目的 研究一个家系中2例非综合征型遗传性聋患者的致病原因。方法 收集该耳聋家系临床检查结果资料,采集静脉血后提取DNA,应用全外显子组测序技术分析可疑致病基因,并通过Sanger测序对变异进行验证。结果 该家系中2代5人,先证者(Ⅱ-2,9...目的 研究一个家系中2例非综合征型遗传性聋患者的致病原因。方法 收集该耳聋家系临床检查结果资料,采集静脉血后提取DNA,应用全外显子组测序技术分析可疑致病基因,并通过Sanger测序对变异进行验证。结果 该家系中2代5人,先证者(Ⅱ-2,9岁)和其弟弟(Ⅱ-3)均为迟发性感音神经性听力损失,患者父母听力正常。先证者弟弟(Ⅱ-3)携带相同突变位点。基因检测结果显示,先证者携带CDH23基因c.4762C>T(p.ARG1588TRP)和c.6604G>A(p.ASP2202ASN)。根据美国医学遗传学与基因组学学会(American College of Medical Genetics and Genomics, ACMG)遗传变异分类标准与指南分析显示,c.4762C>T和c.6604G>A位点均为致病突变。蛋白功能分析发现,变异后氨基酸改变造成蛋白结构发生改变,进而影响蛋白质功能。结论 CDH23基因c.4762C>T和c.6604G>A复合杂合突变位点组合很可能是该家系耳聋致病基因。展开更多
Objectives:Progression to castration-resistant prostate cancer(CRPC)and metastasis are the greatest challenges to effective treatment.Anticancer strategies targeting the key kinases associated with the development of ...Objectives:Progression to castration-resistant prostate cancer(CRPC)and metastasis are the greatest challenges to effective treatment.Anticancer strategies targeting the key kinases associated with the development of CRPC may represent a breakthrough.The tyrosine kinase receptor Erythropoietin-producing hepatocellular(Eph)A2 receptor is highly expressed in CRPC cell lines and may be associated with tumor invasion and metastasis.However,the effects and exact mechanisms of EphA2 in CRPC are only partially understood.This study aimed to investigate the impact of EphA2 on CRPC cell behaviors and underlyingmolecular pathways.Methods:CRISPR/Cas9-mediated gene editing induced EphA2-disrupted in human-derived PC3 andDU145 cells.Single-guideRNAs(sgRNAs)targeting EphA2 were designed,and editing efficiency was validated.Optimal sgRNA sequences were selected to generate EphA2-knockdown(KD)and-overexpressing(OE)cell lines.Cell migration,proliferation,and apoptosis were assessed via functional assays.Transcriptomic analysis,quantitative PCR,and Western blotting were performed to identify downstream effectors.Bioinformatics analyses were used to correlate EphA2 and CDH1 expression with clinical parameters in prostate cancer patients.Results:Editing efficiency was found to vary among different sgRNAs targeting the EphA2 gene.EphA2-KD significantly inhibited CRPC cell migration but did not affect cell proliferation or apoptosis.Conversely,EphA2-OE significantly enhanced the migration of DU145 cells.Molecular analyses revealed that the expression of CDH1(an important marker of the epithelial-mesenchymal transition(EMT)in tumors)was significantly upregulated in PC3-EphA2-KD cells and downregulated in DU145-EphA2-OE cells,indicating that CDH1 is a downstream regulator of EphA2.Bioinformatic analysis revealed that higher EphA2 levels and lower CDH1 expression were both associated with an advanced tumor T stage,higher Gleason scores,and lymph nodemetastases in prostate cancer patients.More importantly,EphA2 was found to be an important predictor of lymph nodemetastasis,in addition to the Gleason score.Adding EphA2 to the Gleason score could significantly improve the detection of lymph node metastasis.Conclusion:CRISPR/Cas9-mediated EphA2-KD significantly suppressed the migration of CRPC cells through the inhibition of the EphA2-CDH1 axis.Strategies targeting the EphA2 genemay be promising for the treatment of CRPC.展开更多
目的:分析宫颈癌和宫颈上皮内瘤样病变(cervical intraepithelial neoplasia, CIN)组织中p16和CDH1基因异常甲基化的变化,评价该指标在宫颈癌中的意义。方法:用甲基化特异性聚合酶链反应法(methylation-specific PCR,MSP)检测CINⅠ40例...目的:分析宫颈癌和宫颈上皮内瘤样病变(cervical intraepithelial neoplasia, CIN)组织中p16和CDH1基因异常甲基化的变化,评价该指标在宫颈癌中的意义。方法:用甲基化特异性聚合酶链反应法(methylation-specific PCR,MSP)检测CINⅠ40例、CINⅡ～Ⅲ40例、宫颈癌40例组织中p16和CDH1基因的异常甲基化。取正常宫颈组织20例作为对照。结果:(1)p16和CDH1甲基化在正常组未见表达;(2)p16、CDH1甲基化阳性率:CINⅡ、Ⅲ组明显高于CINⅠ组,差异有统计学意义(22．4%vs 2．5%,P＜0．05; 35．0%vs 5．0%,P＜0．05),宫颈癌组高于CINⅡ、Ⅲ组,但差异无统计学意义(40．0% vs 22．4%,P＞0．05;57．5% vs 35．0%,P＞0．05);宫颈癌组高于相应CINⅠ组,差异有统计学意义(40．0% vs 2．5%,P＜0．05;57．5% vs 5．0%,P＜0．05);(3)p16和CDH1甲基化总阳性率(任何一个基因出现甲基化即为阳性):CINⅡ、Ⅲ组明显高于CINⅠ组,差异有统计学意义(40．0% vs 5．0%,P＜0．05),宫颈癌组高于CINⅡ、Ⅲ组,差异有统计学意义(70．0% vs 40．0%,P＜0．05)。结论:p16和CDH1基因启动子区异常甲基化与宫颈癌的生物学行为相关,它可能有助于宫颈癌的早期辅助诊断和治疗。展开更多
文摘目的 研究一个家系中2例非综合征型遗传性聋患者的致病原因。方法 收集该耳聋家系临床检查结果资料,采集静脉血后提取DNA,应用全外显子组测序技术分析可疑致病基因,并通过Sanger测序对变异进行验证。结果 该家系中2代5人,先证者(Ⅱ-2,9岁)和其弟弟(Ⅱ-3)均为迟发性感音神经性听力损失,患者父母听力正常。先证者弟弟(Ⅱ-3)携带相同突变位点。基因检测结果显示,先证者携带CDH23基因c.4762C>T(p.ARG1588TRP)和c.6604G>A(p.ASP2202ASN)。根据美国医学遗传学与基因组学学会(American College of Medical Genetics and Genomics, ACMG)遗传变异分类标准与指南分析显示,c.4762C>T和c.6604G>A位点均为致病突变。蛋白功能分析发现,变异后氨基酸改变造成蛋白结构发生改变,进而影响蛋白质功能。结论 CDH23基因c.4762C>T和c.6604G>A复合杂合突变位点组合很可能是该家系耳聋致病基因。
基金funded by the National Natural Science Foundation of China(Grant No.81801754)the Suzhou Science and Technology Bureau Development Plan(Grant No.SYS2020147)the 26th Batch of Extra-Curricular Academic Research Fund Project of Soochow University(Grant No.KY2024235B).
文摘Objectives:Progression to castration-resistant prostate cancer(CRPC)and metastasis are the greatest challenges to effective treatment.Anticancer strategies targeting the key kinases associated with the development of CRPC may represent a breakthrough.The tyrosine kinase receptor Erythropoietin-producing hepatocellular(Eph)A2 receptor is highly expressed in CRPC cell lines and may be associated with tumor invasion and metastasis.However,the effects and exact mechanisms of EphA2 in CRPC are only partially understood.This study aimed to investigate the impact of EphA2 on CRPC cell behaviors and underlyingmolecular pathways.Methods:CRISPR/Cas9-mediated gene editing induced EphA2-disrupted in human-derived PC3 andDU145 cells.Single-guideRNAs(sgRNAs)targeting EphA2 were designed,and editing efficiency was validated.Optimal sgRNA sequences were selected to generate EphA2-knockdown(KD)and-overexpressing(OE)cell lines.Cell migration,proliferation,and apoptosis were assessed via functional assays.Transcriptomic analysis,quantitative PCR,and Western blotting were performed to identify downstream effectors.Bioinformatics analyses were used to correlate EphA2 and CDH1 expression with clinical parameters in prostate cancer patients.Results:Editing efficiency was found to vary among different sgRNAs targeting the EphA2 gene.EphA2-KD significantly inhibited CRPC cell migration but did not affect cell proliferation or apoptosis.Conversely,EphA2-OE significantly enhanced the migration of DU145 cells.Molecular analyses revealed that the expression of CDH1(an important marker of the epithelial-mesenchymal transition(EMT)in tumors)was significantly upregulated in PC3-EphA2-KD cells and downregulated in DU145-EphA2-OE cells,indicating that CDH1 is a downstream regulator of EphA2.Bioinformatic analysis revealed that higher EphA2 levels and lower CDH1 expression were both associated with an advanced tumor T stage,higher Gleason scores,and lymph nodemetastases in prostate cancer patients.More importantly,EphA2 was found to be an important predictor of lymph nodemetastasis,in addition to the Gleason score.Adding EphA2 to the Gleason score could significantly improve the detection of lymph node metastasis.Conclusion:CRISPR/Cas9-mediated EphA2-KD significantly suppressed the migration of CRPC cells through the inhibition of the EphA2-CDH1 axis.Strategies targeting the EphA2 genemay be promising for the treatment of CRPC.
文摘目的:分析宫颈癌和宫颈上皮内瘤样病变(cervical intraepithelial neoplasia, CIN)组织中p16和CDH1基因异常甲基化的变化,评价该指标在宫颈癌中的意义。方法:用甲基化特异性聚合酶链反应法(methylation-specific PCR,MSP)检测CINⅠ40例、CINⅡ～Ⅲ40例、宫颈癌40例组织中p16和CDH1基因的异常甲基化。取正常宫颈组织20例作为对照。结果:(1)p16和CDH1甲基化在正常组未见表达;(2)p16、CDH1甲基化阳性率:CINⅡ、Ⅲ组明显高于CINⅠ组,差异有统计学意义(22．4%vs 2．5%,P＜0．05; 35．0%vs 5．0%,P＜0．05),宫颈癌组高于CINⅡ、Ⅲ组,但差异无统计学意义(40．0% vs 22．4%,P＞0．05;57．5% vs 35．0%,P＞0．05);宫颈癌组高于相应CINⅠ组,差异有统计学意义(40．0% vs 2．5%,P＜0．05;57．5% vs 5．0%,P＜0．05);(3)p16和CDH1甲基化总阳性率(任何一个基因出现甲基化即为阳性):CINⅡ、Ⅲ组明显高于CINⅠ组,差异有统计学意义(40．0% vs 5．0%,P＜0．05),宫颈癌组高于CINⅡ、Ⅲ组,差异有统计学意义(70．0% vs 40．0%,P＜0．05)。结论:p16和CDH1基因启动子区异常甲基化与宫颈癌的生物学行为相关,它可能有助于宫颈癌的早期辅助诊断和治疗。
