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
目的探讨下调泛素样含PHD和环指域1(ubiquitin-like protein containing PHD and RING finger domains 1,UHRF1)基因在喉癌Hep-2细胞中的表达后对其增殖,凋亡,侵袭迁移能力的影响。方法实验分为空白组,阴性对照组和干扰组。利用UHRF1-si...目的探讨下调泛素样含PHD和环指域1(ubiquitin-like protein containing PHD and RING finger domains 1,UHRF1)基因在喉癌Hep-2细胞中的表达后对其增殖,凋亡,侵袭迁移能力的影响。方法实验分为空白组,阴性对照组和干扰组。利用UHRF1-si RNA干扰UHRF1在喉癌Hep-2细胞中的表达,采用q RT-PCR、Western blot检测UHRF1的表达变化。采用MTT法、流式细胞术、Transwell实验检测下调UHRF1表达后Hep-2细胞的增殖、凋亡、侵袭、迁移能力的变化。Western blot检测下调UHRF1表达后Hep-2细胞内Bax、Bcl-2、MMP-2、MMP-9蛋白表达的变化情况。结果与空白组和阴性对照组相比干扰组下调UHRF1的表达后可明显抑制喉癌Hep-2细胞的增殖能力(P<0.001)。空白组,阴性对照组,干扰组凋亡率分别为(5.47±2.77)%,(3.95±0.66)%,(18.95±1.10)%,干扰组凋亡率明显提高(P<0.001)。空白组,阴性对照,干扰组每视野侵袭细胞数分别为(213.7±13.1),(221.3±10.3),(97.7±7.5),干扰组侵袭细胞数明显降低(P<0.001)。空白组,阴性对照,干扰组每视野迁移细胞数分别为(230.7±5.5),(222.7±11.2),(111.7±7.6),干扰组迁移细胞数明显降低(P<0.001)。干扰组细胞Bax蛋白表达上调(P<0.001),Bcl-2蛋白表达下调(P<0.001),MMP-2蛋白、MMP-9蛋白表达下调(P<0.001)。结论在喉癌Hep-2细胞中下调UHRF1的表达后可明显抑制细胞的增殖,促进细胞凋亡,抑制细胞的侵袭迁移能力。展开更多
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
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.
文摘目的探讨下调泛素样含PHD和环指域1(ubiquitin-like protein containing PHD and RING finger domains 1,UHRF1)基因在喉癌Hep-2细胞中的表达后对其增殖,凋亡,侵袭迁移能力的影响。方法实验分为空白组,阴性对照组和干扰组。利用UHRF1-si RNA干扰UHRF1在喉癌Hep-2细胞中的表达,采用q RT-PCR、Western blot检测UHRF1的表达变化。采用MTT法、流式细胞术、Transwell实验检测下调UHRF1表达后Hep-2细胞的增殖、凋亡、侵袭、迁移能力的变化。Western blot检测下调UHRF1表达后Hep-2细胞内Bax、Bcl-2、MMP-2、MMP-9蛋白表达的变化情况。结果与空白组和阴性对照组相比干扰组下调UHRF1的表达后可明显抑制喉癌Hep-2细胞的增殖能力(P<0.001)。空白组,阴性对照组,干扰组凋亡率分别为(5.47±2.77)%,(3.95±0.66)%,(18.95±1.10)%,干扰组凋亡率明显提高(P<0.001)。空白组,阴性对照,干扰组每视野侵袭细胞数分别为(213.7±13.1),(221.3±10.3),(97.7±7.5),干扰组侵袭细胞数明显降低(P<0.001)。空白组,阴性对照,干扰组每视野迁移细胞数分别为(230.7±5.5),(222.7±11.2),(111.7±7.6),干扰组迁移细胞数明显降低(P<0.001)。干扰组细胞Bax蛋白表达上调(P<0.001),Bcl-2蛋白表达下调(P<0.001),MMP-2蛋白、MMP-9蛋白表达下调(P<0.001)。结论在喉癌Hep-2细胞中下调UHRF1的表达后可明显抑制细胞的增殖,促进细胞凋亡,抑制细胞的侵袭迁移能力。
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.
