Aim:Given the encouraging results of the p53-Mdm2 inhibitor RG7388 in clinical trials and the vital function of miR-16-5p in suppressing cell proliferation,the aim of the present study was to investigate the combined ...Aim:Given the encouraging results of the p53-Mdm2 inhibitor RG7388 in clinical trials and the vital function of miR-16-5p in suppressing cell proliferation,the aim of the present study was to investigate the combined impact of RG7388 and miR-16-5p overexpression on the childhood acute lymphoblastic leukemia(chALL).Methods:miRTarBase and miRDB,along with KEGG and STRING databases,were used to predict miR-16-5p target genes and explore protein-protein interaction networks,respectively.B-and T-lymphoblastic cell lines,in addition to patient primary cells,were treated with RG7388.Ectopic overexpression of miR-16-5p in Nalm6 cell line was induced through cell electroporation and transfection of microRNA mimics was confirmed by qRT-PCR.Cell viability was evaluated using the MTT assay.Western blot analyses were performed to evaluate the effects of RG7388 and miR-16-5p upregulation on the protein levels of p53 and its downstream target genes in chALL cells.Paired sample t-test was employed for statistical analyses.Results:MTT assay showed RG7388-induced cytotoxicity in wild-type p53 Nalm6 cell line and p53 functional patient primary cells.However,CCRF-CEM and p53 non-functional leukemic cells indicated drug resistance.Western blot analyses validated the bioinformatics results,confirming the downregulation of WIP1,p53 stabilization,as well as overexpression of p21WAF1 and Mdm2 proteins in Nalm6 cells transfected with miR-16-5p.Moreover,enhanced sensitivity to RG7388 was observed in the transfected cells.Conclusion:This is the first study indicating the mechanistic importance of miR-16-5p overexpression in chALL and its inhibitory role in leukemia treatment when combined with the p53-Mdm2 antagonist,RG7388.These findings might be useful for researchers and clinicians to pave the way for better management of chALL.展开更多
Hepatocellular carcinoma （HCC） is a lethal liver malignancy worldwide. In this study, we reported that protein phosphatase magnesium-dependent 16 （PPM1D） was highly expressed in the majority of HCC cases （approxi...Hepatocellular carcinoma （HCC） is a lethal liver malignancy worldwide. In this study, we reported that protein phosphatase magnesium-dependent 16 （PPM1D） was highly expressed in the majority of HCC cases （approximately 59%） and significantly associated with high serum a-fetoprotein （AFP） level （P = 0.044）. Kaplan- Meier and Cox regression data indicated that PPM1D overexpression was an independent predictor of HCC- specific overall survival （HR, 2.799; 95% CI, 1.346-5.818, P = 0.006）. Overexpressing PPM1D promoted cell viability and invasion, whereas RNA interference-mediated knockdown of PPM1D inhibited proliferation, invasion, and migration of cultured HCC cells. In addition, PPM1D suppression by small interfering RNA decreased the tumorigenicity of HCC cells in vivo. Overall, results suggest that PPM1D is a potential prognostic marker and therapeutic target for HCC.展开更多
To construct a lentiviral shRNA vector targeting human protein phosphatase 1D magnesium-dependent（PPM1D） gene and detect its effectiveness of gene silencing in human gliomas,specific siRNA targets with short hairpin...To construct a lentiviral shRNA vector targeting human protein phosphatase 1D magnesium-dependent（PPM1D） gene and detect its effectiveness of gene silencing in human gliomas,specific siRNA targets with short hairpin frame were designed and synthesized.DNA oligo was cloned into the pFU-GW-iRNA lentiviral expression vector,and then PCR and sequencing analyses were conducted to verify the constructs.After the verified plasmids were transfected into 293T cells,the lentivirus was produced and the titer of virus was determined.Real-time quantitative PCR and Western blot were performed to detect the PPM1D expression level in the infected glioma cells.PCR and Western blot analyses revealed the optimal interfering target,and the virus with a titer of 6×10^8 TU/mL was successfully packaged.The PPM1D expression in human glioma cells was knocked down at both mRNA and protein levels by virus infection.The expression of PPM1D mRNA and protein was decreased by 76.3% and 87.0% respectively as compared with control group.The multiple functions of human glioma cells after PPM1D RNA interference were detected by flow cytometry and cell counting kit-8（CCK-8）.Efficient down-regulation of PPM1D resulted in significantly increased cell apoptosis and reduced cell proliferation and invasion potential in U87-MG cells.We have successfully constructed the lentiviral shRNA expression vector capable of stable PPM1D gene silencing at both mRNA and protein levels in glioma cells.And our data gave evidence that the reduced cell growth observed after PPM1D silencing in glioma cells was at least partly due to increased apoptotic cell death.展开更多
目的探讨甲状腺乳头状癌(papillary carcinoma of thyroid,PTC)中PPM1D蛋白的表达情况及其与临床病理特征和p53的关系。方法应用免疫组化EnVision法检测55例PTC及癌旁正常组织中PPM1D蛋白的表达情况并进行统计处理,分析其与临床病理特...目的探讨甲状腺乳头状癌(papillary carcinoma of thyroid,PTC)中PPM1D蛋白的表达情况及其与临床病理特征和p53的关系。方法应用免疫组化EnVision法检测55例PTC及癌旁正常组织中PPM1D蛋白的表达情况并进行统计处理,分析其与临床病理特征的关系及和p53蛋白表达的相关性。结果 55例PTC中PPM1D的表达(74.5%,41/55)高于癌旁正常组织(10.9%,6/55),差异有统计学意义;PPPM1D的表达与患者年龄、性别、肿瘤大小、组织学类型、淋巴结转移、TNM分期无关,而在间质硬化的病例中表达增强(P<0.05);PTC中PPM1D与p53蛋白表达呈负相关(r=-0.339,P<0.05)。结论高表达的PPM1D与PTC相关,与p53之间可能存在相互作用,在PTC发生、发展中具有重要意义。展开更多
目的 :探讨PPM1D m RNA表达与肝癌预后的相关性。方法:提取86例肝癌患者癌组织及癌旁肝组织总RNA,q PCR法检测PPM1D m RNA表达量,免疫组化检测蛋白表达水平。根据癌旁肝组织中PPM1D m RNA表达量,将肝癌患者分组为高表达组与低表达组,对...目的 :探讨PPM1D m RNA表达与肝癌预后的相关性。方法:提取86例肝癌患者癌组织及癌旁肝组织总RNA,q PCR法检测PPM1D m RNA表达量,免疫组化检测蛋白表达水平。根据癌旁肝组织中PPM1D m RNA表达量,将肝癌患者分组为高表达组与低表达组,对两组患者临床资料及生存时间进行统计分析。结果:PPM1D m RNA在肝癌组织中表达水平显著高于癌旁组织,免疫组化检测蛋白表达水平证实上述结果。以癌旁肝组织PPM1D m RNA表达量为阈值,高表达组56例,低表达组30例。两组患者的AFP水平、肿瘤大小、肿瘤TNM分期以及肿瘤复发、家族史等临床病理因素差异有统计学意义(P<0.01);年龄、性别、门静脉侵犯、淋巴结转移、HBV感染及酒精摄入史等因素差异无统计学意义(P>0.05)。高表达组患者中位生存期为13个月,低表达组为32个月。结论:PPM1D m RNA表达水平可能与肝癌恶性程度相关,可能成为肝癌预后的预测因子。展开更多
Recently,a collaborative research led by Weibo Yang and Jim Sun1 published in Cell Chemical Biology identified a potent and selective small molecule inhibitor(SMIP-30)for human protein phosphatase Mg^(2+)/Mn^(2+)-depe...Recently,a collaborative research led by Weibo Yang and Jim Sun1 published in Cell Chemical Biology identified a potent and selective small molecule inhibitor(SMIP-30)for human protein phosphatase Mg^(2+)/Mn^(2+)-dependent 1 A(PPM1A).They applied this chemical probe to determine the autophagy receptor p62 as a new substrate of PPM1A.展开更多
目的通过NHE1基因敲除模型鼠的海马组织差异蛋白质组学分析,发现并明确Ppp3cb和Ppm1g的表达特征。方法①选取6只2周龄NHE1基因敲除模型鼠作为模型组,同周龄野生型小鼠6只作为对照组,采用琼脂糖凝胶电泳检测其基因型;应用旷场实验和强迫...目的通过NHE1基因敲除模型鼠的海马组织差异蛋白质组学分析,发现并明确Ppp3cb和Ppm1g的表达特征。方法①选取6只2周龄NHE1基因敲除模型鼠作为模型组,同周龄野生型小鼠6只作为对照组,采用琼脂糖凝胶电泳检测其基因型;应用旷场实验和强迫游泳实验对模型组和对照组小鼠进行行为学评估,并按照Racine评分标准对模型鼠进行癫痫发作分级;②通过串联质谱分析技术对模型组和对照组的海马组织进行差异蛋白筛选,基因本体论(Gene Ontology Analysis,GO)分析差异蛋白并进行注释和富集,蛋白网络数据库(search tool for the retrieval of interesting genes,STRING)分析差异蛋白之间的蛋白相互作用(protein-protein interaction,PPI);③应用qPCR和Western blot检测Ppp3cb和Ppm1g的转录和翻译水平,应用免疫组织化学技术分别观察其在组织中的表达量。结果①模型组小鼠NHE1基因未见表达,旷场实验中模型鼠的运动总距离较对照组减少(P=0.0073),跨越的格子数比对照组显著减少(P<0.0001)。强迫游泳实验结果显示,模型鼠不动的时间明显延长(P<0.0001);②以表达倍数(FC)≥1.2倍且P<0.05为筛选标准,检测到海马组织中845个差异表达的蛋白质点,其中有9个蛋白表达上调,7个蛋白表达下调。其中Ppp3cb下调,Ppm1g上调。GO功能注释结果表明,NHE1敲除后,分子功能(MF)富集在蛋白丝氨酸/苏氨酸磷酸酶活性的差异最显著,细胞成分(CC)富集在质膜部分的差异蛋白数量最多,生物过程(BP)富集在负向调节生物过程、免疫系统过程的差异蛋白数量最多。STRING分析显示差异蛋白Ppp3cb和Slc9a1直接作用,Ppm1g通过Ppp3cb和Slc9a1间接作用,Ppp3cb和Ppm1g之间相互作用。③Ppp3cb的转录和翻译水平减少,在组织中的表达量下降,而Ppm1g转录和翻译水平增加,在组织中的表达量上升(P<0.05)。结论本研究确定了NHE1基因敲除小鼠海马组织中差异蛋白Ppp3cb表达下调,而Ppm展开更多
Jansen-de Vries综合征又称为“具有胃肠功能紊乱和高疼痛阈值的智力发育障碍”(Intellectual developmental disorder with gastrointestinal difficulties and high pain threshold,IDDGIP)[(OMIM):617450],是一种由PPM1D基因突变导...Jansen-de Vries综合征又称为“具有胃肠功能紊乱和高疼痛阈值的智力发育障碍”(Intellectual developmental disorder with gastrointestinal difficulties and high pain threshold,IDDGIP)[(OMIM):617450],是一种由PPM1D基因突变导致多系统受累的常染色体显性遗传(Autosomal dominant,AD)或常染色体隐性遗传(Autosomal recessive,AR)的罕见疾病.展开更多
基金This work was supported by the University of Isfahan(96/100000/4000).
文摘Aim:Given the encouraging results of the p53-Mdm2 inhibitor RG7388 in clinical trials and the vital function of miR-16-5p in suppressing cell proliferation,the aim of the present study was to investigate the combined impact of RG7388 and miR-16-5p overexpression on the childhood acute lymphoblastic leukemia(chALL).Methods:miRTarBase and miRDB,along with KEGG and STRING databases,were used to predict miR-16-5p target genes and explore protein-protein interaction networks,respectively.B-and T-lymphoblastic cell lines,in addition to patient primary cells,were treated with RG7388.Ectopic overexpression of miR-16-5p in Nalm6 cell line was induced through cell electroporation and transfection of microRNA mimics was confirmed by qRT-PCR.Cell viability was evaluated using the MTT assay.Western blot analyses were performed to evaluate the effects of RG7388 and miR-16-5p upregulation on the protein levels of p53 and its downstream target genes in chALL cells.Paired sample t-test was employed for statistical analyses.Results:MTT assay showed RG7388-induced cytotoxicity in wild-type p53 Nalm6 cell line and p53 functional patient primary cells.However,CCRF-CEM and p53 non-functional leukemic cells indicated drug resistance.Western blot analyses validated the bioinformatics results,confirming the downregulation of WIP1,p53 stabilization,as well as overexpression of p21WAF1 and Mdm2 proteins in Nalm6 cells transfected with miR-16-5p.Moreover,enhanced sensitivity to RG7388 was observed in the transfected cells.Conclusion:This is the first study indicating the mechanistic importance of miR-16-5p overexpression in chALL and its inhibitory role in leukemia treatment when combined with the p53-Mdm2 antagonist,RG7388.These findings might be useful for researchers and clinicians to pave the way for better management of chALL.
基金This study was supported by grants from the National Natural Science Foundation of China （Nos. 81000880 and 81370057 to Zhi Xu）, the Key Project supported by Medical Science and Technology Development Foundation, Nanjing Department of Health （JQX12007 to Zhi Xu）, the clinical special project for Natural Science Foundation of Jiangsu Province （BL2012016 to Jinfei Chen）, and the Nanjing 12th Five-Year Key Scientific Project of Medicine to Jinfei Chen.
文摘Hepatocellular carcinoma （HCC） is a lethal liver malignancy worldwide. In this study, we reported that protein phosphatase magnesium-dependent 16 （PPM1D） was highly expressed in the majority of HCC cases （approximately 59%） and significantly associated with high serum a-fetoprotein （AFP） level （P = 0.044）. Kaplan- Meier and Cox regression data indicated that PPM1D overexpression was an independent predictor of HCC- specific overall survival （HR, 2.799; 95% CI, 1.346-5.818, P = 0.006）. Overexpressing PPM1D promoted cell viability and invasion, whereas RNA interference-mediated knockdown of PPM1D inhibited proliferation, invasion, and migration of cultured HCC cells. In addition, PPM1D suppression by small interfering RNA decreased the tumorigenicity of HCC cells in vivo. Overall, results suggest that PPM1D is a potential prognostic marker and therapeutic target for HCC.
基金supported by a grant from National Natural Sciences Foundation of China (No. 30772240)
文摘To construct a lentiviral shRNA vector targeting human protein phosphatase 1D magnesium-dependent（PPM1D） gene and detect its effectiveness of gene silencing in human gliomas,specific siRNA targets with short hairpin frame were designed and synthesized.DNA oligo was cloned into the pFU-GW-iRNA lentiviral expression vector,and then PCR and sequencing analyses were conducted to verify the constructs.After the verified plasmids were transfected into 293T cells,the lentivirus was produced and the titer of virus was determined.Real-time quantitative PCR and Western blot were performed to detect the PPM1D expression level in the infected glioma cells.PCR and Western blot analyses revealed the optimal interfering target,and the virus with a titer of 6×10^8 TU/mL was successfully packaged.The PPM1D expression in human glioma cells was knocked down at both mRNA and protein levels by virus infection.The expression of PPM1D mRNA and protein was decreased by 76.3% and 87.0% respectively as compared with control group.The multiple functions of human glioma cells after PPM1D RNA interference were detected by flow cytometry and cell counting kit-8（CCK-8）.Efficient down-regulation of PPM1D resulted in significantly increased cell apoptosis and reduced cell proliferation and invasion potential in U87-MG cells.We have successfully constructed the lentiviral shRNA expression vector capable of stable PPM1D gene silencing at both mRNA and protein levels in glioma cells.And our data gave evidence that the reduced cell growth observed after PPM1D silencing in glioma cells was at least partly due to increased apoptotic cell death.
文摘目的探讨甲状腺乳头状癌(papillary carcinoma of thyroid,PTC)中PPM1D蛋白的表达情况及其与临床病理特征和p53的关系。方法应用免疫组化EnVision法检测55例PTC及癌旁正常组织中PPM1D蛋白的表达情况并进行统计处理,分析其与临床病理特征的关系及和p53蛋白表达的相关性。结果 55例PTC中PPM1D的表达(74.5%,41/55)高于癌旁正常组织(10.9%,6/55),差异有统计学意义;PPPM1D的表达与患者年龄、性别、肿瘤大小、组织学类型、淋巴结转移、TNM分期无关,而在间质硬化的病例中表达增强(P<0.05);PTC中PPM1D与p53蛋白表达呈负相关(r=-0.339,P<0.05)。结论高表达的PPM1D与PTC相关,与p53之间可能存在相互作用,在PTC发生、发展中具有重要意义。
文摘目的 :探讨PPM1D m RNA表达与肝癌预后的相关性。方法:提取86例肝癌患者癌组织及癌旁肝组织总RNA,q PCR法检测PPM1D m RNA表达量,免疫组化检测蛋白表达水平。根据癌旁肝组织中PPM1D m RNA表达量,将肝癌患者分组为高表达组与低表达组,对两组患者临床资料及生存时间进行统计分析。结果:PPM1D m RNA在肝癌组织中表达水平显著高于癌旁组织,免疫组化检测蛋白表达水平证实上述结果。以癌旁肝组织PPM1D m RNA表达量为阈值,高表达组56例,低表达组30例。两组患者的AFP水平、肿瘤大小、肿瘤TNM分期以及肿瘤复发、家族史等临床病理因素差异有统计学意义(P<0.01);年龄、性别、门静脉侵犯、淋巴结转移、HBV感染及酒精摄入史等因素差异无统计学意义(P>0.05)。高表达组患者中位生存期为13个月,低表达组为32个月。结论:PPM1D m RNA表达水平可能与肝癌恶性程度相关,可能成为肝癌预后的预测因子。
文摘Recently,a collaborative research led by Weibo Yang and Jim Sun1 published in Cell Chemical Biology identified a potent and selective small molecule inhibitor(SMIP-30)for human protein phosphatase Mg^(2+)/Mn^(2+)-dependent 1 A(PPM1A).They applied this chemical probe to determine the autophagy receptor p62 as a new substrate of PPM1A.
文摘目的通过NHE1基因敲除模型鼠的海马组织差异蛋白质组学分析,发现并明确Ppp3cb和Ppm1g的表达特征。方法①选取6只2周龄NHE1基因敲除模型鼠作为模型组,同周龄野生型小鼠6只作为对照组,采用琼脂糖凝胶电泳检测其基因型;应用旷场实验和强迫游泳实验对模型组和对照组小鼠进行行为学评估,并按照Racine评分标准对模型鼠进行癫痫发作分级;②通过串联质谱分析技术对模型组和对照组的海马组织进行差异蛋白筛选,基因本体论(Gene Ontology Analysis,GO)分析差异蛋白并进行注释和富集,蛋白网络数据库(search tool for the retrieval of interesting genes,STRING)分析差异蛋白之间的蛋白相互作用(protein-protein interaction,PPI);③应用qPCR和Western blot检测Ppp3cb和Ppm1g的转录和翻译水平,应用免疫组织化学技术分别观察其在组织中的表达量。结果①模型组小鼠NHE1基因未见表达,旷场实验中模型鼠的运动总距离较对照组减少(P=0.0073),跨越的格子数比对照组显著减少(P<0.0001)。强迫游泳实验结果显示,模型鼠不动的时间明显延长(P<0.0001);②以表达倍数(FC)≥1.2倍且P<0.05为筛选标准,检测到海马组织中845个差异表达的蛋白质点,其中有9个蛋白表达上调,7个蛋白表达下调。其中Ppp3cb下调,Ppm1g上调。GO功能注释结果表明,NHE1敲除后,分子功能(MF)富集在蛋白丝氨酸/苏氨酸磷酸酶活性的差异最显著,细胞成分(CC)富集在质膜部分的差异蛋白数量最多,生物过程(BP)富集在负向调节生物过程、免疫系统过程的差异蛋白数量最多。STRING分析显示差异蛋白Ppp3cb和Slc9a1直接作用,Ppm1g通过Ppp3cb和Slc9a1间接作用,Ppp3cb和Ppm1g之间相互作用。③Ppp3cb的转录和翻译水平减少,在组织中的表达量下降,而Ppm1g转录和翻译水平增加,在组织中的表达量上升(P<0.05)。结论本研究确定了NHE1基因敲除小鼠海马组织中差异蛋白Ppp3cb表达下调,而Ppm
文摘Jansen-de Vries综合征又称为“具有胃肠功能紊乱和高疼痛阈值的智力发育障碍”(Intellectual developmental disorder with gastrointestinal difficulties and high pain threshold,IDDGIP)[(OMIM):617450],是一种由PPM1D基因突变导致多系统受累的常染色体显性遗传(Autosomal dominant,AD)或常染色体隐性遗传(Autosomal recessive,AR)的罕见疾病.
