KAI1基因转染ATRA诱导对小细胞肺癌细胞株恶性特征的抑制作用

背景与目的:探讨新抑癌基因KAI1与全反式维甲酸(all-trans-retinoic acid,ATRA)对小细胞肺癌NCI-H446细胞株抑制增殖和诱导分化的作用.材料与方法:用脂质体介导的基因转染方法,借助质粒表达载体(PCMV-NEO-XhoI),将抑癌基因KAI1转入小细胞肺癌NCI-H446细胞中,经G418筛选,获得稳定表达的细胞克隆.用10-6mol/L ATRA作用于转染及未转染KAI1基因的小细胞肺癌NCI-H446细胞株,集落形成率检测细胞体外增殖能力,流式细胞仪进行细胞周期和凋亡分析,间接免疫荧光染色结合流式细胞仪检测转染前后细胞CD82蛋白的表达.免疫组化测定MYC的表达,放射免疫测定LN(层连蛋白)表达.结果:ATRA处理脂质体-KAI1基因转染的小细胞肺癌细胞CD82表达降低,细胞增殖能力下降,凋亡增加,更多的细胞被阻止于G1/G0期,MYC及LN表达下降.结论:抑癌基因KAI1与ATRA对抑制小细胞肺癌NCI-H446细胞株的增殖和促分化有协同作用.     

脂质体介导KAI1基因转染对小细胞肺癌NCI-H446细胞株的抑制作用

 目的　探讨新抑癌基因KAI1对小细胞肺癌NCI H4 4 6细胞株抑制增殖和浸润转移的作用。方法　用脂质体介导的基因转染方法 ,借助真核质粒表达载体 (PCMV NEO XhoI) ,将抑癌基因KAI1转入小细胞肺癌NCI H4 4 6细胞中 ,经G4 18筛选 ,获得稳定表达的细胞克隆。观察NCI H4 4 6细胞的生长 ,MTT法检测细胞体外增殖能力 ,流式细胞仪进行细胞凋亡分析 ,间接免疫荧光染色结合流式细胞仪检测转染前后细胞CD82蛋白的表达。免疫组化测定Myc及MMP 1(基质金属蛋白酶 1)的表达 ,放射免疫测定LN(层黏蛋白 )表达。结果　脂质体 KAI1基因转染的小细胞肺癌细胞CD82表达增加 ,细胞增殖能力下降 ,凋亡增加 ,癌基因Myc ,MMP 1及LN表达下降。 结论　抑癌基因KAI1抑制小细胞肺癌NCI H4 4 6细胞株的增殖 ,降低Myc ,MMP 1及LN的表达。     

转染外源CC10对肺腺癌细胞系A549的CyclinD1蛋白及mRNA的影响(英文)

目的研究转染外源性CC10基因对肺腺癌A549细胞株的细胞周期及周期蛋白CyclinD1蛋白和mRNA表达影响。方法用脂质体法分别将外源性抑癌基因CC10转染到人肺腺癌细胞株A549中,8h、16h、24h、36h、48h后,分别用Western blot法检测转染CC10基因的A549细胞CC10蛋白表达,用流式细胞仪观察细胞周期的变化,用免疫细胞化学及RT-PCR检测转染CC10基因对周期蛋白CyclinD1蛋白、mRNA的影响。结果转染外源CC10基因对A549细胞生长具有抑制作用,细胞生长阻止于G0／G1,细胞周期S期+G2／M期比例下降。转染外源 CC10基因的A549细胞CC10蛋白表达增高,而CyclinD1的蛋白及mRNA表达明显降低。结论转染外源CC10基因对肺癌细胞G0／G1周期的抑制作用可能与CyclinD1基因表达下调有关。     

促红细胞生成素对肺腺癌A549细胞生长和凋亡作用的实验研究

背景与目的 促红细胞生成素(erythropoietin,EPO)对肿瘤细胞的具体作用存在争议.本研究旨在探讨细胞因子超家族成员EPO对肺腺癌AS49细胞株生物学行为的影响.方法 构建真核表达质粒pcDNA3.1(-)-hEPO,并转染肺腺癌A549细胞株.采用酶联免疫吸附(ELISA)法检洲转染后目的基因的蛋白表达水平;MTT法和流式细胞仪检测转染后A549细胞生长、周期及凋亡率等生物学行为的变化;ELISA法检测转染后细胞VEGF表达水平的变化.结果 成功构建真核表达质粒pcDNA3.1(-)-hEPO;转染后各检测时间点,EPO转染组细胞生长均明显受抑(P<0.01);流式细胞术分析表明,EPO转染组S期细胞比例明显高于对照组(P<0.05).且凋亡率也明显增高(P<0.01);EPO转染组VEGF表达量明显降低(P<0.01).结论 EPO基因转染A549后有抑制细胞生长、促进凋亡的作用,并且还可抑制细胞表达VEGF.     

CD147 siRNA对人肺腺癌细胞株A2增殖的影响

目的:研究CD147 siRNA转染对肺腺癌细胞系A2中CD147表达及细胞增殖的影响,探讨CD147 siRNA转染对肺腺癌细胞系的作用。方法:用脂质体LipofectamineTM2000介导CD147 siRNA转染人肺腺癌细胞株A2,用RT-PCR检测CD147 mRNA表达,并用MTT法检测CD147 siRNA转染对A2细胞增殖的影响。结果:转染后的肺腺癌细胞株A2中CD147 mRNA表达水平明显下调,转染了CD147 siRNA的细胞在24、48和72 h的增殖能力较A2分别下降了48.3%、51.8%和53.2%(P均<0.05)。结论:CD147 siRNA转染能有效降低肺腺癌细胞株A2中CD147的表达,且能抑制肿瘤细胞的增殖能力。CD147 RNA干扰有可能成为肺腺癌基因治疗的新途径。     

HDAC1 siRNA转染对肺腺癌A549细胞增殖及凋亡的影响

背景与目的:多项研究表明组蛋白去乙酰化酶(histone deacetylases,HDACs)抑制剂对人非小细胞肺癌细胞株有抑制增殖和诱导凋亡作用.而HDAC抑制剂对HDAC Ⅰ型和Ⅱ型均有抑制作用,在非小细胞肺癌细胞株中尚未明确HDACs中哪一类受抑制能影响肿瘤生长.本研究通过HDAC1 siRNA转染肺腺癌细胞株A549,观察HDACs中HDAC1对肺腺癌细胞增殖、周期和凋亡的影响,明确HDAC1在肺腺癌细胞生长中的作用.方法:MTT法检测不同时间点HDAC1 si RNA转染对A549细胞体外增殖的影响,流式细胞术检测RNA干扰后细胞周期及凋亡率的变化;Western blot法检测细胞内组蛋白H4乙酰化水平的变化.结果:HDAC1 siRNA转染A549细胞后,HDAC1在转录和表达水平均下降,组蛋白H4乙酰化表达增高.siRNA干扰后A549细胞的体外生长抑制呈时间依赖性,流式细胞术检测结果显示细胞阻滞于G2/M期,细胞凋亡增加.结论:HDAC1 siRNA转染能有效地抑制HDAC1在A549细胞中的表达,增加A549细胞中组蛋白的乙酰化,并影响A549细胞相关生物学行为,抑制肺腺癌细胞的生长,为HDAC1基因治疗应用于肺腺癌奠定了基础.     

全反式维A酸联合c-myc RNA干扰抑制肺腺癌细胞增殖并诱导凋亡

目的 探讨全反式维A酸(ATRA)联合c-myc RNA干扰后肺腺癌细胞A549增殖和凋亡的改变及对survivin 基因表达的影响.方法 构建c-myc特异RNA小干扰(siRNA)的表达载体,转染A549细胞后G418(Geneticin)筛选出稳定表达c-myc特异siRNA的细胞株,荧光实时定量RT-PCR和免疫印迹 (Western blot)检测c-myc基因表达水平.ATRA作用于c-myc RNA干扰细胞,四甲基偶氮唑蓝(MTT)法检测吸光度值,流式细胞仪测凋亡率,定量RT-PCR和免疫印迹检测survivin基因表达.结果 成功构建c-myc-siRNA表达载体.c-myc基因mRNA和蛋白表达下降71.9%和85.6%.ATRA联合c-myc-siRNA细胞组吸光度值在各时间点较单用ATRA组,单用c-myc-siRNA组以及对照组均下降,差异有显著性(P＜0.01),凋亡率增加,差异有显著性(P＜0.01).ATRA联合c-myc-siRNA细胞, survivin表达下降49.2%,差异有显著性(P＜0.01).结论 ATRA联合c-myc-siRNA较单用ATRA或c-myc siRNA能更有效地抑制A549细胞的增殖,促进细胞的凋亡.survivin基因表达降低.     

β－arrestin2基因转染对人肺腺癌细胞 A549的凋亡影响

目的：观察外源性β－arrestin2基因对人肺腺癌细胞株 A549凋亡的影响。方法：将真核表达重组体pcDNA3．1－β－arrestin2质粒和空载体 pcDNA3．1质粒采用脂质体转染技术分别导入人肺腺癌 A549细胞,然后采用 G418筛选方法获得稳定转染的细胞系,采用 qRT －PCR 和 Western blot 分别在基因水平和蛋白水平检测β－arrestin2基因的表达。采用流式细胞仪分析转染细胞的凋亡情况。结果：经脂质体转染和筛选,建立了稳定表达β－arrestin2基因的 A549细胞系。转染β－arrestin2基因的细胞生长速度较未转染组和转染空白载体组相比明显减慢（P ＜0．05）。结论：β－arrestin2基因可促进人肺腺癌细胞 A549的细胞凋亡。     

KAI1基因对人鼻咽癌细胞株HNE-1增殖、侵袭及转移能力的影响

目的:研究转移抑制基因KAI1对人鼻咽癌细胞体外增殖、侵袭及转移能力等生物学效应的影响.方法:通过腺病毒载体将KAI1基因转染人鼻咽癌细胞株HNE-1, RT-PCR法检测转染效果; MTT法检测鼻咽癌细胞转染KAI1基因后体外增殖能力的变化;FCM法检测细胞生长周期分布; Transwell侵袭小室检测细胞侵袭能力的改变;细胞同质黏附实验检测细胞黏附力的变化.结果:当腺病毒载体 rAd-KAI1效价为 30 MOI时,对HNE-1细胞的转染率可达92%;转染48 和72 h 时,rAd- KAI1转染组细胞的增殖明显受到抑制,抑制率分别为17%和30%;G0/G1期细胞数量较对照组增多,S期细胞数量减少;转染组的穿膜细胞数为(52.5±4.2)个,明显低于对照组的(167.4±3.5)个,差异有统计学意义(P<0.05);转染组的细胞同质黏附作用高于对照组(P<0.05).结论:腺病毒载体成功介导了抑癌基因KAI1转染进入鼻咽癌HNE-1细胞,并取得较高的转染率.KAI1基因可能通过阻滞HNE-1细胞生长周期,从而抑制细胞增殖.KAI1基因能够抑制HNE-1细胞侵袭能力,其机制可能为KAI1基因增强了鼻咽癌细胞的黏附力所致.     

KAI1基因对乳腺癌细胞体外增殖的抑制作用

目的研究KAI1基因对乳腺癌细胞体外增殖的抑制作用。方法应用脂质体法将pCMV-KAI1质粒转染人高转移性人乳腺癌细胞株MDA-MB-231中,免疫印迹方法检测蛋白表达;利用四甲基偶氮唑盐(MTT)法、平板克隆形成实验、体外黏附及侵袭力实验判断细胞增殖能力及黏附、侵袭力的变化。流式细胞术检测细胞生长周期的变化。结果获得了稳定表达KAI1基因的MDA- MB-231乳腺癌细胞克隆。MTT法显示,转染KAI1基因组的集落形成率(25.33%±2.36%)较转染前(43.17%±2.75%)明显降低(P<0.05)。体外黏附及侵袭力实验表明,转染组的细胞黏附侵袭能力低于未转染组和转染空载体组(P<0.05)。流式细胞术显示,转染KAI1后,G_0/G_1期细胞数量增高,从36.78%±0.61%升高至64.00%±7.56%;G_2/M期数量降低,由17.88%±0.76%降至7.63%±0.60%,差异有统计学意义。结论KAI1基因可以抑制乳腺癌细胞的增殖黏附和侵袭能力,并可能通过调节细胞周期来影响细胞的增殖。     

KAI1/CD82通过Wnt/β-catenin通路对肾癌调控机制的研究

目的:检测KAI1/CD82和p-catenin在肾癌组织及细胞中的表达情况,探讨KAI1/CD82通过Wnt/β-catenin通路对肾癌的调控机制.方法:通过Western blotting检测我院近2年收集的30例肾癌及癌旁组织中CD82及β-catenin的表达;构建KAI1/CD82低表达质粒,转染肾透明细胞癌细胞系786-0和OSRC,划痕实验检测细胞迁移能力;CCK-8法检测细胞增殖能力;Transwell检测细胞侵袭能力;Western blotting检测β-catenin、Axin2、GSK-3β的表达.结果:肾癌组织中CD82表达低于癌旁组织,β-catenin的表达高于癌旁组织.PLTHR-SHKAI1质粒转染786-0和OSRC后,肾癌细胞的迁移、增殖、侵袭能力增强,β-catenin表达增高,Axin2、GSK-3β表达降低.结论:KAI1/CD82和β-catenin在肾癌组织中表达负相关,KAI1抑制肾癌细胞的迁移、增殖、侵袭能力;KAI1/CD82通过Wnt/β-catenin通路相关上游蛋白调控肾癌的发生、发展和转移.     

Nuclear factor-kappaB-dependent expression of metastasis suppressor KAI1/CD82 gene in lung cancer cell lines expressing mutant p53

KAI1/CD82 has been shown to be a metastasis suppressor for several human cancers, and a recent study revealed that wild-type tumor suppressor p53 can directly activate KAI1/CD82 gene expression. However, the response of KAI1/CD82 expression in cancer cells to exogenous stimulants has not been investigated. The present study examined whether tumor necrosis factor (TNF), which mediates many of the cellular responses associated with inflammatory reactions or cancer progression, can affect the KAI1/CD82 expression in lung cancer cells and, if so, whether nuclear factor (NF)-kappaB, a key molecule in TNF-mediated gene expression, is involved in the mechanism of KAI1/CD82 induction. Our results demonstrated that expression of KAI1/CD82 in PC-14 cells expressing mutant p53 could be augmented by TNF-alpha, and that transfer of the gene for a specific inhibitor of NF-kappaB, IkappaB alphaSR (mutant IkappaB alpha; NF-kappaB super-repressor), into PC-14 cells could inhibit this augmentation. The amount of NF-kappaB in the nucleus of PC-14/IkappaB alphaSR cells correlated well with KAI1/CD82 mRNA and protein expression. In addition, IkappaB alphaSR gene transfer inhibited the spontaneous expression of KAI1/CD82 protein in KAI1/CD82-high-expressing RERF-LC-OK cells, which contain a mutant-type p53. These observations indicate that NF-kappaB activation may play a role in the regulation of KAI1/CD82 expression in lung cancer cells independently of wild-type p53, and suggest that KAI1/CD82 expression may be regulated by interaction with the host microenvironment.     

Mutation and expression of the metastasis suppressor gene KAI1 in esophageal squamous cell carcinoma

BACKGROUND: KAI1/CD82, a tumor metastasis suppressor gene, is correlated inversely with the progression and invasion of several tumors. It also has been reported that the KAI1 gene is related to the tumor suppressor gene p53. This study was performed to clarify the correlation between KAI1/CD82 expression and clinicopathologic characteristics and p53 expression in patients with esophageal squamous cell carcinoma (ESCC). The authors also investigated mutation of the KAI1 gene coding region to determine whether this may reduce KAI1 expression in ESCC. METHODS: Using immunohistochemistry with anti-KAI1 polyclonal antibody and monoclonal antibody against p53, KAI1/CD82 and p53 expression were detected in 55 patients with ESCC who had undergone surgery. The authors examined the KAI1 gene mutation in 22 patients with ESCC by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and DNA sequencing. RESULTS: KAI1/CD82 expression was positive in 36 of 55 patients (65.5%). There was a significant inverse correlation between KAI1/CD82 expression and regional lymph node metastasis (P = 0.0045), distant metastasis (P = 0.0092), the number of lymph node metastases (P = 0.0019), and pathologic stage (P = 0.0046). The survival rates of KAI1/CD82 negative patients were poorer than those of positive patients (P = 0. 024). The correlation between KAI1 positive and p53 positive tumors was not statistically significant. None of the 22 patients with ESCC showed mutation of the KAI1 gene by PCR-SSCP. In one patient, there was polymorphism in the SSCP assay and DNA sequencing. CONCLUSIONS: The authors demonstrated immunohistochemically that the expression of KAI1 protein appeared to be correlated with lymph node metastasis. Mutation does not seem to be a mechanism for dysregulation of the KAI1 protein in ESCC.     

慢病毒介导shRNA沉默QKI-6基因对人肺腺癌A549细胞生物学行为的影响

目的:探讨慢病毒介导shRNA沉默QKI-6基因对人肺腺癌A549细胞QKI-6基因表达、细胞周期、克隆形成和细胞迁移的影响。方法:构建QKI-6-shRNA慢病毒表达载体,并用其转染A549细胞,阴性对照组转染无意义序列,空白对照组为A549细胞。运用RT-PCR及Western blot检测各组细胞QKI-6 mRNA及蛋白的表达,流式细胞仪检测细胞周期,琼脂成瘤实验检测细胞克隆形成能力,Transwell实验检测细胞迁移能力。结果:实验组QKI-6 mRNA及蛋白表达均较阴性对照组及空白对照组降低。实验组较阴性对照组及空白对照组,G0/G1期细胞比例降低（P<0.05）,S期细胞比例增高（P<0.05）。琼脂成瘤实验显示,实验组与空白对照组及阴性对照组比较,克隆形成率明显提高（P<0.05）。细胞迁移实验结果显示,实验组与空白对照组及阴性对照组比较,细胞迁移数量明显增多（P<0.05）。结论:QKI-6能够抑制肺腺癌细胞生长、克隆形成及迁移,QKI-6有望成为肺癌治疗的新靶点。     

Rb94对人肺腺癌细胞系A549放射增敏作用的实验研究

目的 构建人视网膜母细胞瘤基因(Rb94)真核表达载体,观察其对人肺腺癌细胞系A549的放射增敏作用并探讨其机制.方法 构建重组表达质粒pIPES-Rb94,经脂质体转染A549细胞、G418筛选获得稳定转染的细胞系.细胞计数法绘制生长曲线、计算细胞倍增时间;实时RT-PCR检测hTERT、Bcl-2mRNA的表达;成克隆实验检测pIRES-Rb94对A549细胞放射敏感性影响,计算放射增敏比(SER);流式细胞仪分析细胞周期.结果 成功构建pIRES-Rb94质粒,转染A549细胞后获得稳定转染细胞系pIRES-Rb94-A549.与A549细胞相比,pIRES-Rb94-A549细胞倍增时间延长(31.5h:39.5 h;t=5.15,P<0.01);hTERT、Bcl-2 mRNA表达下降(0.02:1.00;t=18.99,P<0.01;0.01:1.00;t=13.73,P<0.01);G2+M期细胞增加(35.91%:4.53%;t=36.78,P=0.00),G0+G1期和S期细胞减少(47.02%:74.07%;t=11.71,P=0.00和17.07%:22.32%;t=2.30,P<0.05);由D0值计算的SER为1.30.结论 Rb94对A549细胞具有明显放射增敏作用,其机制可能是G2+M期阻滞作用以及下调hTERT、Bcl-2 mRNA表达,并可能通过调节细胞周期来影响细胞增殖.     

Effects of SASH1 on lung cancer cell proliferation, apoptosis, and invasion in vitro

The purposes of this study were to investigate the effects of the SASH1gene on the growth, proliferation, apoptosis, invasiveness, and metastatic potential of lung cancer cells and explore the potential use of SASH1 for the treatment of human lung cancer. The SASH1 gene was cloned into the pcDNA3.1 eukaryotic expression vector, and SASH1 shRNA were designed and constructed. The resulting constructs were transfected into A549 human lung cancer cells, and the changes in the relevant biological characteristics of the cells overexpressing SASH1 and cells with downregulated expression of SASH1 were analyzed using the MTT assay, transwell invasion assay, and flow cytometry. The effects of the SASH1 gene on the expression of cyclin D1, Bcl-2, and MMP-2/9 were also concurrently examined. In the A549 cells from the pcDNA3.1-SASH1 transfected group, cell viability, proliferation, and migration were significantly reduced compared to the control cells (p?=?0.039, p?=?0.013), and a cell cycle arrest in G1 was observed. The A549 cells transfected with the SASH1 shRNA demonstrated significantly higher cell viabilities, proliferation, and migration compared to the control cells (p?=?0.012, p?=?0.045). Additionally, the percentage of A549 cells undergoing apoptosis was significantly higher in the pcDNA3.1-SASH1 transfected cells and significantly lower in the SASH1 shRNA transfected cells compared to the control cells (p?=?0.010, p?=?0.000). The cyclin D1, Bcl-2, and MMP-9/2 protein expression levels were significantly lower in the pcDNA3.1-SASH1-transfected cells and were significantly higher in the SASH1 shRNA-transfected cells than that in the control cells. The SASH1 gene may inhibit A549 cell growth and proliferation as well as promote cellular apoptosis. The overexpression of the SASH1 gene may also be related to the decreased migration of A549 human lung cancer cells.     

KAI1/CD82 expression as a prognosic factor in sporadic colorectal cancer

BACKGROUND: Since its identification as a suppressor gene for prostate cancer metastasis, down-regulation of KAI1/CD82 in a variety of malignancies has been reported. MATERIALS AND METHODS: Using immunohistochemistry, we examined KAI1/CD82 expression in surgical specimens obtained from 70 patients with advanced colorectal cancer and its correlation with clinicopathological factors, to clarify their prognostic significance. RESULTS: KAI1/CD82 expression was positive in 55% of the 70 colorectal cancers. There were statistically significant correlations between KAI1/CD82 expression and Dukes' stage, venous invasion, lymph node metastasis, tumor differentiation and liver metastasis. The significant correlation between KAI1/CD82 expression and outcome among patients with Dukes' C cancer (p=0.024) is particularly noteworthy. On multivariate analysis, KAI1/CD82 expression and Dukes' stage were identified as significant and independent prognostic factors (p=0.006 and 0.045, respectively). CONCLUSION: KAI1/CD82 expression closely correlates with clinicopathological factors for colorectal cancers. KAI1/CD82 expression appears to be a useful prognostic marker.