目的基于生物信息学研究PKMYT1基因在乳腺癌进展中的作用及影响以及在乳腺癌各细胞系中的表达情况。方法将癌症基因组图谱(the cancer genome atlas,TCGA)乳腺癌RNA转录组数据中的PKMYT1进行单基因分析,其中包括差异分析、配对差异分析...目的基于生物信息学研究PKMYT1基因在乳腺癌进展中的作用及影响以及在乳腺癌各细胞系中的表达情况。方法将癌症基因组图谱(the cancer genome atlas,TCGA)乳腺癌RNA转录组数据中的PKMYT1进行单基因分析,其中包括差异分析、配对差异分析、临床相关性分析、GSEA富集分析及生存分析,同时还利用GEPIA 2、TIMER2.0、The Human Protein Atlas及Kaplan-Meier Plotter数据库进行了多次验证。且通过实时荧光定量PCR检测了PKMYT1在乳腺癌各细胞系中的表达情况,为后续研究筛选合适的细胞系。结果经生物信息学分析发现在乳腺癌中与正常样本比较,PKMYT1在肿瘤样本中的表达明显升高(P<0.05);临床相关性分析结果表明,PKMYT1在乳腺癌中与年龄、肿瘤分期及原发肿瘤直径相关(P<0.05),而与性别、远处转移、淋巴结转移无显著相关性(P>0.05);GSEA富集分析显示在乳腺癌中PKMYT1高表达时,其功能富集与细胞周期、DNA复制、同源重组等功能密切相关,低表达时则与TGF-β信号通路、MAPK信号通路等相关;生存分析发现PKMYT1在乳腺癌中HR=1.22提示为高风险因素,且与生存相关(P<0.05)。同时细胞实验验证了PKMYT1在人乳腺癌细胞系中以正常乳腺细胞系为参照在MDA-MB-231中表达最高,在小鼠乳腺癌细胞系中4T1细胞系表达最高。结论PKMYT1是一种与乳腺癌恶性肿瘤相关的基因,是一种新的肿瘤标志物,或可成为乳腺癌新的治疗靶点。展开更多
Cisplatin(DDP)remains a cornerstone therapy for osteosarcoma(OS);however,pervasive resistance severely limits its clinical efficacy and worsens patient outcomes.Developing strategies to enhance the chemotherapeutic re...Cisplatin(DDP)remains a cornerstone therapy for osteosarcoma(OS);however,pervasive resistance severely limits its clinical efficacy and worsens patient outcomes.Developing strategies to enhance the chemotherapeutic responsiveness of os cells is therefore of critical importance.Here,we conducted a kinome-wide clustered regularly interspaced short palindromic repeats(CRISPR)screen,coupled with transcriptome sequencing,to identify regulators of DDP sensitivity.This approach revealed protein kinase membrane-associated tyrosine/threonine 1(PKMYT1)as a key regulator of DDP sensitivity in OS.Subsequent analysis of patient-derived clinical specimens,along with in vitro functional assays,demonstrated that DDP treatment induces the activation of PKMYT1 in OS cells.Importantly,PKMYT1 silencing markedly enhances cellular sensitivity to DDP,indicating its role in promoting chemoresistance.Mechanistically,PKMYT1 induces phosphorylation of nucleophosmin 1(NPM1)at the S260 site,which competitively impairs NPM1 SUMOylation.This modification interferes with the recruitment of essential DNA damage response factors,including breast cancer suppressor gene 1(BRCA1),receptor-associated protein 80(RAP80),and RADiation sensitive protein 51(RAD51),ultimately affecting double-strand break(DSB)repair.Furthermore,the selective PKMYT1 inhibitor RP6306 was found to synergize with DDP,amplifying its cytotoxic effects in OS cells.Collectively,these findings highlight PKMYT1 as a promising therapeutic target and provide a rationale for combinatorial strategies to overcome DDP resistance in OS.展开更多
Objective:Osteosarcoma is the most common primary malignant bone tumor.However,the survival of patients with osteosarcoma has remained unchanged during the past 30 years,owing to a lack of efficient therapeutic target...Objective:Osteosarcoma is the most common primary malignant bone tumor.However,the survival of patients with osteosarcoma has remained unchanged during the past 30 years,owing to a lack of efficient therapeutic targets.Methods:We constructed a kinome-targeting CRISPR-Cas9 library containing 507 kinases and 100 nontargeting controls and screened the potential kinase targets in osteosarcoma.The CRISPR screening sequencing data were analyzed with the Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout(MAGeCK)Python package.The functional data were applied in the 143B cell line through lenti-CRISPR-mediated gene knockout.The clinical significance of kinases in the survival of patients with osteosarcoma was analyzed in the R2:Genomics Analysis and Visualization Platform.Results:We identified 53 potential kinase targets in osteosarcoma.Among these targets,we analyzed 3 kinases,TRRAP,PKMYT1,and TP53RK,to validate their oncogenic functions in osteosarcoma.PKMYT1 and TP53RK showed higher expression in osteosarcoma than in normal bone tissue,whereas TRRAP showed no significant difference.High expression of all 3 kinases was associated with relatively poor prognosis in patients with osteosarcoma.Conclusions:Our results not only offer potential therapeutic kinase targets in osteosarcoma but also provide a paradigm for functional genetic screening by using a CRISPR-Cas9 library,including target design,library construction,screening workflow,data analysis,and functional validation.This method may also be useful in potentially accelerating drug discovery for other cancer types.展开更多
文摘目的基于生物信息学研究PKMYT1基因在乳腺癌进展中的作用及影响以及在乳腺癌各细胞系中的表达情况。方法将癌症基因组图谱(the cancer genome atlas,TCGA)乳腺癌RNA转录组数据中的PKMYT1进行单基因分析,其中包括差异分析、配对差异分析、临床相关性分析、GSEA富集分析及生存分析,同时还利用GEPIA 2、TIMER2.0、The Human Protein Atlas及Kaplan-Meier Plotter数据库进行了多次验证。且通过实时荧光定量PCR检测了PKMYT1在乳腺癌各细胞系中的表达情况,为后续研究筛选合适的细胞系。结果经生物信息学分析发现在乳腺癌中与正常样本比较,PKMYT1在肿瘤样本中的表达明显升高(P<0.05);临床相关性分析结果表明,PKMYT1在乳腺癌中与年龄、肿瘤分期及原发肿瘤直径相关(P<0.05),而与性别、远处转移、淋巴结转移无显著相关性(P>0.05);GSEA富集分析显示在乳腺癌中PKMYT1高表达时,其功能富集与细胞周期、DNA复制、同源重组等功能密切相关,低表达时则与TGF-β信号通路、MAPK信号通路等相关;生存分析发现PKMYT1在乳腺癌中HR=1.22提示为高风险因素,且与生存相关(P<0.05)。同时细胞实验验证了PKMYT1在人乳腺癌细胞系中以正常乳腺细胞系为参照在MDA-MB-231中表达最高,在小鼠乳腺癌细胞系中4T1细胞系表达最高。结论PKMYT1是一种与乳腺癌恶性肿瘤相关的基因,是一种新的肿瘤标志物,或可成为乳腺癌新的治疗靶点。
基金supported by the National Natural Science Foundation of China(82272664,82172500)The Science and Technology Innovation Program of Hunan Province(2023RC3085)+8 种基金The Hunan Provincial Health High-Level Talent Scientific Research Project(R2023054)Key Project of Scientific Research of the Education Department of Hunan Province(24Aoo08)The Hunan Provincial Administration of Traditional Chinese Medicine Project(D2022117)The Scientifc Research Program of Hunan Provincial Health Commission(B202304077077)The Hunan Provincial Natural Science Foundation of China(2023JJ40822,2024JJ6559,2024JJ6572,2025JJ60590)The State-funded postdoctoral researchers plan of China(No.GZC20233188)The Fundamental Research Funds for the Central Universities of Central South University(2024ZZTS0174)The Changsha Municipal Natural Science Foundation(Grant No.kq2403077)The Health Research Project of Hunan Provincial Health Commission(202105012224)。
文摘Cisplatin(DDP)remains a cornerstone therapy for osteosarcoma(OS);however,pervasive resistance severely limits its clinical efficacy and worsens patient outcomes.Developing strategies to enhance the chemotherapeutic responsiveness of os cells is therefore of critical importance.Here,we conducted a kinome-wide clustered regularly interspaced short palindromic repeats(CRISPR)screen,coupled with transcriptome sequencing,to identify regulators of DDP sensitivity.This approach revealed protein kinase membrane-associated tyrosine/threonine 1(PKMYT1)as a key regulator of DDP sensitivity in OS.Subsequent analysis of patient-derived clinical specimens,along with in vitro functional assays,demonstrated that DDP treatment induces the activation of PKMYT1 in OS cells.Importantly,PKMYT1 silencing markedly enhances cellular sensitivity to DDP,indicating its role in promoting chemoresistance.Mechanistically,PKMYT1 induces phosphorylation of nucleophosmin 1(NPM1)at the S260 site,which competitively impairs NPM1 SUMOylation.This modification interferes with the recruitment of essential DNA damage response factors,including breast cancer suppressor gene 1(BRCA1),receptor-associated protein 80(RAP80),and RADiation sensitive protein 51(RAD51),ultimately affecting double-strand break(DSB)repair.Furthermore,the selective PKMYT1 inhibitor RP6306 was found to synergize with DDP,amplifying its cytotoxic effects in OS cells.Collectively,these findings highlight PKMYT1 as a promising therapeutic target and provide a rationale for combinatorial strategies to overcome DDP resistance in OS.
基金This work was funded by the National Key Research and Development Program of China(Grant No.2016YFA0500304to T.K.)the Science and Technology Program of Guangzhou,(Grant Nos.202002020092 and 201607020038 to T.K.)+2 种基金the National Nature Science Foundation in China(NSFC)(Grant Nos.81772922 to Y.W.,81702890 to X.W.,81530081,31571395 to T.K.)the Guangdong Natural Science Foundation Team Project(Grant No.2014A030312015 to T.K.)the Natural Science Foundation of Guangdong Province(Grant No.2016A030310218 to W.Y.).
文摘Objective:Osteosarcoma is the most common primary malignant bone tumor.However,the survival of patients with osteosarcoma has remained unchanged during the past 30 years,owing to a lack of efficient therapeutic targets.Methods:We constructed a kinome-targeting CRISPR-Cas9 library containing 507 kinases and 100 nontargeting controls and screened the potential kinase targets in osteosarcoma.The CRISPR screening sequencing data were analyzed with the Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout(MAGeCK)Python package.The functional data were applied in the 143B cell line through lenti-CRISPR-mediated gene knockout.The clinical significance of kinases in the survival of patients with osteosarcoma was analyzed in the R2:Genomics Analysis and Visualization Platform.Results:We identified 53 potential kinase targets in osteosarcoma.Among these targets,we analyzed 3 kinases,TRRAP,PKMYT1,and TP53RK,to validate their oncogenic functions in osteosarcoma.PKMYT1 and TP53RK showed higher expression in osteosarcoma than in normal bone tissue,whereas TRRAP showed no significant difference.High expression of all 3 kinases was associated with relatively poor prognosis in patients with osteosarcoma.Conclusions:Our results not only offer potential therapeutic kinase targets in osteosarcoma but also provide a paradigm for functional genetic screening by using a CRISPR-Cas9 library,including target design,library construction,screening workflow,data analysis,and functional validation.This method may also be useful in potentially accelerating drug discovery for other cancer types.
