Pancreatic cancer is one of the deadliest cancers with a very poor prognosis. Recently, there has been a significant increase in research directed towards identifying potential biomarkers that can be used to diagnose ...Pancreatic cancer is one of the deadliest cancers with a very poor prognosis. Recently, there has been a significant increase in research directed towards identifying potential biomarkers that can be used to diagnose and provide prognostic information for pancreatic cancer. These markers can be used clinically to optimize and personalize therapy for individual patients. In this review, we focused on 3 biomarkers involved in the DNA damage response pathway and the necroptosis pathway: Chromodomainhelicase-DNA binding protein 5, chromodomain-helicaseDNA binding protein 7, and mixed lineage kinase domain-like protein. The aim of this article is to review present literature provided for these biomarkers and current studies in which their effectiveness as prognostic biomarkers are analyzed in order to determine their future use as biomarkers in clinical medicine. Based on the data presented, these biomarkers warrant further investigation,and should be validated in future studies.展开更多
染色质域解旋酶DNA结合蛋白5(chromodomain helicase DNA binding protein 5,CHD5)是人类肿瘤中一个重要的抑癌基因,是肿瘤抑制网络的控制开关,但是它在前列腺癌发生发展中的作用却少有研究。本研究利用CRISPR/Cas9技术敲除前列腺癌细胞...染色质域解旋酶DNA结合蛋白5(chromodomain helicase DNA binding protein 5,CHD5)是人类肿瘤中一个重要的抑癌基因,是肿瘤抑制网络的控制开关,但是它在前列腺癌发生发展中的作用却少有研究。本研究利用CRISPR/Cas9技术敲除前列腺癌细胞PC-3中CHD5基因,探索敲除CHD5基因对PC-3细胞增殖能力的影响。研究根据CRISPR/Cas9靶点设计规则设计了3组特异性sgRNA,连接到pLenti-U6-sgRNA-SFFV-Cas9-2A-Puro质粒构建表达载体。测序鉴定后将重组表达载体转染293T细胞包装慢病毒。包装慢病毒滴度分别为CHD5-sgRNA-17.84×10^9 TU/mL,CHD5-sgRNA-25.83×10^9 TU/mL,CHD5-sgRNA-35.23×10^9 TU/mL。用CHD5-sgRNA慢病毒感染PC-3细胞,嘌呤霉素筛选阳性细胞,Western印迹检测CHD5蛋白的表达水平,CCK-8、克隆形成实验以及细胞周期检测对PC-3细胞增值能力的影响。Western印迹结果显示,CHD5-sgRNA-1组CHD5蛋白质水平无明显变化,CHD5-sgRNA-2组和CHD5-sgRNA-3组CHD5蛋白质表达水平显著低于空白组和阴性对照组,成功获得稳定敲除CHD5基因的PC-3细胞系,其中感染CHD5-sgRNA-2的PC-3细胞中CHD5蛋白水平最低,用于后续研究。CCK-8结果显示,敲除CHD5促进PC-3细胞的增殖能力。空白组、阴性对照组以及CHD5-gRNA-2组的克隆数分别为(32.50±1.50)、(35.33±2.02)和(50.83±4.25)。细胞周期分析结果显示,G2/M期细胞分别为(7.56±0.59)%、(8.33±0.61)%和(27.21±1.04)%,差异均有统计学意义(P<0.01)。Western印迹证实,CHD5敲除后,G2/M期周期相关蛋白质细胞周期蛋白B1、cdc25C和cdc2的表达量增加,phospho-cdc25C、phospho-cdc2以及p21的表达水平下降(P<0.05)。以上结果表明,CHD5敲除的PC-3细胞系构建成功,CHD5表达缺失通过上调细胞周期蛋白B1、去磷酸化cdc25C、cdc2以及下调p21表达促进前列腺癌PC-3细胞增殖。展开更多
目的:探讨染色质解旋酶DNA结合蛋白1样基因(chromodomain helicase/ATPase DNA binding protein 1-like gene,CHD1L)对前列腺癌细胞侵袭、迁移能力的影响及其可能的作用机制。方法:采用实时荧光定量PCR技术检测前列腺癌细胞株LNCAP、PC3...目的:探讨染色质解旋酶DNA结合蛋白1样基因(chromodomain helicase/ATPase DNA binding protein 1-like gene,CHD1L)对前列腺癌细胞侵袭、迁移能力的影响及其可能的作用机制。方法:采用实时荧光定量PCR技术检测前列腺癌细胞株LNCAP、PC3、DU145以及前列腺上皮细胞株RWPE-1中CHD1L mRNA表达水平;转染siRNA干扰前列腺癌PC3细胞CHD1L的表达,并用Transwell侵袭实验和划痕实验分析沉默CHD1L对前列腺癌细胞侵袭和迁移能力的影响;Western blotting检测PC3细胞MMP-9、N-钙黏蛋白和E-钙黏蛋白的表达水平。结果:CHD1L mRNA在前列腺癌细胞中的表达水平明显高于前列腺上皮细胞(P<0.01),其中以前列腺癌PC3细胞的表达水平最高。侵袭实验中,干扰组的穿膜细胞数明显低于阴性对照组和空白对照组[(49.67±6.67)vs(113.67±5.69)和(112.00±12.49)个,P<0.05)。划痕实验中,干扰组48 h伤口愈合率也低于阴性对照组和空白对照组[(21.27±3.27)%vs(48.47±5.72)%和(49.93±3.35)%,P<0.05]。干扰组细胞MMP-9和N-钙黏蛋白表达下调,E-钙黏蛋白表达上调。结论:沉默CHD1L可降低前列腺癌PC3细胞的侵袭迁移能力,该作用可能是通过调控MMP-9和EMT相关蛋白表达实现的。展开更多
基金Supported by The National Center for Advancing Translational Sciences of the National Institutes of Health under award numbers ULl TR000454 previously awarded to Dr.Colbert and Dr.Fisher and TLlT R000456 to Dr.ColbertPancreatic Cancer Action Network(Pan-CAN)&sol American Association for Cancer Research(AACR)award 16982+1 种基金Department of Defense(DOD)/Peer Reviewed Cancer Research Program(PRCRP)award CA110535Georgia Cancer Coalition award 11072,all to Dr.Yu
文摘Pancreatic cancer is one of the deadliest cancers with a very poor prognosis. Recently, there has been a significant increase in research directed towards identifying potential biomarkers that can be used to diagnose and provide prognostic information for pancreatic cancer. These markers can be used clinically to optimize and personalize therapy for individual patients. In this review, we focused on 3 biomarkers involved in the DNA damage response pathway and the necroptosis pathway: Chromodomainhelicase-DNA binding protein 5, chromodomain-helicaseDNA binding protein 7, and mixed lineage kinase domain-like protein. The aim of this article is to review present literature provided for these biomarkers and current studies in which their effectiveness as prognostic biomarkers are analyzed in order to determine their future use as biomarkers in clinical medicine. Based on the data presented, these biomarkers warrant further investigation,and should be validated in future studies.
文摘染色质域解旋酶DNA结合蛋白5(chromodomain helicase DNA binding protein 5,CHD5)是人类肿瘤中一个重要的抑癌基因,是肿瘤抑制网络的控制开关,但是它在前列腺癌发生发展中的作用却少有研究。本研究利用CRISPR/Cas9技术敲除前列腺癌细胞PC-3中CHD5基因,探索敲除CHD5基因对PC-3细胞增殖能力的影响。研究根据CRISPR/Cas9靶点设计规则设计了3组特异性sgRNA,连接到pLenti-U6-sgRNA-SFFV-Cas9-2A-Puro质粒构建表达载体。测序鉴定后将重组表达载体转染293T细胞包装慢病毒。包装慢病毒滴度分别为CHD5-sgRNA-17.84×10^9 TU/mL,CHD5-sgRNA-25.83×10^9 TU/mL,CHD5-sgRNA-35.23×10^9 TU/mL。用CHD5-sgRNA慢病毒感染PC-3细胞,嘌呤霉素筛选阳性细胞,Western印迹检测CHD5蛋白的表达水平,CCK-8、克隆形成实验以及细胞周期检测对PC-3细胞增值能力的影响。Western印迹结果显示,CHD5-sgRNA-1组CHD5蛋白质水平无明显变化,CHD5-sgRNA-2组和CHD5-sgRNA-3组CHD5蛋白质表达水平显著低于空白组和阴性对照组,成功获得稳定敲除CHD5基因的PC-3细胞系,其中感染CHD5-sgRNA-2的PC-3细胞中CHD5蛋白水平最低,用于后续研究。CCK-8结果显示,敲除CHD5促进PC-3细胞的增殖能力。空白组、阴性对照组以及CHD5-gRNA-2组的克隆数分别为(32.50±1.50)、(35.33±2.02)和(50.83±4.25)。细胞周期分析结果显示,G2/M期细胞分别为(7.56±0.59)%、(8.33±0.61)%和(27.21±1.04)%,差异均有统计学意义(P<0.01)。Western印迹证实,CHD5敲除后,G2/M期周期相关蛋白质细胞周期蛋白B1、cdc25C和cdc2的表达量增加,phospho-cdc25C、phospho-cdc2以及p21的表达水平下降(P<0.05)。以上结果表明,CHD5敲除的PC-3细胞系构建成功,CHD5表达缺失通过上调细胞周期蛋白B1、去磷酸化cdc25C、cdc2以及下调p21表达促进前列腺癌PC-3细胞增殖。
