背景:溶质载体家族1成员5(solute carrier family 1 member 5,SLC1A5)在多种疾病中发挥了潜在作用,但确切作用机制尚不清楚。构建稳定的SLC1A5过表达和敲低细胞模型可为深入研究SLC1A5在疾病中的确切作用机制以及发现潜在治疗靶点提供...背景:溶质载体家族1成员5(solute carrier family 1 member 5,SLC1A5)在多种疾病中发挥了潜在作用,但确切作用机制尚不清楚。构建稳定的SLC1A5过表达和敲低细胞模型可为深入研究SLC1A5在疾病中的确切作用机制以及发现潜在治疗靶点提供有力的实验工具。目的:构建小鼠SLC1A5过表达和敲低的慢病毒载体,以建立稳定转染的RAW264.7细胞株,为深入探讨SLC1A5在炎症中的作用提供实验基础。方法:根据SLC1A5基因序列设计合成引物并使用聚合酶链反应扩增该基因片段。将目的基因定向接入经Age I/Nhe I酶切的载体质粒GV492中构建重组慢病毒质粒,对阳性克隆进一步筛选后测序比对结果;pHelper1.0质粒载体、pHelper2.0质粒载体、目的质粒载体与293T细胞共同培养并转染,获得慢病毒原液进行包装和滴度测定;在此基础上,通过体外培养RAW264.7细胞,确定嘌呤霉素工作质量浓度;不同滴度的慢病毒分别与RAW264.7细胞共同培养,根据荧光强度确定转染效率;用嘌呤霉素挑选出稳定转染细胞,实时荧光定量聚合酶链反应和蛋白免疫印迹方法检测稳定转染细胞株的SLC1A5基因和蛋白表达水平。结果与结论:(1)测序序列与目的序列一致提示重组慢病毒载体构建成功;(2)过表达SLC1A5慢病毒的滴度为1×10~9 TU/mL,敲低SLC1A5慢病毒的滴度为3×10~9 TU/mL;(3)确定RAW264.7细胞嘌呤霉素工作质量浓度为3μg/mL;(4)过表达/敲低SLC1A5慢病毒转染RAW264.7细胞的最佳条件皆为HiTransG P转染增强液且感染复数值等于50;(5)过表达SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量明显上调,而敲低SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量显著下调。结果表明,成功构建了小鼠SLC1A5过表达和敲低的慢病毒载体并获得稳定转染的RAW264.7细胞株。展开更多
目的通过生物信息学分析SLC7A5在乳腺癌中的表达及其与临床病理特征的相关性,为乳腺癌的治疗及预后评估提供参考。方法在Timer2.0、癌症基因组图谱(the cancer genome atlas,TCGA)、UALAN在基因表达谱交互式分析(gene expression profil...目的通过生物信息学分析SLC7A5在乳腺癌中的表达及其与临床病理特征的相关性,为乳腺癌的治疗及预后评估提供参考。方法在Timer2.0、癌症基因组图谱(the cancer genome atlas,TCGA)、UALAN在基因表达谱交互式分析(gene expression profiling interactive analysis 2,GEPIA2)和人类蛋白质表达图谱(human protein atlas,HPA)等数据库中获取SLC7A5在乳腺癌中的表达特征。使用Western blot法检测SLC7A5基因在正常乳腺细胞MCF-10A和乳腺癌细胞株MCF-7中的表达情况。使用R软件分析SLC7A5在乳腺癌中的预后价值,在Kaplan-Meier Plotter和基因表达数据库(gene expression omnibus,GEO)中进行双重验证,同时分析其表达与乳腺癌患者不同病理特征之间的相关性。运用R语言分析SLC7A5与肿瘤突变负荷(tumor mutation burden,TMB)及免疫检测点相关基因的相关性,并进行基因集富集分析(gene set enrichment analysis,GSEA),并对分析结果进一步可视化处理。结果SLC7A5在包括乳腺癌在内的多种实体恶性肿瘤中表达升高,高表达SLC7A5的乳腺癌患者总生存期缩短。SLC7A5在乳腺癌中的表达水平与乳腺癌雌激素受体、孕激素受体、淋巴结转移及分子分型相关。TMB结果提示,SLC7A5在乳腺癌中的表达与TMB呈正相关(r=0.33,P<0.001)。免疫检查点分析结果表明,SLC7A5的表达量与程序性死亡受体1(programmed death-1,PD-1)、细胞毒性T淋巴细胞相关蛋白4(cytotoxic T lymphocyte-associated antigen 4,CTLA4)、B和T细胞衰减因子(B and T lymphocyte attenuator,BTLA)等呈正相关。GSEA富集分析结果显示SLC7A5主要与干扰素γ反应相关通路相关。结论SLC7A5在乳腺癌中表达上调,其表达水平与不良预后相关,可能通过参与干扰素γ反应相关通路促进免疫检查点的表达影响乳腺癌的进展,其或可作为为乳腺癌患者预后评估和指导治疗的生物学标志物。展开更多
Objectives:Triple-negative breast cancer(TNBC)is the breast cancer subtype with the poorest prognosis.This study aimed to elucidate the molecular pathways through which isoliquiritigenin(ISL),a natural chalcone compou...Objectives:Triple-negative breast cancer(TNBC)is the breast cancer subtype with the poorest prognosis.This study aimed to elucidate the molecular pathways through which isoliquiritigenin(ISL),a natural chalcone compound derived fromlicorice and other plant roots,targets interferon regulatory factor 5(IRF5)in TNBC.Methods:TNBC cell lines were cultured and subjected to IRF5 knockdown using short hairpin RNA.Cell proliferation was assessed by cell counting kit-8(CCK-8)assay and colony formation assays.Western blotting and quantitative reverse transcription polymerase chain reaction(RT-PCR)were employed to measure expression levels of IRF5,solute carrier family 7 member 5(SLC7A5),and indoleamine 2,3-dioxygenase 1(IDO1).Intracellular tryptophan and its metabolites were quantified using commercially available assay kits and high-performance liquid chromatography(HPLC).TNBC cells were treated with various concentrations of ISL to evaluate its effects on proliferation and tryptophanmetabolism.Results:IRF5 was highly expressed in TNBC cell lines.Silencing IRF5 significantly inhibited cellular proliferation and growth.Knockdown of IRF5 reduced the expression of SLC7A5 and IDO1,leading to decreased intracellular levels of tryptophan and its metabolites.ISL markedly suppressed TNBC cell proliferation and disrupted tryptophan metabolism in tumor cells.Conclusion:ISL may inhibit TNBC progression by downregulating IRF5 and interfering with SLC7A5/IDO1-mediated tryptophan metabolic reprogramming,suggesting a potential therapeutic mechanism for TNBC treatment.展开更多
采用PCR测序与PCR-RFLP技术,研究了SLC24A5基因变异与他留乌骨鸡羽色及肤色的关系。通过对他留乌骨鸡麻羽、白羽和黑羽3个品系以及乌骨与非乌骨2个类型的SLC24A5基因个体测序,共检测到19个SNP位点,其中3个位点引起氨基酸变异。采用每个...采用PCR测序与PCR-RFLP技术,研究了SLC24A5基因变异与他留乌骨鸡羽色及肤色的关系。通过对他留乌骨鸡麻羽、白羽和黑羽3个品系以及乌骨与非乌骨2个类型的SLC24A5基因个体测序,共检测到19个SNP位点,其中3个位点引起氨基酸变异。采用每个品系或类型PCR扩增池测序技术,确定了每个SNP位点在不同品系或类型的基因频率,在分析基因频率差异及SNP变异特性的基础上,针对G482C位点,建立了Nhe I mismatch PCR-RFLP分子标记。利用该遗传标记对所有受试个体进行基因分型,并分析其与羽色肤色性状的相关性。结果表明,SLC24A5基因Nhe I mismatch PCR-RFLP分子标记与肤色性状显著相关。展开更多
文摘目的通过生物信息学分析SLC7A5在乳腺癌中的表达及其与临床病理特征的相关性,为乳腺癌的治疗及预后评估提供参考。方法在Timer2.0、癌症基因组图谱(the cancer genome atlas,TCGA)、UALAN在基因表达谱交互式分析(gene expression profiling interactive analysis 2,GEPIA2)和人类蛋白质表达图谱(human protein atlas,HPA)等数据库中获取SLC7A5在乳腺癌中的表达特征。使用Western blot法检测SLC7A5基因在正常乳腺细胞MCF-10A和乳腺癌细胞株MCF-7中的表达情况。使用R软件分析SLC7A5在乳腺癌中的预后价值,在Kaplan-Meier Plotter和基因表达数据库(gene expression omnibus,GEO)中进行双重验证,同时分析其表达与乳腺癌患者不同病理特征之间的相关性。运用R语言分析SLC7A5与肿瘤突变负荷(tumor mutation burden,TMB)及免疫检测点相关基因的相关性,并进行基因集富集分析(gene set enrichment analysis,GSEA),并对分析结果进一步可视化处理。结果SLC7A5在包括乳腺癌在内的多种实体恶性肿瘤中表达升高,高表达SLC7A5的乳腺癌患者总生存期缩短。SLC7A5在乳腺癌中的表达水平与乳腺癌雌激素受体、孕激素受体、淋巴结转移及分子分型相关。TMB结果提示,SLC7A5在乳腺癌中的表达与TMB呈正相关(r=0.33,P<0.001)。免疫检查点分析结果表明,SLC7A5的表达量与程序性死亡受体1(programmed death-1,PD-1)、细胞毒性T淋巴细胞相关蛋白4(cytotoxic T lymphocyte-associated antigen 4,CTLA4)、B和T细胞衰减因子(B and T lymphocyte attenuator,BTLA)等呈正相关。GSEA富集分析结果显示SLC7A5主要与干扰素γ反应相关通路相关。结论SLC7A5在乳腺癌中表达上调,其表达水平与不良预后相关,可能通过参与干扰素γ反应相关通路促进免疫检查点的表达影响乳腺癌的进展,其或可作为为乳腺癌患者预后评估和指导治疗的生物学标志物。
基金supported by the Science and Technology Program of Guangzhou(No.202103000020).
文摘Objectives:Triple-negative breast cancer(TNBC)is the breast cancer subtype with the poorest prognosis.This study aimed to elucidate the molecular pathways through which isoliquiritigenin(ISL),a natural chalcone compound derived fromlicorice and other plant roots,targets interferon regulatory factor 5(IRF5)in TNBC.Methods:TNBC cell lines were cultured and subjected to IRF5 knockdown using short hairpin RNA.Cell proliferation was assessed by cell counting kit-8(CCK-8)assay and colony formation assays.Western blotting and quantitative reverse transcription polymerase chain reaction(RT-PCR)were employed to measure expression levels of IRF5,solute carrier family 7 member 5(SLC7A5),and indoleamine 2,3-dioxygenase 1(IDO1).Intracellular tryptophan and its metabolites were quantified using commercially available assay kits and high-performance liquid chromatography(HPLC).TNBC cells were treated with various concentrations of ISL to evaluate its effects on proliferation and tryptophanmetabolism.Results:IRF5 was highly expressed in TNBC cell lines.Silencing IRF5 significantly inhibited cellular proliferation and growth.Knockdown of IRF5 reduced the expression of SLC7A5 and IDO1,leading to decreased intracellular levels of tryptophan and its metabolites.ISL markedly suppressed TNBC cell proliferation and disrupted tryptophan metabolism in tumor cells.Conclusion:ISL may inhibit TNBC progression by downregulating IRF5 and interfering with SLC7A5/IDO1-mediated tryptophan metabolic reprogramming,suggesting a potential therapeutic mechanism for TNBC treatment.
文摘采用PCR测序与PCR-RFLP技术,研究了SLC24A5基因变异与他留乌骨鸡羽色及肤色的关系。通过对他留乌骨鸡麻羽、白羽和黑羽3个品系以及乌骨与非乌骨2个类型的SLC24A5基因个体测序,共检测到19个SNP位点,其中3个位点引起氨基酸变异。采用每个品系或类型PCR扩增池测序技术,确定了每个SNP位点在不同品系或类型的基因频率,在分析基因频率差异及SNP变异特性的基础上,针对G482C位点,建立了Nhe I mismatch PCR-RFLP分子标记。利用该遗传标记对所有受试个体进行基因分型,并分析其与羽色肤色性状的相关性。结果表明,SLC24A5基因Nhe I mismatch PCR-RFLP分子标记与肤色性状显著相关。
