Background:Clear cell renal cell carcinoma(ccRCC)stands as the most prevalent form of kidney cancer,accounting for a significant proportion of malignancies affecting the kidneys.ccRCC is well known as a type of tumour...Background:Clear cell renal cell carcinoma(ccRCC)stands as the most prevalent form of kidney cancer,accounting for a significant proportion of malignancies affecting the kidneys.ccRCC is well known as a type of tumour with immunogenicity.Immune checkpoint inhibitors(ICIs)aim to enhance the anticancer immune response in ccRCC by blocking programmed cell death 1 ligand 1/programmed death 1(PD-L1/PD-1)pathways.In a previous study,we showed that RING finger protein 26(RNF26)degrades chromobox 7(CBX7)to activate the tumor necrosis factor(TNF)in ccRCC.Methods:We analyzed The Cancer Genome Atlas(TCGA)database using the R package ESTIMATE and found that RNF26 was significantly associated with ccRCC immune infiltration.The relationship between RNF26 and the PD1 checkpoint signaling pathway was detected by enrichment analysis.In addition,the molecular mechanism of RNF26 up-regulation of PD-L1 was detected by transcriptome sequencing,RT-qPCR,and Western Blot in ccRCC cell lineages 786-O and A498 cells.The transplantation tumor experiments in C57BL/6 mice were used to test the efficacy of anti-PD1 and knockdown of RNF26 in vivo.Results:We showed that RNF26 suppressed the immune response to ccRCC.Next,we revealed that RNF26 activated the PD-1 checkpoint pathway to suppress the immune response to ccRCC,possibly via the CBX7/PD-L1 axis.Conclusion:The suggestion derived from our results is that targeting RNF26 holds the potential to amplify the efficacy of anticancer immunotherapies in the treatment of ccRCC.展开更多
利用生物信息学数据库分析泛素样含PHD和环指结构域蛋白1(UHRF1)在恶性胸膜间皮瘤(MPM)中的表达水平及临床意义。基于TCGA数据库和GTEx数据库差异表达分析UHRF1 m RNA在MPM组织和正常肺组织中的表达水平;使用R软件分析UHRF1 mRNA表达量...利用生物信息学数据库分析泛素样含PHD和环指结构域蛋白1(UHRF1)在恶性胸膜间皮瘤(MPM)中的表达水平及临床意义。基于TCGA数据库和GTEx数据库差异表达分析UHRF1 m RNA在MPM组织和正常肺组织中的表达水平;使用R软件分析UHRF1 mRNA表达量与临床病理参数的相关性;构建Kaplan-Meier模型和单因素多因素COX回归模型分析UHRF1基因在MPM中的预后;利用TIMER2.0数据库分析UHRF1基因与免疫细胞浸润的关系;GSEA分析UHRF1基因发挥功能的主要富集通路。选取8例MPM组织及4例非MPM胸膜组织,通过RT-q PCR的方法验证UHRF1在MPM与非MPM胸膜组织的表达情况。数据库分析结果表明,与正常肺组织相比,UHRF1 m RNA在MPM组织中高表达;UHRF1高表达患者提示MPM患者预后不良;UHRF1基因表达量与CD4^(+)辅助型T细胞2、CD4^(+)效应记忆性T细胞、巨噬细胞等多种免疫细胞浸润水平具有显著的相关性(P<0.01),且显著影响MPM患者的预后。功能富集分析显示,UHRF1主要在DNA复制、蛋白酶体、同源重组等通路中起作用。在收集到的病例样本中,与非MPM胸膜组织相比,UHRF1 mRNA在MPM组织中的表达显著增高(P<0.001)。UHRF1在MPM组织中高表达,可能通过调节DNA甲基化和免疫细胞浸润来影响MPM患者预后,有望成为MPM治疗和预后评估的潜在靶点。展开更多
Background: The mitogen-activated extracellular signal-regulated kinase 1/2(MEK1/2) inhibitor trametinib has shown promising therapeutic effects on melanoma, but its efficacy on colorectal cancer(CRC) is limited. Synt...Background: The mitogen-activated extracellular signal-regulated kinase 1/2(MEK1/2) inhibitor trametinib has shown promising therapeutic effects on melanoma, but its efficacy on colorectal cancer(CRC) is limited. Synthetic lethality arises with a combination of two or more separate gene mutations that causes cell death, whereas individual mutations keep cells alive. This study aimed to identify the genes responsible for resistance to trametinib in CRC cells,using a synthetic lethal short hairpin RNA(shRNA) screening approach.Methods: We infected HT29 cells with a pooled lentiviral shRNA library and applied next-generation sequencing to identify shRNAs with reduced abundance after 8-day treatment of 20 nmol/L trametinib. HCT116 and HT29 cells were used in validation studies. Stable ring finger protein 183(RNF183)-overexpressing cell lines were generated by pcDNA4-myc/his-RNF183 transfection. Stable RNF 183-knockdown cell lines were generated by infection of lentiviruses that express RNF183 shRNA, and small interference RNA(siRNA) was used to knock down RNF183 transiently.Quantitative real-time PCR was used to determine the mRNA expression. Western blotting, immunohistochemical analysis, and enzyme-linked immunosorbent assay(ELISA) were used to evaluate the protein abundance. MTT assay,colony formation assay, and subcutaneous xenograft tumor growth model were used to evaluate cell proliferation.Results: In the primary screening, we found that the abundance of RNF183 shRNA was markedly reduced after treatment with trametinib. Trametinib induced the expression of RNF183, which conferred resistance to drug-induced cell growth repression and apoptotic and non-apoptotic cell deaths. Moreover, interleukin-8(IL-8) was a downstream gene of RNF183 and was required for the function of RNF183 in facilitating cell growth. Additionally, elevated RNF183 expression partly reduced the inhibitory effect of trametinib on IL-8 expression. Finally, xenograft tumor model showed the synergism of RNF183 knockdown and trametinib in repressing the growth of CRC cells in vivo.Conclusion: The RNF183-IL-8 axis is responsible for the resistance of CRC cells to the MEK1/2 inhibitor trametinib and may serve as a candidate target for combined therapy for CRC.展开更多
目的·探索RBX1(ring-box protein 1)在葡萄膜黑色素瘤(uveal melanoma,UVM)肿瘤细胞中对免疫相关基因的调控作用。方法·通过检索癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库分析RBX1在肿瘤中的表达水平以及与临床分...目的·探索RBX1(ring-box protein 1)在葡萄膜黑色素瘤(uveal melanoma,UVM)肿瘤细胞中对免疫相关基因的调控作用。方法·通过检索癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库分析RBX1在肿瘤中的表达水平以及与临床分期、生存预后的相关性。使用靶向RBX1的小干扰RNA(small interfering RNA,siRNA)分别在UVM细胞系92.1、OMM2.3和MEL290中瞬时敲低RBX1,并对瞬时敲低RBX1的92.1细胞进行转录组测序,分析si RBX1转染细胞与对照细胞的差异表达基因,并采用基因集富集分析(gene set enrichment analysis,GSEA)对差异基因进行分析,探究RBX1与肿瘤免疫相关基因的关系。在分析结果的基础上,通过实时荧光定量PCR(qPCR)分别检测瞬时敲低RBX1的92.1、OMM2.3和MEL290细胞系中信号转导与转录激活因子1(signal transducer and activator of transcription 1,STAT1)及其下游的CXC趋化因子配体9(C-X-C motif chemokine ligand 9,CXCL9)和CXCL10的mRNA表达水平,并通过Western blotting检测92.1细胞中STAT1及p-STAT1蛋白表达水平。在瞬时敲低RBX1的细胞系OMM2.3和MEL290中,分别加入5 nmol/L或10 nmol/L的STAT1抑制剂fludarabine,处理48 h后通过qPCR检测CXCL9和CXCL10的mRNA表达水平。结果·TCGA数据库分析表明:与正常组织相比,RBX1在多种肿瘤中高表达,在肾上腺皮质癌和UVM中显著高表达;且这2种肿瘤分期晚的患者,RBX1表达水平更高,同时RBX1表达水平高的患者的总生存期更短(均P<0.05)。对瞬时敲低RBX1的92.1细胞和对照细胞进行转录组测序,获得差异基因,并且GSEA结果显示,RBX1参与调控肿瘤免疫相关通路。热图分析显示RBX1敲低后STAT1表达水平上升。在92.1、OMM2.3和MEL290细胞系中,qPCR结果显示RBX1敲低后STAT1、CXCL9和CXCL10的mRNA表达水平上升,Western blotting结果显示在92.1细胞系中敲低RBX1后STAT1及p-STAT1表达水平上升。加入STAT1抑制剂后,OMM2.3和MEL290细胞系中的CXCL9和CXCL10 mRNA上调表达被抑制。结论·RBX1在UVM细胞中可能通过STAT1调控CXCL9和CXCL10的表达,参与肿瘤免疫调控。展开更多
基金supported by Natural Science Foundation of Hunan Province of China(Grant No.2022JJ30870(Liang Zhu)).
文摘Background:Clear cell renal cell carcinoma(ccRCC)stands as the most prevalent form of kidney cancer,accounting for a significant proportion of malignancies affecting the kidneys.ccRCC is well known as a type of tumour with immunogenicity.Immune checkpoint inhibitors(ICIs)aim to enhance the anticancer immune response in ccRCC by blocking programmed cell death 1 ligand 1/programmed death 1(PD-L1/PD-1)pathways.In a previous study,we showed that RING finger protein 26(RNF26)degrades chromobox 7(CBX7)to activate the tumor necrosis factor(TNF)in ccRCC.Methods:We analyzed The Cancer Genome Atlas(TCGA)database using the R package ESTIMATE and found that RNF26 was significantly associated with ccRCC immune infiltration.The relationship between RNF26 and the PD1 checkpoint signaling pathway was detected by enrichment analysis.In addition,the molecular mechanism of RNF26 up-regulation of PD-L1 was detected by transcriptome sequencing,RT-qPCR,and Western Blot in ccRCC cell lineages 786-O and A498 cells.The transplantation tumor experiments in C57BL/6 mice were used to test the efficacy of anti-PD1 and knockdown of RNF26 in vivo.Results:We showed that RNF26 suppressed the immune response to ccRCC.Next,we revealed that RNF26 activated the PD-1 checkpoint pathway to suppress the immune response to ccRCC,possibly via the CBX7/PD-L1 axis.Conclusion:The suggestion derived from our results is that targeting RNF26 holds the potential to amplify the efficacy of anticancer immunotherapies in the treatment of ccRCC.
文摘利用生物信息学数据库分析泛素样含PHD和环指结构域蛋白1(UHRF1)在恶性胸膜间皮瘤(MPM)中的表达水平及临床意义。基于TCGA数据库和GTEx数据库差异表达分析UHRF1 m RNA在MPM组织和正常肺组织中的表达水平;使用R软件分析UHRF1 mRNA表达量与临床病理参数的相关性;构建Kaplan-Meier模型和单因素多因素COX回归模型分析UHRF1基因在MPM中的预后;利用TIMER2.0数据库分析UHRF1基因与免疫细胞浸润的关系;GSEA分析UHRF1基因发挥功能的主要富集通路。选取8例MPM组织及4例非MPM胸膜组织,通过RT-q PCR的方法验证UHRF1在MPM与非MPM胸膜组织的表达情况。数据库分析结果表明,与正常肺组织相比,UHRF1 m RNA在MPM组织中高表达;UHRF1高表达患者提示MPM患者预后不良;UHRF1基因表达量与CD4^(+)辅助型T细胞2、CD4^(+)效应记忆性T细胞、巨噬细胞等多种免疫细胞浸润水平具有显著的相关性(P<0.01),且显著影响MPM患者的预后。功能富集分析显示,UHRF1主要在DNA复制、蛋白酶体、同源重组等通路中起作用。在收集到的病例样本中,与非MPM胸膜组织相比,UHRF1 mRNA在MPM组织中的表达显著增高(P<0.001)。UHRF1在MPM组织中高表达,可能通过调节DNA甲基化和免疫细胞浸润来影响MPM患者预后,有望成为MPM治疗和预后评估的潜在靶点。
基金supported by the National Natural Science Foundation of China(Nos.81672744,81472252)Science and Technology Project of Guangdong Province(No.2016A020217007)Guangdong Esophageal Cancer Institute(No.M201606)
文摘Background: The mitogen-activated extracellular signal-regulated kinase 1/2(MEK1/2) inhibitor trametinib has shown promising therapeutic effects on melanoma, but its efficacy on colorectal cancer(CRC) is limited. Synthetic lethality arises with a combination of two or more separate gene mutations that causes cell death, whereas individual mutations keep cells alive. This study aimed to identify the genes responsible for resistance to trametinib in CRC cells,using a synthetic lethal short hairpin RNA(shRNA) screening approach.Methods: We infected HT29 cells with a pooled lentiviral shRNA library and applied next-generation sequencing to identify shRNAs with reduced abundance after 8-day treatment of 20 nmol/L trametinib. HCT116 and HT29 cells were used in validation studies. Stable ring finger protein 183(RNF183)-overexpressing cell lines were generated by pcDNA4-myc/his-RNF183 transfection. Stable RNF 183-knockdown cell lines were generated by infection of lentiviruses that express RNF183 shRNA, and small interference RNA(siRNA) was used to knock down RNF183 transiently.Quantitative real-time PCR was used to determine the mRNA expression. Western blotting, immunohistochemical analysis, and enzyme-linked immunosorbent assay(ELISA) were used to evaluate the protein abundance. MTT assay,colony formation assay, and subcutaneous xenograft tumor growth model were used to evaluate cell proliferation.Results: In the primary screening, we found that the abundance of RNF183 shRNA was markedly reduced after treatment with trametinib. Trametinib induced the expression of RNF183, which conferred resistance to drug-induced cell growth repression and apoptotic and non-apoptotic cell deaths. Moreover, interleukin-8(IL-8) was a downstream gene of RNF183 and was required for the function of RNF183 in facilitating cell growth. Additionally, elevated RNF183 expression partly reduced the inhibitory effect of trametinib on IL-8 expression. Finally, xenograft tumor model showed the synergism of RNF183 knockdown and trametinib in repressing the growth of CRC cells in vivo.Conclusion: The RNF183-IL-8 axis is responsible for the resistance of CRC cells to the MEK1/2 inhibitor trametinib and may serve as a candidate target for combined therapy for CRC.
