管癌和正常食管组织中的表达情况,和预后的预测分析。结论在NIH/3T3细胞中沉默Smo抑制Hedgehog通路可能会通过细胞因子CXCL12、IL-33、TGF-β2和FGF10影响肿瘤细胞的增殖。展开更多
Fibroblast-like synoviocytes （FLSs） contribute to synovial hyperplasia in rheumatoid arthritis （RA）. Smoothened （Smo） is a key component of sonic hedgehog （Shh） signaling and contributes to tumor cell prolifer...Fibroblast-like synoviocytes （FLSs） contribute to synovial hyperplasia in rheumatoid arthritis （RA）. Smoothened （Smo） is a key component of sonic hedgehog （Shh） signaling and contributes to tumor cell proliferation. The objective of this study was to investigate the role of Smo in RA synoviocyte proliferation. FLSs were isolated from RA synovium. Shh signaling was studied using a Smo antagonist （GDC-0449） and small interfering RNA （siRNA） targeting the Smo gene in FLSs. Cell proliferation was quantified by using kit-8 assay and cell cycle distribution and apoptosis were evaluated by flow cytometry. Cell cycle-related genes and proteins were detected by real-time PCR and western blot. FLSs treated with GDC-0449 or Smo-siRNA showed significantly decreased proliferation compared to controls （P〈0.05）. Incubation with GDC-0449 or transfection with Smo-siRNA resulted in a significant increase of G1 phase cells compared to controls （P 〈 0.05）. Cell cycle arrest was validated by the significant increase in cyclin D1 and E1 mRNA expression, decrease in cyclin-dependent kinase p21 mRNA expression in Smo-siRNA transfected cells （P 〈 0.05）. Protein expression of cyclin D1 was also downregulated after Smo gene knockdown （P 〈0.05）.The results suggest that Shh signaling plays an important role in RA-FLSs proliferation in a Smo-dependent manner and may contribute to synovial hyperplasia. Targeting Shh signaling may help control joint damage in patients with RA.展开更多
Emerging evidence suggests that neuro-inflammation begins early and drives the pathogenesis of Alzheimer＇s disease （AD）, and anti-inflammatory therapies are under clinical development. However, several anti-inflamm...Emerging evidence suggests that neuro-inflammation begins early and drives the pathogenesis of Alzheimer＇s disease （AD）, and anti-inflammatory therapies are under clinical development. However, several anti-inflammatory compounds failed to improve memory in clinical trials, indicating that reducing inflammation alone might not be enough. On the other hand, neuro-inflammation is implicated in a number of mental disorders which share the same therapeutic targets. Based on these observations, we screened a batch of genes related with mental disorder and neuro-inflammation in a classical olfactory conditioning in an amyloid beta （Aβ） overexpression fly model. A Smoothened （SMO） mutant was identified as a genetic modifier of Aβ toxicity in 3-min memory and downregulation of SMO rescued Aβ- induced 3-min and 1-h memory deficiency. Also, Aβ activated innate inflammatory response in fly by increasing the expression of antimicrobial peptides, which were alleviated by downregulating SMO. Furthermore, pharmaceutical administration of a SMO antagonist LDE rescued Aβ-induced upregulation of SMO in astrocytes of mouse hippocampus, improved memory in Morris water maze （MWM）, and reduced expression ofastrocyte secreting pro-inflammatory factors IL-1β, TNFα and the microglia marker IBA-1 in an APP/PS1 transgenic mouse model. Our study suggests that SMO is an important conserved modulator of Aβ toxicity in both flv and mouse models of AD.展开更多
基金Supported by National Natural Science Foundation of China,No.81101896
文摘AIM:To investigate the regulative effect of miRNA-3383p(miR-338-3p) on cell growth in colorectal carcinoma(CRC).METHODS:The lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed.The recombinant viral vector encoding the pre-miR338-3p or miR-338-3p-inhibitor and the two packaging plasmids psPAX2 and pMD2.G were cotransfected into human embryonic kidney 293T cells to package lentivirus.The supernatant containing the lentivirus particles was harvested to determine the viral titer,and this supernatant was then used to transduce CRCderived cell line,SW-620.Flow cytometry was utilized for sorting the green fluorescent protein(GFP) + cells to establish the SW-620 cell line stably expressing premiR-338-3p or miR-338-3p-inhibitor.Moreover,the expression of miR-338-3p was determined by real-time reverse transcriptase polymerase chain reaction,andWestern blotting was used to detect the expression of the smoothened(SMO,the possible target of miR-3383p) protein in SW-620 cells.Furthermore,the status of CRC cell proliferation and apoptosis were detected by 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay and flow cytometry,respectively.RESULTS:Restriction enzyme digestion and DNA sequencing demonstrated that the lentiviral vector pLVTHM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed successfully.GFP was expressed after the SW-620 cells were transduced by the lentivirus.Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p was significantly increased(relative expression 3.91 ± 0.51 vs 2.36 ± 0.44,P < 0.01).Furthermore,overexpression of miR-338-3p inhibited the expression of SMO protein in SW-620 cells,which showed obviously suppressed proliferation ability [cellular proliferation inhibition rate(CPIR) 61.9% ± 5.2% vs 41.6% ± 4.8%,P < 0.01].Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p-inhibitor was significantly decreased(relative expression 0.92 ± 0.29 vs 2.36 ± 0.44,P < 0.01).Moreover,the downregulated expression of miR-338-3p caused upregulated expression of the SMO protein in SW-620 cells,which showed significantly enhanced proliferation ability(CPIR 19.2% ± 3.8% vs 41.6% ± 4.8%,P < 0.01).However,anti-SMO-siRNA largely,but not completely,reversed the effects induced by blockage of miR-338-3p,suggesting that the regulative effect of miR-338-3p on CRC cell growth was indeed mediated by SMO.CONCLUSION:miR-338-3p could suppress CRC growth by inhibiting SMO protein expression.
文摘目的探讨通过沉默Smoothened(Smo)基因抑制Hedgehog通路后,小鼠胚胎成纤维细胞NIH/3T3中mRNA表达谱的变化。方法提取Smo沉默和对照NIH/3T3细胞的总RNA,并进行测序。与对照组比较,分析差异表达的mRNA,通过BLAST2GO和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)分析和预测这些差异表达mRNA的相关功能。选取差异表达mRNA进行实时定量PCR验证。结果通过测序在Smo沉默的NIH/3T3细胞中发现有2289个mRNA差异表达,其中1646个mRNA在Smo沉默后表达下调,而643个mRNA表达上调。荧光实时定量PCR结果与测序结果一致。生物信息学分析显示,差异表达mRNA参与细胞过程、单有机体过程、生物学调节。差异表达mRNA富集在轴突导向(ko04360)、癌症通路(ko05200)、癌症中的microRNA(ko05206)等通路。利用GEPIA数据库分析CXCL12、IL-33、TGF-β2和FGF10及其受体在食管癌和正常食管组织中的表达情况,和预后的预测分析。结论在NIH/3T3细胞中沉默Smo抑制Hedgehog通路可能会通过细胞因子CXCL12、IL-33、TGF-β2和FGF10影响肿瘤细胞的增殖。
基金This work was supported in part by grants from the National Natural Science Foundation of China （81072480）, from the Natural Science Foundation of Guangdong Province （$2012020010927）, and from the Science and Technology Program of Guangdong Province （2013B021800076） （Jian-lin Huang） the major projects from Science and Technology Program of Guangzhou City, from the National Natural Science Foundation of Guangdong Province, from the Department of Education of Guangdong Province, and grants from NIH AR059103 and NIH AI084359 （Song Guo Zheng）.
文摘Fibroblast-like synoviocytes （FLSs） contribute to synovial hyperplasia in rheumatoid arthritis （RA）. Smoothened （Smo） is a key component of sonic hedgehog （Shh） signaling and contributes to tumor cell proliferation. The objective of this study was to investigate the role of Smo in RA synoviocyte proliferation. FLSs were isolated from RA synovium. Shh signaling was studied using a Smo antagonist （GDC-0449） and small interfering RNA （siRNA） targeting the Smo gene in FLSs. Cell proliferation was quantified by using kit-8 assay and cell cycle distribution and apoptosis were evaluated by flow cytometry. Cell cycle-related genes and proteins were detected by real-time PCR and western blot. FLSs treated with GDC-0449 or Smo-siRNA showed significantly decreased proliferation compared to controls （P〈0.05）. Incubation with GDC-0449 or transfection with Smo-siRNA resulted in a significant increase of G1 phase cells compared to controls （P 〈 0.05）. Cell cycle arrest was validated by the significant increase in cyclin D1 and E1 mRNA expression, decrease in cyclin-dependent kinase p21 mRNA expression in Smo-siRNA transfected cells （P 〈 0.05）. Protein expression of cyclin D1 was also downregulated after Smo gene knockdown （P 〈0.05）.The results suggest that Shh signaling plays an important role in RA-FLSs proliferation in a Smo-dependent manner and may contribute to synovial hyperplasia. Targeting Shh signaling may help control joint damage in patients with RA.
基金supported by grants from the National Science Foundation of China(Nos.91332207 and 91632301,to Yi Zhong)the Beijing Municipal Science and Technology Commission(Z161100002616010,to Yi Zhong)+1 种基金the National Basic Research Project(973 program)of the Ministry of Science and Technology of China(2013cb835100,to Yi Zhong)the Tsinghua-Peking Joint Center for Life Sciences
文摘Emerging evidence suggests that neuro-inflammation begins early and drives the pathogenesis of Alzheimer＇s disease （AD）, and anti-inflammatory therapies are under clinical development. However, several anti-inflammatory compounds failed to improve memory in clinical trials, indicating that reducing inflammation alone might not be enough. On the other hand, neuro-inflammation is implicated in a number of mental disorders which share the same therapeutic targets. Based on these observations, we screened a batch of genes related with mental disorder and neuro-inflammation in a classical olfactory conditioning in an amyloid beta （Aβ） overexpression fly model. A Smoothened （SMO） mutant was identified as a genetic modifier of Aβ toxicity in 3-min memory and downregulation of SMO rescued Aβ- induced 3-min and 1-h memory deficiency. Also, Aβ activated innate inflammatory response in fly by increasing the expression of antimicrobial peptides, which were alleviated by downregulating SMO. Furthermore, pharmaceutical administration of a SMO antagonist LDE rescued Aβ-induced upregulation of SMO in astrocytes of mouse hippocampus, improved memory in Morris water maze （MWM）, and reduced expression ofastrocyte secreting pro-inflammatory factors IL-1β, TNFα and the microglia marker IBA-1 in an APP/PS1 transgenic mouse model. Our study suggests that SMO is an important conserved modulator of Aβ toxicity in both flv and mouse models of AD.
