Anti-proliferation effects of Twist gene silencing in gastric cancer SGC7901 cells

AIM:To study the role of Twist gene in gastric cancer by gene silencing,including the potential of induction of apoptosis,cell cycle arrest,and proliferation inhibition in human malignant gastric SGC7901 cells.METHODS:The expression level of Twist in gastric cancer samples was measured by immunohistochemistry.The effects of Twist gene silencing were detected at both m RNA and protein levels by RT-PCR and Western blot.We also evaluated the cell proliferation and apoptosis by CCK-8 assay and flow cytometry.We determined the activity of caspase-3 and caspase-9 with a caspase activity assay kit.Cell cycle distribution was analyzed by flow cytometry.Cell migration and invasion ability was evaluated by wound scratch assay and Boyden chamber assay.RESULTS:Twist protein was highly expressed in gastric cancer samples.Twist gene silencing significantly induced apoptosis,cell cycle arrest at G0/G1 phase,proliferation inhibition,and reduced the ability of migration and invasion in human gastric cancer SGC7901 cells.Meanwhile,both caspase-3 and caspase-9 were activated.CONCLUSION:The Twist gene could serve as a potential molecular target for gene therapy of gastric cancer with targeted small interfering RNA.     

Claudin 1 mediates tumor necrosis factor alpha-induced cell migration in human gastric cancer cells

AIM: To investigate the role of claudin 1 in the regulation of genes involved in cell migration and tumor necrosis factor alpha (TNF-α)-induced gene expression in human gastric adenocarcinoma cells.METHODS: Knockdown experiments were conducted with claudin 1 small interfering RNA (siRNA), and the effects on the cell cycle, apoptosis, migration and invasion were analyzed in human gastric adenocarcinoma MKN28 cells. The gene expression profiles of cells were analyzed by microarray and bioinformatics.RESULTS: The knockdown of claudin 1 significantly inhibited cell proliferation, migration and invasion, and increased apoptosis. Microarray analysis identified 245 genes whose expression levels were altered by the knockdown of claudin 1. Pathway analysis showed that the top-ranked molecular and cellular function was the cellular movement related pathway, which involved MMP7, TNF-SF10, TGFBR1, and CCL2. Furthermore, TNF- and nuclear frctor-κB were the top-ranked upstream regulators related to claudin 1. TNF-α treatment increased claudin 1 expression and cell migration in MKN28 cells. Microarray analysis indicated that the depletion of claudin 1 inhibited 80% of the TNF-α-induced mRNA expression changes. Further, TNF-α did not enhance cell migration in the claudin 1 siRNA transfected cells.CONCLUSION: These results suggest that claudin 1 is an important messenger that regulates TNF-α-induced gene expression and migration in gastric cancer cells. A deeper understanding of these cellular processes may be helpful in establishing new therapeutic strategies for gastric cancer.     

PSME1基因对人胃腺癌细胞株SGC-7901生物学特性的影响

背景:PSME1基因编码蛋白PA28α是26S蛋白酶体的重要组成部分,研究显示其在多种人类恶性肿瘤中表达下调,可能影响肿瘤细胞的生物学特性。目的:探讨PSME1基因对胃癌细胞恶性表型相关生物学特性的影响。方法:将PSME1基因克隆入pcDNA3.1载体,构建PC-PSME1表达载体,以脂质体法转染人胃腺癌细胞株SGC-7901,筛选并鉴定稳定转染细胞株SGC-PSME1。以生长曲线法、克隆形成实验、流式细胞术和细胞迁徙/侵袭实验分析SGC-PSME1细胞株的生长、增殖、细胞周期、细胞凋亡和侵袭转移情况。结果:与转染空载体的SGC-PC细胞和空白对照组SGC-7901细胞相比,SGC-PSME1细胞的生长速度和克隆形成率降低,G0/G1期细胞比例增高,S期细胞比例降低,细胞凋亡率增高,差异均有统计学意义(P0.05);SGC-PSME1细胞的迁徙率与两对照组相比无明显差异。结论:PSME1基因对抑制胃癌细胞的恶性表型有一定意义。其可抑制胃癌细胞的生长、增殖,同时影响细胞周期,对细胞的侵袭转移能力可能无明显影响。     

大黄酸通过miR-29c-3p调节FSCN1的表达影响胃癌细胞的生物学行为

目的 探讨大黄酸(Rhein)对人胃癌细胞(SGC-7901)增殖、迁移、侵袭、凋亡的影响及其机制.方法 通过qRT-PCR检测大黄酸处理后细胞中miR-29c-3p的表达以及miR-29c-3p的转染效率;miR-29c-3p mimics组、NC mimics组、Rhein+miR-29c-3p inhibitor...     

Growth inhibitory effect of wild-type Kras2 gene on a colonic adenocarcinoma cell line

AIM: To observe the growth inhibitory effect of wild-type Kras2 gene on a colonic adenocarcinoma cell line Caco-2. METHODS: Recombinant plasmid pCI-neo-Kras2 with wild type Kras2 open reading frame was constructed. The Caco-2 cells were transfected with either pCI-neo or pCI-neo-Kras2 using Lipofectamine 2000. The expression of wild type Kras 2 was examined by Northern blot analysis. And the expression of wild type Kras2 protein was examined by Western blot analysis. The effects of wild-type Kras2 on cell proliferation were analyzed by monotetrazolium (MTT) assay, meanwhile analyses of cell cycle and spontaneous apoptosis rate were carried out by flow cytometry (FCM). RESULTS: The plasmid of pCI-neo-Kras 2 was successfully established. The growth rate of cells transfected with pCI-neo-Kras2 was significantly lower than the control cells transfected with the empty pCI-neo vector (P < 0.05). Cell cycle analysis revealed arrest of the pCI-neo-Kras2 transfected cells in G0/G1 phases, decreased DNA synthesis and decreased fractions of cells in S phase. The proliferative index of cells transfected with pCI-neo-Kras2 was decreased compared with the control cells (49.78% vs 64.21%),while the apoptotic rate of Caco-2 cells with stable Kras 2 expression increased (0.30% vs 0.02%). CONCLUSION: The wild-type Kras2 gene effectively inhibits the growth of the colonic adenocarcinoma cell line Caco-2.     

Anticancer effects of suberoylanilide hydroxamic acid in esophageal squamous cancer cells in vitro and in vivo

The effects of suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, have not been studied in esophageal squamous cell cancer (ESCC). Cell viability assay; flow cytometry for cell cycle and annexin V apoptosis assays; assays for cell migration, invasion, and adhesion to extracellular matrix (ECM); and immunoblotting and immunofluorescence staining were performed in three ESCC cell lines. Tumor xenograft with semiquantitative immunohistochemistry was used to study the effects of SAHA in vivo. SAHA effectively inhibited growth of ESCC cells with half‐inhibitory concentrations (IC₅₀) ranging from 2.6 to 6.5 μmol/L. SAHA restored acetylation of histone 3 lysine 9 (H3K9Ac) and histone 4 lysine 12 (H4K12Ac) with an induction of G1 or G2 cell cycle arrest and apoptosis. Expression of cell cycle checkpoint regulatory proteins including cyclin‐dependent kinases (CDKs) and cyclins was decreased, whereas expression of cell cycle suppressors, p21, p27, and Rb was increased in ESCC cells after SAHA treatment. SAHA inhibited migration, invasion, and ECM adhesion in ESCC cells with an induction of E‐cadherin expression. SAHA significantly inhibited growth of ESCC tumors with increased expression of p21, p27, Rb, and E‐cadherin while decreasing expression of CDK4 and cyclin D1 within the murine tumors. In conclusion, SAHA had antigrowth activity against ESCC cells in vitro and in vivo while inhibiting cell migration, cell invasion, and ECM adhesion, suggesting its potential as an epigenetic therapeutic agent for ESCC.     

Effects of adenoviral-mediated gene transduction of NK4 on proliferation, movement, and invasion of human colonic LS174T cancer cells in vitro

AIM: To investigate the inhibitory effects of a recombinant adenovirus vector that expresses NK4, a truncated form of human hepatocyte growth factor (HGF), on human colonic adenocarcinoma cells in vitro to establish a basis for future NK4 gene cancer therapy. METHODS: Cells from the LS174T human colonic adenocarcinoma cell line were infected with recombinant adenovirus rvAdCMV/NK4 and the effects of the manipulation on tumor cell proliferation, scatter, migration, and basement membrane invasion were assessed. Cells infected with a recombinant adenovirus vector (Ad-LacZ) expressingβ-galactosidase served as the controls. RESULTS: We found that rvAdCMV/NK4 expression attenuated HGF-induced tumor cell scatter, migration, and basement membrane invasion (P<0.05), but did not inhibit tumor cell proliferation. CONCLUSION: HGF-induced LS174T tumor cell scatter, migration, and invasion can be antagonized by the recombinant NK4-expressing adenovirus.     

miR-1180在胃癌中的表达及其对胃癌增殖、凋亡的影响

目的研究miR-1180在胃腺癌组织及胃癌细胞系中的表达及其对胃癌细胞增殖、凋亡的影响,探讨miR-1180在胃癌中可能的作用机制。方法应用qRT-PCR检测58例胃腺癌及20例癌旁正常组织中miR-1180的表达,分析其表达与胃腺癌临床病理特征的关系。检测miR-1180在胃癌细胞系中的表达,慢病毒干扰技术下调SGC-7901中miR-1180的表达,检测下调后对SGC-7901增殖、凋亡及细胞周期的影响。结果与癌旁正常组织比较,58例胃腺癌组织中miR-1180的表达明显增加(t=16.463,P=0.000),miR-1180的表达与患者的肿瘤大小、TNM分期及淋巴结转移有关(P均<0.05)。miR-1180在胃癌细胞系中表达升高(P=0.000),下调SGC-7901中miR-1180的表达,可见细胞增殖减少(3113±74 vs 1673±51,P=0.000),凋亡增加(4.313±0.220 vs 7.717±0.125,P=0.000);细胞周期G 1期细胞明显增加(45.89±0.33 vs 60.44±0.390,P=0.000),S期细胞明显减少(35.523±0.354 vs 21.953±0.444,P=0.000),G 2期细胞变化不大(18.587±0.672 vs 17.603±0.731,P=0.162)。结论miR-1180的表达促进胃癌的进展,胃癌中miR-1180的高表达与预后不良有关。     

Effect and mechanism of miR-34a on proliferation,apoptosis and invasion of laryngeal carcinoma cells

Objective: To discuss the effect and mechanism of miR-34 a on the proliferation, apoptosis and invasion of laryngeal carcinoma cells. Methods: The laryngeal squamous carcinoma Hep2 cells were transiently transfected with miR-34 a mimics and miR-34 a NC. The MTT, colony-forming assay, Hoechst staining and Annexin V-PI double staining flow cytometry were employed to detect the effect of miR-34 a on the viability and apoptosis of laryngeal squamous carcinoma Hep2 cells; Transwell assay to defect the effect of miR-34 a on the migration and invasion of laryngeal squamous carcinoma Hep2 cells; western blot and RTPCR assay to defect the effect of miR-34 a mimics on the expression of survivin and Ki-67 m RNA in laryngeal squamous carcinoma Hep2 cells. Results: Compared with miR-34 a NC group, the cell viability in miR-34 mimics group was significantly decreased(P0.01), the cell apoptosis rate was significantly increased(P0.01), the abilities of cell migration and invasion were significantly reduced(P0.01) and the expression of survivin and Ki-67 m RNA was significantly decreased(P0.01). Conclusions: The increased expression of miR-34 a can induce the apoptosis of Hep2 laryngeal carcinoma cells and inhibit the cell proliferation and invasion, which is related to the down-regulated expression of survivin and Ki-67.     

核抗原Mina53在人胃癌细胞中的作用及其意义探讨

背景:核抗原Mina53基因为原癌基因Myc的下游直接靶基因之一,在一些消化系统恶性肿瘤中呈高表达,并与肿瘤增殖、侵袭、转移或患者生存期相关。目的:研究Mina53在人胃癌细胞中的作用及其对胃癌发生、发展的意义。方法:选择Mina53表达水平较高的人胃癌细胞株SGC7901和AGS,应用RNA干扰技术下调其Mina53表达,以转染无关序列siRNA的细胞作为对照组。采用CCK-8实验检测细胞增殖,流式细胞术检测细胞周期和细胞凋亡,细胞侵袭和迁移实验检测细胞侵袭、迁移能力。结果:与相应对照组相比,Mina53 siRNA转染组SGC7901、AGS细胞增殖受抑(96 h相对增殖率:60%和68%),并发生明显细胞周期G1期阻滞(SGC7901细胞G1/G2:2.76±0.12对1.86±0.06,P<0.05;AGS细胞G1/G2:1.78±0.13对1.34±0.05,P<0.05),细胞凋亡率分别增加9.8%±1.2%和10.6%±1.5%(P<0.05),穿透Transwell小室基质胶细胞数(SGC7901细胞:11.67±0.88对24.33±1.45,P<0.05;AGS细胞:8.00±1.15对20.33±1.73,P<0.05)和穿透Transwell小室微孔膜细胞数(SGC7901细胞:7.00±1.53对14.67±2.03,P<0.05;AGS细胞:8.00±1.16对15.33±1.45,P<0.05)均显著减少。结论:Mina53对人胃癌细胞的增殖、细胞周期、细胞凋亡以及侵袭、迁移能力具有调控作用,可能影响胃癌的生长、浸润和转移,有望作为胃癌基因治疗的靶点。     

EGLN3在肺腺癌中表达的Meta分析

目的 分析EGLN3在肺腺癌(lung adenocarcinoma, LUAD)中的作用。方法 (1)通过癌症基因组图谱(the cancer genome atlas, TCGA)数据库下载535例肺腺癌样本和59例正常样本,分析EGLN3在肺腺癌和正常肺组织中的表达差异;(2)使用R语言分析EGLN3的表达水平及其在肺腺癌中的临床意义;(3)通过Kaplan-Meier方法、单因素和多因素COX回归分析和生存预测列线图确定EGLN3的表达水平与肺腺癌预后的关系;(4)通过基因集富集分析(GSEA)筛选EGLN3相关的生物学途径;(5)细胞实验：通过蛋白质印迹法(WB)验证EGLN3在肺腺癌细胞中是否存在差异表达;对EGLN3基因进行过表达和干扰表达,采用CCK-8增殖实验、划痕实验和Transwell侵袭实验,明确表达水平对肺腺癌细胞增殖、迁移和侵袭的影响。结果 (1)EGLN3在肺腺癌组织中表达水平高于正常肺组织细胞;(2)EGLN3表达水平与肺腺癌病理分期相关,病理分期越高,对应EGLN3表达水平越高;(3)EGLN3表达水平与肺腺癌总生存期(OS)独立预后相关,EGLN3高...     

槲皮素对人胃癌细胞侵袭和MMP-2表达的影响

目的:观察槲皮素(Quercetin,Que)对人胃癌细胞侵袭的影响,并探讨其可能机制.方法:采用不同浓度的槲皮素处理胃癌BGC-823细胞后,以软琼脂集落培养试验检测癌细胞锚着不依赖性增殖,以Boyden小室模型方法检测癌细胞侵袭能力,采用荧光实时定量PCR检测癌细胞基质金属蛋白酶-2(matrix metallopeptidase-2,MMP-2)基因mRNA水平,以Western blot方法检测癌细胞MMP-2基因蛋白水平变化.结果:不同浓度的胃癌BGC-823细胞经槲皮素处理后,恶性增殖和侵袭能力均明显下降,且呈剂量依赖性(P<0.005,P<0.005).槲皮素处理组MMP-2基因mRNA和蛋白水平均明显下调,且呈时间和浓度依赖性,即随着作用时间的延长和槲皮素作用浓度的增加,MMP-2的mRNA和蛋白水平逐渐下降,差异均有统计学意义(P<0.001,P<0.001).结论:槲皮素可明显抑制胃癌BGC-823细胞侵袭能力,其机制可能与下调MMP-2基因表达有关.     

厄洛替尼联合放疗对MKN45细胞株的影响

目的:观察厄洛替尼联合放疗对人胃癌MKN45细胞周期和凋亡的影响,了解厄洛替尼对放疗增敏的作用机制.方法:通过MTT法和集落形成实验,检测厄洛替尼和放射线对MKN45细胞的生长抑制作用,计算出半数抑制浓度(50%inhibitory concentration,IC50)和放射生物学参数平均致死剂量(mean lethal dose,D0)、准阈剂量(quasi-threshold,Dq)值,得出放射增敏比;流式细胞仪检测MKN45细胞经厄洛替尼及联合放射线处理后细胞的凋亡率及周期分布情况;Westernblot法检测厄洛替尼及联合放射线对MKN45细胞的Bax与Bcl-2蛋白表达的影响.结果:厄洛替尼及放射线均能抑制MKN45细胞的生长,随用药浓度或剂量的增高,抑制作用增强(P<0.01).厄洛替尼与放射线联合对MKN45细胞的抑制作用大于单药和单纯照射(P<0.01);两者联合使S期细胞比率明显降低,放射敏感的G2/M期和G0/G1期细胞比率明显增加(71.87±0.77vs60.72±0.26,P<0.01),细胞凋亡率增加;厄洛替尼联合放射线作用于细胞后,Bcl-2蛋白表达明显减少,Bax蛋白表达则明显增加.结论:厄洛替尼通过增加G2/M和G0/G1期细胞比率,降低Bcl-2、升高Bax蛋白表达,从而降低Bcl-2/Bax比率,增加细胞凋亡,以此提高MKN45细胞的放射敏感性.     

Paris chinensis dioscin induces G2/M cell cycle arrest and apoptosis in human gastric cancer SGC-7901 cells

AIM:To investigate the anti-tumor effects of Paris chinensis dioscin(PCD)and mechanisms regarding cell cycle regulation and apoptosis in human gastric cancer SGC-7901 cells.METHODS:Cell viability was analyzed by the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay.Cell apoptosis was evaluated by flow cytometry and laser scanning confocal microscope(LSCM)using Annexin-V/propidium iodide(PI)staining,and the cell cycle was evaluated using PI staining with flow cytom-etry.Intracellular calci...     

SphK1对结肠癌lovo细胞的增殖、迁移和凋亡的影响

目的:探讨鞘胺醇激酶1(SphK1)对结肠癌lovo细胞增殖、迁移和凋亡的影响及其作用机制.方法:培养人结肠癌lovo细胞株,实验分3组:对照组、PMA组和DMS组.PMA组加入佛波醇-12-豆蔻酸酯-13-乙酸酯(PMA,100nmol/L),DMS组加入N,N-二甲基鞘胺醇(DMS,50μmol/L),对照组加入等量的培养基.采用MTT法和克隆形成实验检测细胞生长增殖的变化,流式细胞术检测细胞凋亡,Tranwell小室模型观察细胞迁移能力的变化,RT-PCR检测mRNA的表达,Westernblot检测蛋白的表达.结果:PMA显著促进细胞的增殖、迁移并抑制细胞的凋亡,DMS则显著抑制细胞的增殖、迁移并促进细胞的凋亡(对照组、PMA组和DMS组的细胞增殖活力:0.71±0.03vs1.05±0.05vs0.46±0.04;克隆形成率:1.32%±0.26%vs2.17%±0.17%vs0.73%±0.13%;凋亡率:16.25%vs9.15%vs32.58%;迁移细胞数:72.19±3.36vs98.46±6.25vs40.48±4.27;均P<0.05).PMA显著促进黏着斑激酶(FAK)的活性和表达,相反DMS则抑制FAK的活性和表达[对照组、PMA组和DMS组FAKmRNA的表达强度:0.151±0.008vs0.212±0.014vs0.114±0.021;蛋白:0.332±0.022vs0.374±0.029vs0.296±0.018;磷酸化FAK(p-FAKTyr397)蛋白:0.186±0.032vs0.234±0.017vs0.112±0.023;均P<0.05].结论:SphK1可促进lovo细胞的增殖和迁移能力并抑制细胞的凋亡,其机制可能是通过激活FAK通路而发挥作用.     

硝呋齐特对A549细胞增殖、凋亡、迁移、侵袭的影响及其作用机制

目的探讨硝呋齐特(nifuroxazide)对A549细胞增殖、凋亡、迁移、侵袭的影响及其可能的作用机制。方法用不同浓度(2.5、5、10μmol/L)的nifuroxazide处理肺腺癌A549细胞,采用甲基噻唑基四唑(MTT)法观察nifuroxazide对细胞增殖的影响;流式细胞术观察nifuroxazide对细胞凋亡的影响;采用Transwell小室观察nifuroxazide对细胞迁移及侵袭的影响。实时荧光定量聚合酶链反应(qRT-PCR)检测nifuroxazide对微小RNA-219-5p(miR-219-5p)表达的影响;观察抑制miR-219-5p表达对细胞增殖、凋亡、迁移及侵袭能力的影响;蛋白免疫印迹法(Western blot)检测B淋巴细胞瘤-2(Bcl-2)、B淋巴细胞瘤-2相关蛋白(Bax)蛋白表达。结果nifuroxazide处理A549细胞后细胞增殖能力受到明显抑制(P<0.05),随浓度增加其抑制作用逐渐增强(P<0.05);nifuroxazide作用于A549细胞后,细胞凋亡率显著增加(P<0.05),迁移与侵袭细胞数显著减少(P<0.05),miR-219-5p与Bax的表达水平显著升高(P<0.05),而Bcl2表达水平显著降低(P<0.05);抑制miR-219-5p的表达可逆转nifuroxazide对A549细胞增殖、迁移、侵袭、凋亡的作用。结论硝呋齐特可通过上调miR-219-5p的表达诱导A549细胞凋亡,抑制细胞增殖、迁移及侵袭。