miR-210对肾细胞癌细胞迁移、增殖和凋亡能力的影响

目的探讨miR-210在肾细胞癌(RCC)中的功能。方法采用细胞计数试剂盒(CCK)-8和噻唑蓝(MTT)试验评估细胞活力和增殖能力,采用细胞划痕和Transwell试验检测细胞迁移和侵袭能力。结果 miR-210在RCC细胞系中的表达水平上调,miR-210的上调促进了ACHN和786-O细胞增殖,miR-210的上调促进了ACHN和786-O细胞侵袭和迁移能力。流式细胞仪分析证实,miR-210的上调抑制了ACHN和786-O细胞的凋亡。结论 miR-210可能在RCC中充当致癌基因。     

红景天苷对人恶性黑色素瘤细胞A375增殖和凋亡的影响

目的：观察红景天苷对人恶性黑色素瘤细胞A375增殖及凋亡的影响。方法分别用0、0．01、0．1、1、10、100μg/mL红景天苷处理人恶性黑色素瘤细胞A375后,MTT法检测各组细胞增殖率,Annexin V-FITC/PI双染色流式细胞术检测细胞凋亡率,RT-PCR检测各组细胞周期依赖性激酶CDk4和CDk2 mRNA、细胞周期素CyclinE和CyclinD1 mRNA的影响。结果与阴性对照组比较,各给药组细胞增殖抑制率明显增多（P＜0．05）。给药组细胞以G0/G1期为主,S期细胞增加,G2/M期细胞减少（P＜0．05）。给药组中的CDk2、 CDk4、CyclinD1和CyclinE mRNA表达较阴性对照组明显降低（P＜0．05）。结论红景天苷能够抑制人恶性黑色素瘤细胞A375增殖和促进其凋亡。     

亚砷酸钠对黑色素瘤细胞A375和G361色素代谢的影响

目的 探讨亚砷酸钠(NaAsO2)暴露对黑色素瘤细胞A375(以下简称A375)和G361(以下简称G361)的色素水平及酪氨酸酶(TYR)活力的影响,以及两种细胞色素代谢能力的差异.方法 以0.0(对照)、0.1、1.0 μmol/L NaAsO2作用于A375和G361细胞,72 h后,以阿尔玛蓝还原法检测细胞活力水平,同时测定细胞中黑色素水平及TYR的活力水平.结果 NaAsO2作用72h后,0.1 μmol/L剂量组的A375和G361细胞均呈轻度增殖表现,细胞活力[A375:(103.32±1.26)％;G361:(104.10±1.76)％]与对照组[A375:(100.00±1.08)％;G361:(100.00±1.79)％]比较,差异无统计学意义(P均＞0.05);1.0μmol/L剂量组细胞活力[A375:(75.32±1.59)％;G361:(78.26±2.10)％]与对照组相比均显著下降(P均＜0.05).对照、0.1、1.0μmol/L剂量组的G361细胞的黑色素水平[(7.19±0.35)、(7.34±0.83)、(8.19±0.86) pg/细胞]均高于A375细胞[(4.35±0.72)、(4.54±0.01)、(4.60±0.59)pg/细胞,P均＜0.05],G361细胞的TYR活力水平[(54.13±1.21)、(54.56±0.21)、(56.25±0.85)贝克勒尔(Bq)]均高于A375细胞[(42.00±0.21)、(42.90±0.54)、(42.91±0.01)Bq,P均＜0.05].2种细胞的黑色素生成水平及TYR活力水平均随NaAsO2剂量增高而呈增高趋势,但组间比较,差异均无统计学意义(P均＞ 0.05).结论 砷致色素代谢异常可能与砷暴露致黑色素水平及TYR活力增高有关;不同个体间色素代谢差异,可能与个体的黑色素代谢本底水平及TYR本底活力差异有关.     

血清miR-663、miR-126和miR-370对冠心病患者支架内再狭窄的预测价值

目的研究血清微小RNA(miRNA)中的miR-663、miR-126和miR-370在冠心病患者支架植入术后的表达情况,分析以上3种miRNA对支架内再狭窄(ISR)的预测价值。方法研究纳入175例冠心病支架植入术后并随访复查冠状动脉造影的患者,根据造影结果分为ISR组(n=66)和无ISR组(n=109)。分析比较两组患者的临床资料。收集所有患者的血清,通过实时荧光定量PCR检测血清miR-663、miR-126和miR-370的表达情况。应用统计学方法分析以上3个miRNA预测ISR的价值。结果 175例支架植入术后冠心病患者中发生ISR的共66例。ISR组吸烟率更高(P0.05);ISR组血糖、低密度脂蛋白胆固醇、高敏C反应蛋白(hs-CRP)水平均高于无ISR组(P0.01)。ISR组血清miR-663、miR-126表达量低于无ISR组,而miR-370表达量高于无ISR组(均P0.01)。Logistic回归分析表明,miR-663、miR-126表达水平与ISR发生相关,吸烟、hs-CRP是ISR的独立危险因素。受试者工作特征曲线结果表明,miR-663、miR-126和miR-370预测ISR的曲线下面积(AUC)分别为0.772、0.720和0.650;而miR-663联合miR-126预测ISR的AUC为0.909,优于单一miRNA的预测效能。结论冠心病支架植入术后ISR患者血清miR-663、miR-126表达下降,miR-370表达增加。miR-663、miR-126和miR-370可用于预测ISR的发生,miR-663联合miR-126可明显提高预测ISR的效能。     

Notch1 siRNA 对小鼠恶性黑色素瘤细胞体内外增殖的抑制作用研究

目的：探讨靶向Notch1基因小干扰RNA（ Notch1 siRNA,以下简称siNotch）对小鼠恶性黑色素瘤细胞体内外增殖的抑制作用。方法将siNotch 转染小鼠恶性黑色素瘤细胞株B16F1,诱导RNA干扰,采用RT-PCR、Western blot、MTT法及细胞克隆试验体外检测细胞Notch1基因和蛋白表达及细胞增殖、克隆形成变化。随后分别将转染及对照细胞皮下接种于同基因C57BL/6小鼠（分别为转染组及对照组）,在成瘤过程中瘤内注射相应si-Notch1,记录肿瘤生长情况并绘制生长曲线,处死动物后取肿瘤比较各组肿瘤体积,并行HE及免疫组化染色观察肿瘤组织病理学变化和Notch1蛋白表达情况。结果 siNotch1转染B16F1细胞后,细胞增殖能力、克隆形成率及小鼠体内成瘤等均受到抑制（P＜0．01）,免疫组化显示转染组Notch1蛋白表达阳性细胞百分比明显低于对照组（P均＜0．01）。结论 siNotch1可降低细胞Notch1的表达,从而抑制小鼠MM细胞的增殖与生长,可作为治疗恶性黑色素瘤的新方法。     

亚砷酸钠对人类黑色素瘤G361细胞系酪氨酸酶mRNA表达的影响

目的 研究亚砷酸钠(NaAsO2)对人类黑色素瘤G361细胞系酪氨酸酶(TYR)mRNA表达的影响.方法 将体外培养的G361细胞分别暴露于短时(12、24、48 h)低剂量(0.1、1.0μmol/L)、短时(3、6、12、24、48 h)高剂量(5.0、10.0μmol/L)及长时(8、16周)低剂量(0.1μmol/L)下,以荧光实时定量PCR方法测定细胞TYR mRNA表达水平.结果 染毒剂量较低(0.1、1.0μmol/L)、作用时间较短(12、24、48 h)时,黑色素瘤G361细胞TYRmRNA表达水平同一时段各剂量组问比较差异无统计学意义(F值分别为0.173、0.687、2.869,P＞0.05).染毒剂量较高(5.0、10.0μmol/L)、作用12、24、48 h时,细胞TYR mRNA表达水平同一时段各剂量组间比较差异有统计学意义(F值分别为81.056、7.616、24.132,P＜0.05或＜0.01);其中,与对照组比较,除5.0、10.0μmol/L组3、6 h及5.0μmol/L组12 h外,其余各组TYR mRNA相对表达水平比同一时段的对照组明显降低(P＜0.05),0.1μmol/L组染毒8周时细胞TYR mRNA表达水平(138.71±4.56)明显高于对照组(100.00±8.06),两组比较差异有统计学意义(t=-7.238,P＜0.01).结论 NaAsO2暴露可通过引起色素代谢关键酶TYR mRNA表达水平改变而影响色素代谢过程.     

高表达微小RNA22对结肠癌细胞HCT-116增殖的影响

目的探讨高表达微小RNA(mir)-22对结肠癌细胞系(HCT-116)增殖的影响及mir-22水平与结肠癌细胞增殖能力的相关性。方法合成mir-22基因序列,构建SD13-hsa-mir-22质粒,质粒提取后完成对大肠癌细胞系HCT-116细胞的转染,确认转染效率后进行Realtime PCR检测,确定高表达模型建立成功。将HCT-116细胞分为高表达mir-22组、空载体转染(NC)组及未经处理的HCT-116组。通过四甲基偶氮唑蓝比色(MTT)法检测细胞生长,将测定结果进行统计学分析。结果 MTT实验转染高表达mir-22的HCT116细胞和转染空载体的细胞及未转染的阴性对照组细胞相比,生长速度明显减慢。而转染空载体组及阴性对照组细胞生长速度无明显差距。结论 Mir22抑制结肠癌细胞系HCT-116的增殖。     

miR-200a对宫颈癌细胞系HeLa增殖、迁移、侵袭调控作用观察及其靶向mRNA和lncRNA预测分析

目的 观察微小RNA-200a(miR-200a)对宫颈癌细胞系HeLa增殖、迁移和侵袭的调控作用,对miR-200a的靶向mRNA及靶向长链非编码RNA(lncRNA)进行预测分析。方法 取人宫颈癌细胞系HeLa分为1、2、3及4组,分别转染miRNA模拟物miR-200a mimics、miRNA抑制物miR-200a inhibitor、对照物mimics NC及inhibitor NC。培养48 h时采用qRT-PCR法检测各组细胞miR-200a表达;采用CCK8法检测各组细胞增殖能力,采用Transwell侵袭实验检测细胞侵袭能力,采用Transwell迁移实验和细胞划痕实验检测各组细胞迁移能力。使用miRNA数据库在线预测miR-200a靶向mRNA和靶向lncRNA,采用基因本体论功能注释和京都基因和基因组百科全书富集分析法分析宫颈组织中miR-200a的靶向mRNA和靶向lncRNA的生物学功能。结果 与3组比较,1组细胞miR-200a相对表达量高;与4组比较,2组细胞miR-200a相对表达量低（P均<0.05）。与3组比较,培养72、96 h时1组细胞增殖...     

三氧化二砷对人恶性黑色素瘤A375细胞增殖及新癌基因PPO表达的影响

目的 探讨三氧化二砷对人恶性黑素瘤A375细胞株细胞周期、增殖、凋亡及新癌基因PPO(proliferation and phosphorylation oncogene)表达的影响.方法 培养人恶性黑色素瘤A375细胞株,采用 MTT法检测三氧化二砷在不同浓度和不同时间下对A375细胞增殖的影响,流式细胞术分析三氧化二砷对细胞周期及凋亡的影响,倒置相差显微镜观察药物处理后对人A375细胞的细胞分化及形态的影响,免疫细胞化学(SP)法检测三氧化二砷对PPO蛋白表达的影响.结果 MTT法证实三氧化二砷在1～10 μmol/L浓度范围内对A375细胞有明显的增殖抑制作用,呈现明显的剂量和时间依赖效应关系.流式细胞术检测发现,随着药物浓度的增加,G0/G1的细胞逐渐减少(P＜0.01),S期的比例显著增加,出现S期阻滞.倒置显微镜观察发现细胞出现典型凋亡细胞形态学改变.免疫细胞化学法检测PPO蛋白主要表达于A375细胞胞浆内,染色呈棕黄色.三氧化二砷作用48 h后,随着药物浓度的增加,PPO的染色程度逐渐减弱,差异具有显著性(P＜0.01).结论 三氧化二砷能够显著抑制A375细胞株增殖,并诱导凋亡发生,且与三氧化二砷的浓度和作用时间成正相关,其作用机制可能是通过下调新癌基因PPO的表达发挥作用.     

法舒地尔通过Rho/ROCK信号通路抑制肝星状细胞黏附、迁移和增殖

目的观察Rho／Rho激酶（ROCK）信号转导通路抑制剂——法舒地尔，对肝星状细胞（1ISC）黏附、迁移和增殖的影响。方法将培养的HSC分为以下5组：对照组；法舒地尔12.5μmol／L组；法舒地尔25μmol／L组；法舒地尔50μmol／L组；法舒地尔100μmol／L组。采用甲苯胺蓝染色法测定细胞黏附率，Boyden Chamber小室测定HSC跨膜迁移数量，用四甲基偶氮唑盐法检测细胞增殖，Western blot检测HSC RhoA、p-MLC（Thr18／Ser19）和α-平滑肌肌动蛋白的表达。结果法舒地尔对HSC的黏附、迁移和增殖均具有抑制作用，随着浓度增加，抑制作用加强；法舒地尔抑制HSC的α-平滑肌肌动蛋白表达，并且对Rho／ROCK信号转导通路关键信号分子P-MLC（Thr18／Ser19）的蛋白表达有抑制作用。结论法舒地尔通过抑制Rho／ROCK信号通路细胞骨架的调节作用抑制1ISC黏附、迁移和增殖。     

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.     

New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway

The most recently discovered PTEN tumor suppressor gene has been found to be defective in a large number of human cancers. In addition, germ-line mutations in PTEN result in the dominantly inherited disease Cowden syndrome, which is characterized by multiple hamartomas and a high proclivity for developing cancer. A series of publications over the past year now suggest a mechanism by which PTEN loss of function results in tumors. PTEN appears to negatively control the phosphoinositide 3-kinase signaling pathway for regulation of cell growth and survival by dephosphorylating the 3 position of phosphoinositides.     

Downregulated RPL6 inhibits lung cancer cell proliferation and migration and promotes cell apoptosis by regulating the AKT signaling pathway

BackgroundLung cancer is still one of the most common causes of cancer-related mortality, and the overall survival is less than 5%. It is important and necessary to elucidate the mechanism of lung cancer progression. Recently, more and more research has demonstrated that many ribosomal proteins (RPs) are dysregulated in tumors. Among these RPs, ribosomal protein L6 (RPL6) is reported to promote cell growth and cell cycle progression in gastric cancer cells through upregulating cyclin E. However, its function in lung cancer is still unknown.MethodsWe first explored RPL6 expression in lung cancer samples. Next, we used gene knockdown to analyze the unknown regulatory role of RPL6 in lung cancer carcinoma and lung cancer cell lines. Furthermore, we explored the potential signaling pathway of RPL6 with Western blotting.ResultsIn this study, we demonstrated that RPL6 expression was highly expressed in lung cancer tissues and lung cancer cell lines. RPL6 downregulation inhibited H1299 and H2975 cell proliferation, migration and induced cell apoptosis. Also RPL6 downregulation increased the protein levels of cleaved caspase-3 and Bax, while decreasing the protein level of B-cell lymphoma 2 (Bcl-2). Western blotting results showed that proteins activating the AKT signaling pathway, such as p-AKT and p-S6, were downregulated in RPL6 knockdown lung cancer cells.ConclusionsThese findings show that RPL6 can be used as a potential therapeutic and preventive biomarker in lung cancer treatment and prognosis by regulating the AKT signaling pathway.     

Lysophosphatidylcholine inhibits endothelial cell migration and proliferation via inhibition of the extracellular signal-regulated kinase pathway

Lysophosphatidylcholine (lysoPC), a major lipid component of oxidized low density lipoprotein, inhibits endothelial cell (EC) migration and proliferation, which are critical processes during angiogenesis and the repair of injured vessels. However, the mechanism(s) of lysoPC-induced inhibition of EC migration and proliferation has not been clarified. In this report, we demonstrate the critical role of extracellular signal-regulated kinase (ERK) in growth factor-stimulated EC migration and proliferation as well as their inhibition by lysoPC. EC migration and proliferation stimulated by basic fibroblast growth factor (FGF-2) were blocked by inhibition of ERK activity by both the specific mitogen-activated protein kinase kinase (MEK) 1 inhibitor PD98059 and the overexpression of a dominant-negative mutant of MEK1. Conversely, overexpression of a constitutively active mutant of MEK1 increased EC migration and proliferation, which were comparable to those of ECs stimulated with FGF-2. LysoPC inhibited FGF-2-induced ERK activation via prevention of Ras activation without inhibiting tyrosine phosphorylation of phospholipase C-gamma. Taken together, our data demonstrate that ERK activity is required for FGF-2-induced EC migration and proliferation and suggest that inhibition of the Ras/ERK pathway by lysoPC contributes to the reduced EC migration and proliferation.     

三氧化二砷对人恶性黑素瘤细胞株A375生长和凋亡影响的研究

目的研究三氧化二砷（As2O3）对人恶性黑素瘤细胞株A375的生长抑制与促凋亡作用。方法采用四甲基偶氮唑蓝（MTT）还原法检测As2O3对黑素瘤细胞的抑制作用；倒置显微镜、电镜观察细胞形态变化和凋亡特征；流式细胞仪分析DNA含量和检测细胞周期。结果1．0、2．0、4．0、8．0、16．0μmol／L As2O3均能抑制A375细胞的生长，且抑制率具有浓度和时间依赖性。8．0和16．0μmol／L组主要表现细胞毒性作用；4．0μmol／L组作用72h后，A375细胞出现较典型的凋亡形态特征，流式细胞仪检测到亚二倍体凋亡峰，同时出现S期阻滞。结论As2O3能有效的抑制人恶性黑素瘤细胞株A375的生长并促进其凋亡。     

circZFR promotes cell proliferation and migration by regulating miR-511/AKT1 axis in hepatocellular carcinoma

BackgroundEmerging data suggest the crucial regulatory roles of circular RNAs (circRNAs) in hepatocellular carcinoma (HCC). However, the pathophysiology role of circZFR in HCC remains largely unknown.AimsThis study aims to disclose the functions of circZFR in HCC progression and its potential molecular mechanism.MethodscircZFR and miR-511 were identified by qRT-PCR. Colony formation assay, wound-healing assay, transwell assay, and flow cytometry assay were performed to determine the cell proliferation, migration, invasion and apoptosis. Western blotting and immunohistochemistry (IHC) were utilized to evaluate the expression level of AKT1, GSK3β, β-catenin and cascades of proliferation-related proteins both in vitro and in vivo. Dual luciferase reporter assay was conducted to evaluate the interactions among circZFR, miR-511 and AKT1.ResultsThe expression of circZFR was enhanced and the expression of miR-511 was down-regulated in HCC tissues and cells. Functionally, circZFR silencing or miR-511 overexpression suppressed cell proliferation, migration and invasion, and induced apoptosis of HCC cells. Mechanistically, circZFR acted as a miR-511 sponge to up-regulate its target gene AKT1, which activated cascades of proliferation-related proteins (c-Myc, cyclin D1, Survivin and Bcl-2). Furthermore, depletion of circZFR inhibited tumorigenesis and decreased the expression level of AKT1 in xenograft models.ConclusioncircZFR promotes HCC progression by directly down-regulating miR-511 to activate AKT1 signaling, suggesting that circZFR is a potential target in HCC treatment. Targeting circZFR may provide therapeutic benefits for HCC.