MiR-451靶向调控SIRT1通路对胃癌细胞增殖的作用机制研究

摘 要：［目的］ 探讨miR-451靶向调控SIRT1通路对胃癌细胞增殖的作用机制研究。［方法］ 通过转染胃癌HepG2细胞实验进行分组,胃癌组、模拟物组和抑制物组,采用RT-PCR法检测HepG2细胞中miR-451、SIRT1 mRNA表达,流式细胞术检测HepG2细胞凋亡,Western blot检测细胞中SIRT1蛋白水平,细胞克隆测定细胞存活率,裸鼠皮下成瘤实验检测小鼠肿瘤体积。［结果］模拟物组miR-451表达量高于其他两组,与抑制物组相比,胃癌组miR-451表达水平较高（t=8.651,P＜0.001）。胃癌组SIRT1表达高于模拟物组,与抑制物组相比,胃癌组和模拟物组SIRT1表达水平明显降低（t=6.362,P＜0.001）,三组比较差异有显著性（F=3.565,P＜0.001）。与胃癌组相比,模拟物组HepG2细胞凋亡明显增加,胃癌组和抑制物组细胞凋亡情况均弱于模拟物组,三组比较差异有显著性（?字2=9.797,P=0.007）。胃癌组和抑制物组HepG2细胞单克隆形成率有上升趋势,模拟物组明显下降,三组比较组间差异有显著性（F=6.031,P＜0.001）。模拟物组小鼠肿瘤体积较抑制物组明显变小,与胃癌组相比,抑制物组肿瘤体积增加,三组比较差异有显著性（F=9.967,P＜0.001）。［结论］ miR-451过表达可以抑制SIRT1通路相关蛋白活性,促进胃癌HepG2细胞凋亡,抑制其增殖和发展。     

沉默信息调节因子1在肿瘤发生发展中的作用

沉默信息调节因子1（SIRT1）是一种依赖于烟酰胺腺嘌呤二核苷酸的组蛋白脱乙酰酶,参与肿瘤的发生发展过程。研究表明,SIRT1在胃癌、乳腺癌、前列腺癌等多种实体肿瘤中呈高表达,可促进肿瘤发生发展;同时发现,SIRT1在其他肿瘤如胶质母细胞瘤、膀胱癌、卵巢癌等中SIRT1表达降低,起抑癌基因的作用。因此,SIRT1的具体作用机制仍未阐明,尚存争议。     

miR-138靶向调控SIRT1表达及其对膀胱癌细胞增殖和凋亡的影响

目的 探讨微小RNA-138（miRNA-138）在膀胱癌细胞中的表达情况,并研究其对膀胱癌细胞增殖和凋亡的影响及其可能的靶基因。方法 采用实时定量PCR（QPCR）法检测膀胱癌细胞T24和正常膀胱上皮细胞SV-HUC-1中miR-138的表达水平。将T24细胞分为3组：未转染组、miR-138对照组（转染阴性对照片段）和miR-138转染组（转染miR-138 mimics）。采用MTT法检测细胞的增殖情况,流式细胞术检测细胞的凋亡情况;Western blotting检测细胞中沉默信息调节因子1（SIRT1）蛋白的表达水平;双荧光素酶报告基因实验验证miR-138与SIRT1间的靶向关系。结果 膀胱癌T24细胞中miR-138的表达量为0.57±0.19,低于SV-HUC-1细胞的1.00±0.26（P＜0.05）。miR-138转染组的miR-138表达量为2.59±0.67,高于未转染组的1.00±0.36和miR-138对照组的1.08±0.49（P＜0.05）。miR-138转染组T24细胞的增殖率显著低于未转染组和miR-138对照组（P＜0.05）。 转染48 h后,miR-138转染组的细胞凋亡率为（29.8±1.9）％,高于未转染组的（5.8±1.2）％和miR-138对照组的（7.7±0.9）％（P＜0.05）。miR-138转染组SIRT1的相对表达量为0.59±0.22,低于未转染组的1.00±0.35和miR-138对照组的1.20±0.42（P＜0.05）。双荧光素酶报告基因实验证明SIRT1是miR-138的直接作用靶点。结论 miR-138在膀胱癌细胞中低表达,可能通过靶向SIRT1调控膀胱癌细胞的增殖和凋亡。     

番茄红素通过SIRT1/NF-κB轴抑制肾癌786-O细胞增殖且促进其凋亡

目的：探讨番茄红素通过沉默信息调节因子1（SIRT1）/核因子-κB（NF-κB）轴对肾癌786-O 细胞增殖、凋亡的影响。方法：常规培养人正常肾细胞HK-2和人肾癌细胞786-O ，实验分为对照组（0.1% DMSO）、顺铂组（40 μg/mL）、番茄红素低质量浓度（2.5 μg/mL）组、番茄红素高质量浓度（5 μg/mL）组、番茄红素（5 μg/mL）+EX527（SIRT1抑制剂）（3 μmol/L）组。CCK-8法、克隆形成实验检测各组HK-2、786-O 细胞的增殖能力，流式细胞术检测各组786-O 细胞的凋亡，RH123、DCFH-DA 染色分别检测各组786-O 细胞的线粒体膜电位（MMP）、活性氧（ROS）水平，WB法检测各组786-O细胞中凋亡相关蛋白 BAX、Bcl-2、C-casp3和SIRT1/NF-κB轴相关蛋白 SIRT1、p-NF- κB 蛋白的表达。786-O 细胞移植瘤实验检测番茄红素低（5 mg/kg）、高质量浓度（20 mg/Kg）、顺铂（2 mg/kg）、番茄红素（20 mg/kg）+EX527（10 mg/kg）对移植瘤生长的影响，TUNEL法检测各组移植瘤组织中的细胞凋亡。结果：番茄红素呈剂量依赖性地抑制786-O细胞的增殖活性，番茄红素、顺铂均明显抑制786-O细胞的克隆形成能力且促进其凋亡，细胞中MMP损伤率升高而ROS 水平降低，凋亡相关蛋白BAX、C-casp3 表达均显著升高（均P<0.05）而Bcl-2表达下调（P<0.05），SIRT1表达显著升高（P<0.05）而p-NF-κB的表达显著降低（P<0.05），上述作用均可被EX527 逆转；番茄红素、顺铂抑制786-O细胞移植瘤的生长且促进其细胞凋亡，其作用也能被EX527 逆转。结论：番茄红素通过上调SIRT1、抑制NF-κB通路的激活进而抑制786-O细胞增殖且诱导其凋亡。     

沉默信息调节因子过表达或谷氨酰胺剥夺对肾透明细胞癌细胞凋亡、增殖的影响

背景与目的：肾透明细胞癌（clear cell renal cell carcinoma,ccRCC）是最常见的肾癌类型,它与代谢密切相关。探讨沉默信息调节因子4（silent information regulator 4,SIRT4）过表达或谷氨酰胺（glutamine,Gln）剥夺对ccRCC细胞增殖、凋亡的影响。方法：慢病毒构建SIRT4和突变体H161Y过表达的Caki-2细胞株,利用无Gln的培养基来构建Gln剥夺模型,并通过体外增殖活力实验［细胞计数试剂盒-8（cell counting kit-8,CCK-8）］和克隆形成实验来分析两者对Caki-2细胞增殖和生长能力的影响;利用DCFH-DA荧光探针检测细胞内活性氧自由基（reactive oxygen species,ROS）水平进而评估Gln代谢对细胞ROS含量的影响;进一步通过线粒体膜电位检测、凋亡检测和蛋白质印迹法（Western blot）检测凋亡相关分子,分析SIRT4过表达以及Gln剥夺对Caki-2细胞凋亡的影响。结果：过表达SIRT4可抑制Gln代谢从而抑制Caki-2细胞增殖,另外还原性物质还原型烟酰胺腺嘌呤二核苷酸磷酸（reduced nicotinamide adenine dinucleotide phostate,NADPH）的生成减少能够增加细胞内ROS含量,促进细胞凋亡。而Gln剥夺抑制细胞增殖和促进细胞凋亡的效果均比过表达SIRT4明显,但长期缺乏Gln将导致细胞无法生长。结论：无论是过表达SIRT4还是Gln剥夺均能抑制ccRCC细胞增殖,促进凋亡。     

苹果多酚通过激活AMPK/SIRT1信号通路对LPS诱导的肺上皮细胞自噬反应的影响

目的:探讨苹果多酚通过调节腺苷酸活化蛋白激酶/沉默信息调节因子1（AMP-activated protein kinase/Sirtuin1,AMPK/SIRT1)信号通路对脂多糖（lipopolysaccharide,LPS）诱导的人肺泡上皮细胞（A549）自噬反应的影响。方法:使用不同浓度的苹果多酚提取物（apple polyphenol extract,APE）预处理A549细胞2 h后,LPS诱导A549细胞培养24 h,MTT法检测增殖活性,筛选APE最佳预处理浓度;将A549细胞分为对照组、LPS组（3 mg/L LPS）、LPS+APE组（3 mg/L LPS+20 μg/mL APE）、APE+Compound C组（3 mg/L LPS+20 μg/mL APE+50 μmol/L Compound C）,免疫荧光染色观察A549细胞自噬;流式细胞术检测A549细胞凋亡;Western blot法检测细胞中自噬相关蛋白及AMPK/SIRT1通路相关蛋白表达水平。结果:与对照组比较,经LPS诱导的A549细胞增殖活性、自噬水平、LC3Ⅱ/LC3Ⅰ、Beclin-1、SIRT1、p-ULK1/ULK1、p-AMPK/AMPK蛋白表达降低,p62蛋白表达及细胞凋亡率升高（P＜0.05）;与LPS组比较,LPS+APE组细胞增殖活性、自噬水平、LC3Ⅱ/LC3Ⅰ、Beclin-1、SIRT1、p-ULK1/ULK1、p-AMPK/AMPK蛋白表达水平显著升高,p62蛋白表达及细胞凋亡率显著降低（P＜0.05）;与LPS+APE组比较,APE+Compound C组A549细胞增殖活性、自噬水平、LC3Ⅱ/LC3Ⅰ、Beclin-1、SIRT1、p-ULK1/ULK1、p-AMPK/AMPK蛋白表达水平显著降低,p62蛋白表达及细胞凋亡率显著升高（P＜0.05）。结论:苹果多酚通过激活AMPK/SIRT1 信号通路提高LPS诱导的肺上皮细胞自噬,降低细胞凋亡。     

组蛋白去乙酰酶SIRT1介导细胞自噬在肿瘤中的研究进展

自噬现象广泛存在于真核细胞中,是细胞的一种自我保护机制。这类分解代谢过程发生在细胞内,进化上具有高度保守性,在多种疾病及生命进程中发挥着重要作用。Sirtuin1（SIRT1）是一种组蛋白去乙酰化酶,具有烟酰胺腺嘌呤二核苷酸（NAD+）依赖性,在细胞生存、衰老、凋亡和代谢等生物学过程中扮演着重要角色。近年来,SIRT1与自噬的关系受到越来越多的关注,且SIRT1被证明与肿瘤的发生发展密切相关。探索SIRT1和自噬间的联系以及SIRT1介导的细胞自噬在肿瘤中的作用,有助于对肿瘤的发生发展机制展开进一步的探索。本文对肿瘤中SIRT1与自噬的作用关系及机制作以综述,希望能够为肿瘤的深入研究及新型靶向药物的研制提供新思路。     

SIRT1通过调节Noxa表达影响非小细胞肺癌细胞株A549对顺铂的敏感性

背景与目的 非小细胞肺癌的顺铂耐药是常见的临床现象,严重制约了患者的化疗效果,是亟待解决的问题.SIRT1和Noxa的表达变化影响肿瘤细胞对化疗药物的敏感性.本研究旨在研究SIRT1表达对非小细胞肺癌对顺铂的敏感性的影响,并探讨其涉及Noxa表达的机制,以求为提高非小细胞肺癌细胞对顺铂敏感性提供希望.方法 利用实时荧光定量PCR和Westem blot分析A549细胞及顺铂耐药的A549/DDP细胞SIRT1及Noxa mRNA和蛋白水平的表达差异.利用siRNA干扰技术抑制A549/DDP细胞的SIRT1表达,进而使用Cell Titer Blue试验、流式细胞术从细胞增殖、细胞周期和细胞凋亡方面分析SIRT1沉默对A549/DPP细胞顺铂敏感性的影响.同时利用实时荧光定量PCR和Western blot分析SIRT1抑制对A549/DPP细胞Noxa表达的影响.结果 A549细胞和A549/DDP细胞对顺铂的敏感性有显著差异,与A549细胞相比,A549/DDP细胞的SIRT1表达较高,但Noxa表达较低.使用siRN抑制A549/DPP细胞的SIRT1表达后,与未抑制SIRT1细胞相比,4μg/mL顺铂处理后的细胞存活率降低,G2期/M期阻滞比例增加,凋亡率提高.同时,SIRT1沉默导致A549/DPP细胞的Noxa表达增加.结论 较高的SIRT1可能引起A549细胞对顺铂的耐药性,抑制SIRT1可以提高A549/DDP细胞对顺铂的敏感性,其机制可能涉及SIRT1对Noxa的调节.     

细胞外信号调节激酶1/2参与三氧化二砷诱导的胶质瘤细胞凋亡

目的:探讨细胞外信号调节激酶1/2(ERK1/2)信号通路在三氧化二砷诱导的胶质瘤细胞U251凋亡中的作用,为应用三氧化二砷治疗胶质瘤奠定基础。方法:50μmol/L三氧化二砷作用U251细胞,不同时间检测胶质瘤细胞增殖活性,半定量PCR检测ERK1/2mRNA表达,Western blot检测ERK1/2蛋白表达;Ho-echst33258染色及流式细胞术(FCM)检测细胞凋亡;转染ERK1/2上游激酶MEK1,应用ERK1/2激酶抑制剂U0126观察ERK1/2通路在肿瘤凋亡及增殖中的作用;比色分析法检测Caspase-3活性的变化。结果:三氧化二砷诱导胶质瘤细胞发生明显凋亡,抑制肿瘤细胞增殖;增加ERK1/2蛋白的表达,呈时间依赖性;阻断ERK1/2信号通路后胶质瘤细胞凋亡受到抑制,Caspase-3活性下降。结论:ERK1/2信号通路在三氧化二砷诱导的胶质瘤细胞凋亡中起重要作用。     

抑制PDK1经由ASK1/JNK/Bim通路诱导慢性粒细胞白血病细胞凋亡

背景与目的：3-磷酸肌醇依赖性蛋白激酶1（3-phosphoinositide-dependent kinase-1,PDK1）在细胞的生长、增殖及存活中具有重要的生理作用,其功能异常参与多种肿瘤的发生。探讨PDK1在慢性粒细胞白血病（chronic myelogenous leukemia,CML）细胞中的表达及参与细胞凋亡的调节机制。方法：通过蛋白质印迹法（Western blot）检测蛋白水平;通过细胞计数试剂盒（cell counting kit-8,CCK-8）及克隆形成实验检测细胞增殖;通过流式细胞术检测细胞凋亡。结果：CML患者外周血白细胞中PDK1蛋白含量明显高于健康体检者（P<0.05）;在细胞株中,抑制PDK1能明显降低K562和KU812的增殖活力及克隆形成能力;同时,细胞凋亡明显（P<0.01）,凋亡执行分子caspase-3及底物多聚ADP核糖聚合酶［poly（ADP-ribose）polymerase,PARP］被切割活化。抑制剂GSK2334470降低PDK1的磷酸化水平（K562：P<0.05;KU812：P<0.01）,但并不影响其表达丰度。蛋白激酶B（protein kinase B,AKT）及下游糖原合成酶激酶-3β（glycogen synthase kinase-3β,GSK-3β）磷酸化水平均明显下降,然而β-catenin及靶基因c-Myc表达均未受影响（P>0.05）。磷酸化凋亡信号调节激酶1（apoptosis signal-regulating kinase 1,ASK1）水平降低（P<0.001）,下游c-Jun氨基末端激酶（c-Jun N-terminal kinase,JNK）及Bcl-2相互作用细胞凋亡介导因子（Bcl-2 interacting mediator of cell death,Bim）磷酸化水平明显升高,同时,Bim表达量也有所增加。最后,抑制PDK1能够增加CML细胞对伊马替尼（imatinib）的敏感性,与imatinib单独作用相比,联合GSK2334470能够显著提高细胞凋亡水平（P<0.01）。结论：PDK1抑制剂GSK2334470通过调节ASK1/JNK/Bim信号通路磷酸化水平激活CML细胞凋亡。     

Silent mating‐type information regulation 2 homolog 1 overexpression is an important strategy for the survival of adapted suspension tumor cells

Characterization of circulating tumor cells (CTC) is important to prevent death caused by the metastatic spread of cancer cells because CTC are associated with distal metastasis and poor prognosis of breast cancer. We have previously developed suspension cells (SC) using breast cancer cell lines and demonstrated their high metastatic potential. As survival of CTC is highly variable from a few hours to decades, herein we cultured SC for an extended time and named them adapted suspension cells (ASC). Silent mating‐type information regulation 2 homolog 1 (SIRT1) expression increased in ASC, which protected the cells from apoptosis. High SIRT1 expression was responsible for the suppression of nuclear factor kappa B (NF‐κB) activity and downregulation of reactive oxygen species (ROS) in ASC. As the inhibition of NF‐κB and ROS production in SIRT1‐depleted ASC contributed to the development of resistance to apoptotic cell death, maintenance of a low ROS level and NF‐κB activity in ASC is a crucial function of SIRT1. Thus, SIRT1 overexpression may play an important role in growth adaptation of SC because SIRT1 expression is increased in long‐term rather than in short‐term cultures.     

胰腺癌细胞PANC-1中LSD1负向调控抑癌基因SIRT3的实验研究

背景与目的：组蛋白赖氨酸去甲基化酶1(lysine specific demethylase 1,LSD1)是重要的染色质修饰蛋白之一,可以通过调节染色质的结构调节基因的转录调控,进而影响肿瘤的发生、发展、侵袭、转移以及代谢异常等恶性潜能,是判断肿瘤预后的生物标志物。Sirtuins家族去乙酰化酶3(sirtuin3,SIRT3)基因是位于线粒体内的抑癌基因,通过调控肿瘤代谢异常以及氧化损伤行使抑癌基因的功能。本研究通过基因转录调控的手段,研究胰腺癌细胞PANC-1中LSD1与SIRT3的关系。方法：通过RNA干扰(RAN interference,RNAi)、免疫共沉淀(co-immunoprecipitation,CoIP)、染色质免疫共沉淀(chromatin immunoprecipitationassay,ChIP)及启动子活性分析等分子生物学实验手段,探讨LSD1与SIRT3在PANC-1细胞中的关系。结果：通过RNAi的手段干扰LSD1的表达,发现SIRT3基因转录水平和蛋白水平明显上升;通过蛋白相互作用的手段,发现LSD1可以与SIRT3转录调控的重要转录因子过氧化物酶增殖体激活受体辅激动子-1α(peroxisomeproliferator-activated receptor gamma,coactivator 1 alpha,PGC-1α)相互作用;通过ChIP方法,发现LSD1与PGC-1α共同募集到SIRT3基因的启动子区域染色质上;通过启动子活性分析,发现LSD1基因可以显著抑制PGC-1α对SIRT3基因的转录调控。结论：LSD1可以表观遗传调控抑癌基因SIRT3的转录,为深入研究LSD1与肿瘤代谢异常以及氧化应激提供了理论依据。     

Recent advances of SIRT1 and implications in chemotherapeutics resistance in cancer

Cancer is a big group of diseases and one of the leading causes of mortality worldwide. Despite enormous studies and efforts are being carried out in understanding the cancer and developing drugs against tumorigenesis, drug resistance is the main obstacle in cancer treatments. Chemotherapeutic treatment is an important part of cancer treatment and drug resistance is getting gradually multidimensional with the advancement of studies in cancer. The underlying mechanisms of drug resistance are largely unknown. Sirtuin1 (SIRT1) is a type of the Class III histone deacetylase family that is distinctively dependent on nicotinamide adenine dinucleotide (NAD+) for catalysis reaction. SIRT1 is a molecule which upon upregulation directly influences tumor progression, metastasis, tumor cell apoptosis, autophagy, DNA repair, as well as other interlinked tumorigenesis mechanism. It is involved in drug metabolism, apoptosis, DNA damage, DNA repair, and autophagy, which are key hallmarks of drug resistance and may contribute to multidrug resistance. Thus, understanding the role of SIRT1 in drug resistance could be important. This study focuses on the SIRT1 based mechanisms that might be a potential underlying approach in the development of cancer drug resistance and could be a potential target for drug development.     

Repositioning antipsychotic chlorpromazine for treating colorectal cancer by inhibiting sirtuin 1

Investigating existing drugs for repositioning can enable overcoming bottlenecks in the drug development process. Here, we investigated the effect and molecular mechanism of the antipsychotic drug chlorpromazine (CPZ) and identified its potential for treating colorectal cancer (CRC). Human CRC cell lines harboring different p53 statuses were used to investigate the inhibitory mechanism of CPZ. CPZ effectively inhibited tumor growth and induced apoptosis in CRC cells in a p53-dependent manner. Activation of c-jun N-terminal kinase (JNK) was crucial for CPZ-induced p53 expression and the subsequent induction of tumor apoptosis. Induction of p53 acetylation at lysine382 was involved in CPZ-mediated tumor apoptosis, and this induction was attenuated by sirtuin 1 (SIRT1), a class III histone deacetylase. By contrast, knocking down SIRT1 sensitized tumor cells to CPZ treatment. Moreover, CPZ induced the degradation of SIRT1 protein participating downstream of JNK, and JNK suppression abrogated CPZ-mediated SIRT1 downregulation. Clinical analysis revealed a significant association between high SIRT1 expression and poor outcome in CRC patients. These data suggest that SIRT1 is an attractive therapeutic target for CRC and that CPZ is a potential repositioned drug for treating CRC.     

High expression of the longevity gene product SIRT1 and apoptosis induction by sirtinol in adult T-cell leukemia cells

Adult T-cell leukemia-lymphoma (ATL) is an aggressive peripheral T-cell neoplasm that develops after long-term infection with human T-cell leukemia virus (HTLV-1). SIRT1, a nicotinamide adenine dinucleotide(+) -dependent histone/protein deacetylase, plays a crucial role in various physiological processes, such as aging, metabolism, neurogenesis and apoptosis, owing to its ability to deacetylate numerous substrates, such as histone and NF-κB, which is implicated as an exacerbation factor in ATL. Here, we assessed how SIRT1 is regulated in primary ATL cells and leukemic cell lines. SIRT1 expression in ATL patients was significantly higher than that in healthy controls, especially in the acute type. Sirtinol, a SIRT1 inhibitor, induced significant growth inhibition or apoptosis in cells from ATL patients and leukemic cell lines, especially HTLV-1-related cell lines. Sirtinol-induced apoptosis was mediated by activation of the caspase family and degradation of SIRT1 in the nucleus. Furthermore, SIRT1 knockdown by SIRT1-specific small interfering RNA caused apoptosis via activation of caspase-3 and PARP in MT-2 cells, HTLV-1-related cell line. These results suggest that SIRT1 is a crucial antiapoptotic molecule in ATL cells and that SIRT1 inhibitors may be useful therapeutic agents for leukemia, especially in patients with ATL.     

SIRT1与肿瘤

SIRT1 (Sirtuin type 1)是一种依赖于烟酰胺腺嘌呤二核苷酸(NAD+)的组蛋白脱乙酰酶,参与组蛋白的共价修饰,并可通过转录、翻译及翻译后修饰等多种途径参与肿瘤的发生发展过程。肿瘤细胞内SIRT1的表达或功能异常是肿瘤发生发展的重要机制之一,并可能成为治疗肿瘤的潜在靶点。     

SIRT1 phosphorylation by AMP-activated protein kinase regulates p53 acetylation

The deacetylase SIRT1 regulates multiple biological processes including cellular metabolism and aging. Importantly, SIRT1 can also inactivate the p53 tumor suppressor via deacetylation, suggesting a role in oncogenesis. Recently, SIRT1 was shown to be released from its endogenous inhibitor DBC1 by a process requiring AMPK and the phosphorylation of SIRT1 by yet undefined kinase(s). Here we provide further evidence that AMPK directly phosphorylates SIRT1 on T344, releasing it from DBC1. Furthermore, a phospho-mimetic SIRT1 (T334E) showed decreased binding to DBC1, supporting the importance of this phosphorylation in AMPK-mediated regulation of SIRT1 activity. In addition, inhibition of AMPK by Compound C led to increased p53 acetylation, suggesting a role for the AMPK/SIRT1 pathway in regulating p53 signaling. Together, our results support a hypothesis that AMPK negatively regulates p53 acetylation via phosphorylation of SIRT1 on T344. Furthermore, our findings also define the AMPK/SIRT1 axis as a possible targetable pathway to regulate p53 function.