A large number of testis-specific genes are involved in the complex process of mammalian spermatogenesis. Identification of these genes and their roles is important for understanding the mechanisms underlying spermato...A large number of testis-specific genes are involved in the complex process of mammalian spermatogenesis. Identification of these genes and their roles is important for understanding the mechanisms underlying spermatogenesis. Here we report on a novel human RING finger protein, ZNF645, which contains a C3HC4 RING finger domain, a C2H2 zinc-finger domain, and a proline-rich region, indicating that it has a structure similar to that of the c-Cbl-like protein Hakai. ZNF645 was exclusively expressed in normal human testicular tissue. Immunohistochemical analysis confirmed that ZNF645 protein was present in spermatocytes, round and elongated spermatids, and Leydig cells. Immunofluorescence staining of mature sperms further showed that the ZNF645 protein was localized over the postacrosomal perinuclear theca region and the entire length of sperm tail. An in vitro ubiquitination assay indicated that the RING finger domain of the ZNF645 protein had E3 ubiquitin ligase activity. Therefore, we suggest that ZNF645 might act as an E3 ubiquitin-protein ligase and play a role in human sperm production and quality control.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
BACKGROUND Gastric cancer(GC)is one of the most prevalent malignant tumors that endangers human health.Early diagnosis is essential for improving the prognosis and survival rate of GC patients.Ring finger protein 180(...BACKGROUND Gastric cancer(GC)is one of the most prevalent malignant tumors that endangers human health.Early diagnosis is essential for improving the prognosis and survival rate of GC patients.Ring finger protein 180(RNF180)is involved in the regulation of cell differentiation,proliferation,apoptosis,and tumorigenesis,and aberrant hypermethylation of CpG islands in the promoter is strongly associated with the occurrence and development of GC.Thus,methylated RNF180 can be used as a potential biomarker for GC diagnosis.AIM To use droplet digital polymerase chain reaction(ddPCR)to quantify the methylation level of the RN180 gene.A reproducible ddPCR assay to detect methylated RNF180 from trace DNA was designed and optimized.METHODS The primer and probe were designed and selected,the conversion time of bisulfite was optimized,the ddPCR system was adjusted by primer concentration,amplification temperature and amplification cycles,and the detection limit of ddPCR was determined.RESULTS The best conversion time for blood DNA was 2 h 10 min,and that for plasma DNA was 2 h 10 min and 2 h 30 min.The results of ddPCR were better when the amplification temperature was 56°C and the number of amplification cycles was 50.Primer concentrations showed little effect on the assay outcome.Therefore,the primer concentration could be adjusted according to the reaction system and DNA input.The assay required at least 0.1 ng of input DNA.CONCLUSION In summary,a ddPCR assay was established to detect methylated RNF180,which is expected to be a new diagnostic biomarker for GC.展开更多
目的研究非肌层浸润膀胱癌(non-muscle invasive bladder cancer,NMIBC)癌组织中结构域蛋白去泛素化酶5(domain protein ubiquitin 5,OTUD5)、环指蛋白186(ring finger protein 186,RNF186)表达与临床病理特征的关系及预后价值研究。方...目的研究非肌层浸润膀胱癌(non-muscle invasive bladder cancer,NMIBC)癌组织中结构域蛋白去泛素化酶5(domain protein ubiquitin 5,OTUD5)、环指蛋白186(ring finger protein 186,RNF186)表达与临床病理特征的关系及预后价值研究。方法收集2019年1月~2020年1月唐山市中医院收治的106例NMIBC患者。免疫组织化学检测NMIBC癌组织及癌旁组织OTUD5,RNF186表达。比较不同临床病理特征NMIBC癌组织OTUD5,RNF186表达差异。Kaplan-Meier曲线分析(Log-Rank检验)OTUD5,RNF186表达对NMIBC无进展生存预后的影响。COX回归模型分析影响NMIBC患者无进展生存预后的因素。结果NMIBC癌组织中OTUD5(62.26%),RNF186(66.04%)的阳性率高于癌旁组织(11.32%,8.49%),差异具有统计学意义(χ^(2)=59.146,75.079,均P<0.05)。NMIBC癌组织中OTUD5与RNF186表达呈显著正相关(r=0.659,P<0.05)。T 1期、高级别尿路上皮癌NMIBC癌组织中OTUD5(72.97%,84.21%),RNF186(77.03,86.84%)阳性率高于Ta/Tis期(37.50%,50.00%)、低级别尿路上皮癌(40.63%,54.41%),差异具有统计学意义(χ^(2)=11.964,13.199;12.143,11.431,均P<0.05)。OTUD5阳性组(40.91%)、RNF186阳性组(45.71%)三年累积无进展生存率低于OTUD5阴性组(90.00%)、RNF186阴性组(86.11%)患者(Log-Ranktestχ^(2)=24.710,14.586,均P<0.05)。OTUD5阳性(OR=1.446,95%CI:1.086~1.925)、RNF186阳性(OR=1.579,95%CI:1.132~2.024)、肿瘤TNM分期T1期(OR=1.582,95%CI:1.219~2.054)、病理分级高级别(OR=1.570,95%CI:1.188~2.074)是影响NMIBC患者无进展生存的独立危险因素(均P<0.001)。结论NMIBC患者癌组织OTUD5,RNF186表达升高,与肿瘤TNM分期、病理分级有关,是影响NMIBC患者预后的独立危险因素。展开更多
目的探讨泛素连接酶肌肉环指状蛋白2(muscle ring finger protein 2,MuRF2)对缺氧心肌细胞活力和自噬的调控作用。方法构建大鼠源MuRF2基因过表达和敲减慢病毒载体,分别感染大鼠心肌细胞H9C2。将H9C2细胞分为阴性对照组(NC)、阳性对照组...目的探讨泛素连接酶肌肉环指状蛋白2(muscle ring finger protein 2,MuRF2)对缺氧心肌细胞活力和自噬的调控作用。方法构建大鼠源MuRF2基因过表达和敲减慢病毒载体,分别感染大鼠心肌细胞H9C2。将H9C2细胞分为阴性对照组(NC)、阳性对照组[MuRF2过表达空载体组(LV-Vector)、MuRF2敲减空载体组(LV-RNAi-Vector)]、Mu RF2过表达组(LV-MuRF2)和MuRF2敲减组(LV-RNAi-MuRF2),缺氧培养(5%CO_(2)、1%O_(2)、94%N_(2))后,采用CCK-8法和ELISA法检测心肌细胞损伤水平,利用透射电子显微镜检测心肌细胞超微结构和自噬水平变化,采用Western blot测定自噬相关蛋白LC3-Ⅱ和P62的表达水平。结果与对照组相比,缺氧组心肌细胞间隙增大、细胞皱缩、多见漂浮的死亡细胞和细胞碎片,心肌细胞活力下降、肌钙蛋白Ⅰ(cTnⅠ)含量增加(P均<0.05);电子显微镜下可观察到部分线粒体发生肿胀,并存在嵴断裂、嵴溶解现象,自噬小体散在胞质内;缺氧组LC3-Ⅱ蛋白表达水平升高,P62蛋白表达水平降低(P均<0.05)。缺氧培养后,与NC组和LV-Vector组相比,LV-MuRF2组细胞活力增加,偶见自噬小体,LC3-Ⅱ蛋白表达水平降低、P62蛋白表达水平增高(P均<0.05);与NC组和LV-RNAi-Vector组相比,LV-RNAi-MuRF2组细胞活力降低,可见大量自噬小体,LC3-Ⅱ蛋白表达水平升高、P62蛋白表达水平降低(P均<0.05)。结论泛素连接酶MuRF2可减轻缺氧所致的心肌细胞损伤,其机制可能与抑制心肌细胞线粒体自噬有关。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
文摘A large number of testis-specific genes are involved in the complex process of mammalian spermatogenesis. Identification of these genes and their roles is important for understanding the mechanisms underlying spermatogenesis. Here we report on a novel human RING finger protein, ZNF645, which contains a C3HC4 RING finger domain, a C2H2 zinc-finger domain, and a proline-rich region, indicating that it has a structure similar to that of the c-Cbl-like protein Hakai. ZNF645 was exclusively expressed in normal human testicular tissue. Immunohistochemical analysis confirmed that ZNF645 protein was present in spermatocytes, round and elongated spermatids, and Leydig cells. Immunofluorescence staining of mature sperms further showed that the ZNF645 protein was localized over the postacrosomal perinuclear theca region and the entire length of sperm tail. An in vitro ubiquitination assay indicated that the RING finger domain of the ZNF645 protein had E3 ubiquitin ligase activity. Therefore, we suggest that ZNF645 might act as an E3 ubiquitin-protein ligase and play a role in human sperm production and quality control.
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.
基金Supported by the National Key Research and Development Program of China,No.2020YFC2002700the National Natural Science Foundation of China,No.81972010+1 种基金the CAMS Initiative for Innovative Medicine,No.2016-I2M-1-007the Science Developing Funds of Navy General Hospital,No.CXPY201810.
文摘BACKGROUND Gastric cancer(GC)is one of the most prevalent malignant tumors that endangers human health.Early diagnosis is essential for improving the prognosis and survival rate of GC patients.Ring finger protein 180(RNF180)is involved in the regulation of cell differentiation,proliferation,apoptosis,and tumorigenesis,and aberrant hypermethylation of CpG islands in the promoter is strongly associated with the occurrence and development of GC.Thus,methylated RNF180 can be used as a potential biomarker for GC diagnosis.AIM To use droplet digital polymerase chain reaction(ddPCR)to quantify the methylation level of the RN180 gene.A reproducible ddPCR assay to detect methylated RNF180 from trace DNA was designed and optimized.METHODS The primer and probe were designed and selected,the conversion time of bisulfite was optimized,the ddPCR system was adjusted by primer concentration,amplification temperature and amplification cycles,and the detection limit of ddPCR was determined.RESULTS The best conversion time for blood DNA was 2 h 10 min,and that for plasma DNA was 2 h 10 min and 2 h 30 min.The results of ddPCR were better when the amplification temperature was 56°C and the number of amplification cycles was 50.Primer concentrations showed little effect on the assay outcome.Therefore,the primer concentration could be adjusted according to the reaction system and DNA input.The assay required at least 0.1 ng of input DNA.CONCLUSION In summary,a ddPCR assay was established to detect methylated RNF180,which is expected to be a new diagnostic biomarker for GC.
文摘目的探讨泛素连接酶肌肉环指状蛋白2(muscle ring finger protein 2,MuRF2)对缺氧心肌细胞活力和自噬的调控作用。方法构建大鼠源MuRF2基因过表达和敲减慢病毒载体,分别感染大鼠心肌细胞H9C2。将H9C2细胞分为阴性对照组(NC)、阳性对照组[MuRF2过表达空载体组(LV-Vector)、MuRF2敲减空载体组(LV-RNAi-Vector)]、Mu RF2过表达组(LV-MuRF2)和MuRF2敲减组(LV-RNAi-MuRF2),缺氧培养(5%CO_(2)、1%O_(2)、94%N_(2))后,采用CCK-8法和ELISA法检测心肌细胞损伤水平,利用透射电子显微镜检测心肌细胞超微结构和自噬水平变化,采用Western blot测定自噬相关蛋白LC3-Ⅱ和P62的表达水平。结果与对照组相比,缺氧组心肌细胞间隙增大、细胞皱缩、多见漂浮的死亡细胞和细胞碎片,心肌细胞活力下降、肌钙蛋白Ⅰ(cTnⅠ)含量增加(P均<0.05);电子显微镜下可观察到部分线粒体发生肿胀,并存在嵴断裂、嵴溶解现象,自噬小体散在胞质内;缺氧组LC3-Ⅱ蛋白表达水平升高,P62蛋白表达水平降低(P均<0.05)。缺氧培养后,与NC组和LV-Vector组相比,LV-MuRF2组细胞活力增加,偶见自噬小体,LC3-Ⅱ蛋白表达水平降低、P62蛋白表达水平增高(P均<0.05);与NC组和LV-RNAi-Vector组相比,LV-RNAi-MuRF2组细胞活力降低,可见大量自噬小体,LC3-Ⅱ蛋白表达水平升高、P62蛋白表达水平降低(P均<0.05)。结论泛素连接酶MuRF2可减轻缺氧所致的心肌细胞损伤,其机制可能与抑制心肌细胞线粒体自噬有关。
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.
