RNA干扰整合素连接激酶基因表达对人舌鳞癌Tb细胞生长、迁移和侵袭能力的影响

目的探讨沉默整合素连接激酶(integrin-linked kinase,ILK)基因表达对人舌鳞癌Tb细胞生长、迁移和侵袭能力的影响。方法将ILK siRNA表达质粒和阴性对照质粒在脂质体介导下转染人舌鳞癌Tb细胞,稳定表达细胞株分别命名为Tb siILK和Tb vector组,并设正常Tb细胞对照组(Tb组)。Western blot法检测细胞中ILK蛋白的表达;细胞免疫荧光法检测细胞中ILK、p-Akt和p-GSK3β的表达;光镜和HE染色观察细胞的形态学变化;划痕试验检测细胞的迁移能力;Transwell法检测细胞的侵袭能力;流式细胞术检测细胞的细胞周期和凋亡情况;MTT法检测细胞的增殖活力;Western blot法检测沉默ILK基因后细胞中p-Akt、Akt、p-GSK3β、GSK3α/β、Snail的表达。结果与Tb和Tb vector组相比,Tb siILK组细胞中ILK蛋白的表达水平显著降低(P<0.01);ILK、p-Akt、p-GSK3β在胞质中的荧光信号明显减弱;大部分细胞的上皮形态特征更为明显;细胞的迁移距离和侵袭细胞数显著减少(P<0.05);G2-M期和G0-G1期细胞比例均显著增加(P<0.01);细胞的增殖活力显著减低;ILK基因沉默后,细胞中p-Akt、p-GSK3β和Snail蛋白的表达水平显著降低(P<0.05)。3组细胞的凋亡率差异无统计学意义(P>0.05)。结论抑制ILK基因的表达可通过Akt/GSK3β/Snail途径显著抑制人舌鳞癌Tb细胞的生长、迁移和侵袭能力,ILK有望作为治疗人舌鳞癌的靶基因。     

野生型着丝粒蛋白-E基因沉默对人结肠癌HCT116细胞增殖、凋亡、迁移和侵袭能力的影响

目的利用RNA干扰技术下调野生型着丝粒蛋白E(Wild type centromere protein E,CENP-EWT)基因的表达,观察CENP-EWT基因沉默对结肠癌HCT116细胞增殖、凋亡、迁移和侵袭能力的影响。方法将HCT116细胞分为空白对照组、空载体转染组和CENP-EWT shRNA转染组,转染48 h后,采用巢式PCR检测细胞中CENP-EWT基因mRNA的转录水平;MTT法检测细胞的增殖活力;Hoechst法检测细胞的凋亡比例;Transwell小室试验检测细胞的迁移和侵袭能力。结果与空白对照组和空载体转染组相比,CENP-EWT shRNA转染组可有效抑制CENP-EWT基因的转录水平(P<0.05);CENP-EWT shRNA转染组细胞的增殖活力明显下降(P<0.05);细胞凋亡比例明显增加(P<0.01),细胞的迁移和侵袭能力明显增强(P<0.01)。结论 CENP-EWT基因沉默能够抑制人结肠癌HCT116细胞增殖,诱导细胞凋亡,但同时能增强细胞的迁移和侵袭能力。     

骨形态发生蛋白9对食管鳞状细胞癌细胞的抑制作用

目的探讨骨形态发生蛋白9(Bone morphogenetic proteins 9,BMP9)对食管鳞状细胞癌细胞增殖、克隆、迁移、侵袭及细胞周期的影响。方法用AdGFP和AdBMP9分别感染3株食管鳞状细胞癌细胞株ECA109、KYSE150和KYSE180,并设不感染病毒的3种细胞作为对照,采用RT-PCR法检测AdBMP9感染的3株细胞中BMP9基因mRNA的转录水平,MTT法检测病毒感染细胞0⁹6 h各组细胞的增殖活力,平板克隆法检测各组细胞的克隆形成能力,划痕试验检测各组细胞的迁移能力,Transwell小室试验检测各组细胞的侵袭能力,并对3组ECA109细胞进行细胞周期的检测。结果 AdBMP9感染的3株细胞中BMP9基因mRNA的转录水平均明显提高。与AdGFP感染和未感染病毒的细胞相比,3种细胞在过表达BMP9后,增殖活力明显降低(P<0.05);细胞克隆形成能力均下降(下降率达70%以上),且克隆细胞团明显变小;细胞迁移和侵袭能力均下降;AdBMP9感染的ECA109细胞处于G1期的细胞比例明显上升,S期比例明显下降(P<0.05)。结论 BMP9可明显抑制食管鳞癌细胞的增殖、克隆、迁移和侵袭能力,并将细胞周期阻滞于G1期。     

人参皂苷单体Rh2(S亚型)对人急性髓系白血病细胞株KG1α增殖、凋亡的影响及其机制

目的探讨人参皂苷单体Rh2(S亚型)对人急性髓系白血病细胞株KG1α增殖、凋亡的影响及其机制。方法取对数生长期的KG1α细胞,分为两组:空白对照组(常规培养)和人参皂苷单体Rh2(S)组,采用MTT法检测细胞的增殖活力,倒置显微镜观察细胞的形态及数量,透射电镜观察细胞的超微结构,流式细胞仪检测细胞周期的分布,Real-time PCR检测细胞中β-catenin基因mRNA水平,免疫细胞化学法检测细胞中β-catenin、TCF4、CyclinD1蛋白的定位及表达,Western blot检测细胞中β-catenin、TCF4、CyclinD1蛋白的表达水平。结果人参皂苷单体Rh2(S)对KG1α细胞的增殖具有明显的抑制作用,且呈浓度和时间依赖性。与空白对照组比较,Rh2(S)作用48 h后,可见KG1α细胞分散生长,数量明显减少;可见数量不等,程度不同的凋亡细胞;G0/G1期细胞比例明显上升(P<0.05),G2+M和S期细胞比例明显下降(P<0.05);细胞中β-catenin基因mRNA水平明显降低(P<0.01)。β-catenin、TCF4、CyclinD1蛋白的表达水平明显降低(P<0.05或P<0.01)。结论人参皂苷单体Rh2(S亚型)对KG1α细胞具有明显的增殖抑制作用,其机制可能抑制Wnt/β-catenin/TCF4/CyclinD1信号通路,使细胞周期阻滞,诱导细胞凋亡。     

人骨形态发生蛋白9对人骨肉瘤细胞MG63和U2OS的抑制作用及其机制

目的探讨人骨形态发生蛋白9(Human bone morphogenetic protein 9,hBMP9)对人骨肉瘤细胞MG63和U2OS的抑制作用及其机制。方法用重组腺病毒AdBMP9分别感染MG63和U2OS细胞,并设空白对照组(不加任何处理因素)和AdGFP感染对照组,免疫细胞化学法(Immunocytochemistry,ICC)和Western blot法检测感染后两种细胞中hBMP9的表达水平;MTT和台盼蓝拒染活细胞计数法检测细胞的增殖活力;Hoechst/PI荧光双染法检测细胞的凋亡情况;划痕愈合试验检测细胞的迁移能力;ICC法检测Wnt/β-catenin信号途径中β-catenin的表达。结果AdBMP9感染的两种细胞中hBMP9的表达水平均明显高于空白对照组和AdGFP感染组(P<0.05);hBMP9表达的上调可抑制MG63和U2OS细胞的增殖,且呈时间依赖性(P<0.01),并使两种细胞的凋亡率明显增加(P<0.01),迁移能力明显下降(P<0.01),β-catenin的表达量明显减少(P<0.01)。结论 hBMP9可能通过下调Wnt/β-catenin信号途径活性,抑制骨肉瘤细胞的增殖、迁移,并促进其凋亡。     

PML(NLS~-)干扰对HL-60细胞增殖与凋亡的影响

目的探讨缺失核定位信号的PML,即PML(NLS-)干扰对急性早幼粒细胞白血病细胞HL-60增殖与凋亡的影响。方法将靶向PML(NLS-)基因的3组干扰质粒pGpu6-PML(NLS-)shRNA和阴性对照质粒pGpu6-NCshRNA分别转染HL-60细胞,转染后48 h,G418筛选阳性克隆,分别命名为Si-1、Si-2、Si-3和NC组,并设空白对照组。采用RT-PCR法和Western blot法检测各组细胞中PML(NLS-)基因mRNA的转录水平和蛋白的表达水平;MTT法检测细胞的增殖活力;流式细胞术分析细胞的细胞周期及凋亡情况。结果 Si-1和Si-2组HL-60细胞PML(NLS-)基因mRNA的转录水平和蛋白的表达水平与空白对照组相比明显减低(P<0.05),有干扰效果;Si-1组HL-60细胞的增殖水平与空白对照组相比明显降低(P<0.05),以转染后48 h降低最为显著(P<0.01);抑制PML(NLS-)表达可引起HL-60细胞S期比例增高,G1和G2期比例下降(P<0.05);Si-1组细胞凋亡率明显高于NC组和空白对照组(P<0.05)。结论干扰PML(NLS-)的表达可促进HL-60细胞的凋亡,抑制其增殖。     

lncRNA ZFAS1在食管癌中的作用及其机制

目的探讨长链非编码RNA(long non-coding RNA,lncRNA)ZFAS1(lncZFAS1)对食管癌细胞增殖、凋亡及迁移的影响,并初步探讨其机制。方法采用qRT-PCR法检测食管癌组织及食管癌细胞中lncZFAS1的表达;shRNA干扰lncZFAS1表达后,采用CCK-8法、流式细胞术、划痕试验、Transwell试验检测其对细胞增殖、凋亡、迁移及侵袭的影响;建立小鼠移植瘤模型,检测敲低lncZFAS1对移植瘤生长的影响。结果 lncZFAS1在食管癌组织及食管癌细胞中的表达均明显高于癌旁组织及正常食管上皮细胞。敲低lncZFAS1可显著抑制食管癌细胞的增殖、迁移及侵袭能力;可使食管癌细胞中miR-150-5P表达上调,HMGA2表达下调;可显著抑制裸鼠移植瘤的生长。结论 lncZFAS1可促进食管癌细胞的增殖及迁移,其机制可能与调控miR-150-5P/HMGA2分子轴相关。     

禽流感病毒NS1A蛋白诱导人肺癌细胞SPC-A1的凋亡

目的研究禽流感病毒NS1A蛋白对人肺癌细胞SPC-A1增殖的影响。方法将含有NS1A基因的重组质粒pVAX1-NS1A经脂质体介导转染人肺癌细胞SPC-A1,用MTT检测其对细胞增殖的抑制作用;Annexin VFITC-PI结合流式细胞仪检测细胞凋亡率;PI结合流式细胞仪检测细胞周期的变化;对转染细胞的表达产物进行Western blot分析。结果NS1A蛋白能抑制人肺癌细胞SPC-A1增殖且呈时间依赖性。随着作用时间延长,重组质粒转染组细胞凋亡率可达38.17%,细胞周期中G0/G1比例升高,G1期阻滞。Western blot分析显示,重组质粒转染组细胞在相对分子质量约30000处可见特异性反应条带,与NS1A蛋白产物大小相符。结论禽流感病毒NS1A蛋白能够抑制SPC-A1细胞增殖,并诱导SPC-A1细胞凋亡。     

Musashi2对白血病K562细胞体外侵袭能力的影响

目的探讨Musashi2(Msi2)对白血病K562细胞体外侵袭能力的影响。方法将靶向Msi2基因的si-RNA1、siRNA2及阴性对照序列转染K562细胞,同时设空白对照组(未转染的K562细胞)。实时荧光定量PCR法及Western blot法分别检测K562细胞中Msi2基因m RNA转录及蛋白的表达水平;经细胞生长曲线观察细胞体外增殖能力;细胞黏附试验检测K562细胞体外黏附能力;Transwell试验检测K562细胞体外迁移及侵袭能力。结果与阴性对照组及空白对照组比较,Msi2-1及Msi2-2组K562细胞中的Msi2基因m RNA转录及蛋白的表达水平显著下降(P<0.05),体外黏附、迁移、侵袭能力明显减弱(P<0.05)。结论抑制Msi2的表达可显著抑制白血病K562细胞的体外侵袭能力,本实验为进一步研究Msi2在白血病发生发展中的调控机制奠定了基础。     

甲基化转移酶3对食管癌细胞增殖、迁移及谷氨酰胺代谢的影响

目的 探讨甲基化转移酶3(methyltransferase like-3,METTL3)在食管癌中的表达及其对细胞增殖、凋亡、迁移和谷氨酰胺代谢的影响。方法 采用qRT-PCR、Western blot和免疫组化法检测食管癌组织及其癌旁组织中METTL3基因的表达;CCK8试验、克隆形成试验、划痕试验和Transwell试验检测METTL3抑制剂STM2457对人食管鳞状癌细胞系Eca109和KYSE150增殖及迁移的影响;流式细胞术、TUNEL染色试验检测其对细胞周期及凋亡的影响;TMT/iTRAQ定量蛋白质组法分析其对下游相关信号通路的影响;谷氨酰胺和谷氨酸检测试验及Western blot法分析其对食管癌细胞谷氨酰胺代谢的影响。结果 METTL3在食管癌组织中的表达显著上调（t=5.024,P <0.000 1）。STM2457抑制Eca109和KYSE150细胞的METTL3表达后,显著抑制了细胞的增殖及迁移能力,细胞周期在G0/G1期发生阻滞,细胞凋亡率增加,同时降低了谷氨酰胺摄取量和谷氨酸生成量,并下调了谷氨酰胺代谢相关蛋白丙氨酸-丝氨酸-半胱氨酸转运载体2(ala...     

YKL-40转染对前列腺癌LNcap细胞增殖及侵袭活性的影响

目的探讨外源性YKL-40基因转染对前列腺癌LNcap细胞增殖、侵袭、迁移及黏附活性的影响。方法 RT-PCR法检测前列腺癌细胞LNcap、PC-3、DU-145 YKL-40基因内源性表达情况;用pcDNA3.1-YKL-40质粒转染LNcap细胞,分别经MTT法、Boyden小室法及黏附试验检测转染前后细胞增殖、侵袭、迁移及黏附活性,并进行体外药敏试验。结果仅DU-145细胞可内源性表达YKL-40基因;pcDNA3.1-YKL-40转染的LNcap细胞在第2～5天的A490值均显著高于对照组(P<0.05),其侵袭(75.11±4.40)和迁移穿膜细胞数(133.00±5.07)及黏附率(107.57%)均显著高于对照组(P<0.05),对5-FU、顺铂和依托泊苷的IC50值分别为(31.15±0.43)、(4.15±0.13)和(55.22±0.57)μmol/L,均显著高于对照组(P<0.05)。结论 YKL-40基因能促进LNcap细胞增殖,提高细胞侵袭、迁移及黏附活性,并使其对5-FU、顺铂和依托泊苷具有一定的耐药性。     

Allopregnanolone Promotes Migration and Invasion of Human Glioblastoma Cells through the Protein Tyrosine Kinase c-Src Activation

Glioblastomas (GBs) are the most aggressive and common primary malignant brain tumors. Steroid hormone progesterone (P4) and its neuroactive metabolites, such as allopregnanolone (3α-THP) are synthesized by neural, glial, and malignant GB cells. P4 promotes cellular proliferation, migration, and invasion of human GB cells at physiological concentrations. It has been reported that 3α-THP promotes GB cell proliferation. Here we investigated the effects of 3α-THP on GB cell migration and invasion, the participation of the enzymes involved in its metabolism (AKR1C1-4), and the role of the c-Src kinase in 3α-THP effects in GBs. 3α-THP 100 nM promoted migration and invasion of U251, U87, and LN229 human-derived GB cell lines. We observed that U251, LN229, and T98G cell lines exhibited a higher protein content of AKR1C1-4 than normal human astrocytes. AKR1C1-4 silencing did not modify 3α-THP effects on migration and invasion. 3α-THP activated c-Src protein at 10 min (U251 cells) and 15 min (U87 and LN229 cells). Interestingly, the pharmacological inhibition of c-Src decreases the promoting effects of 3α-THP on cell migration and invasion. Together, these data indicate that 3α-THP promotes GB migration and invasion through c-Src activation.     

The Role of TKS5 in Chromosome Stability and Bladder Cancer Progression

TKS5 promotes invasion and migration through the formation of invadopodia in some tumour cells, and it also has an important physiological function in cell migration through podosome formation in various nontumour cells. To date, the role of TKS5 in urothelial cells, and its potential role in BC initiation and progression, has not yet been addressed. Moreover, the contribution of TKS5 to ploidy control and chromosome stability has not been reported in previous studies. Therefore, in the present study, we wished to address the following questions: (i) Is TKS5 involved in the ploidy control of urothelial cells? (ii) What is the mechanism that leads to aneuploidy in response to TKS5 knockdown? (iii) Is TKS5 an oncogene or tumour-suppressor gene in the context of BC? (iv) Does TKS5 affect the proliferation, migration and invasion of BC cells? We assessed the gene and protein expressions via qPCR and Western blot analyses in a set of nontumour cell strains (Y235T, HBLAK and UROtsa) and a set of BC cell lines (RT4, T24, UMUC3 and J82). Following the shRNA knockdown in the TKS5-proficient cells and the ectopic TKS5 expression in the cell lines with low/absent TKS5 expression, we performed functional experiments, such as metaphase, invadopodia and gelatine degradation assays. Moreover, we determined the invasion and migration abilities of these genetically modified cells by using the Boyden chamber and wound-healing assays. The TKS5 expression was lower in the bladder cancer cell lines with higher invasive capacities (T24, UMUC3 and J82) compared to the nontumour cell lines from human ureter (Y235T, HBLAK and UROtsa) and the noninvasive BC cell line RT4. The reduced TKS5 expression in the Y235T cells resulted in augmented aneuploidy and impaired cell division. According to the Boyden chamber and wound-healing assays, TKS5 promotes the invasion and migration of bladder cancer cells. According to the present study, TKS5 regulates the migration and invasion processes of bladder cancer (BC) cell lines and plays an important role in genome stability.     

Hypoxia-Induced FAM13A Regulates the Proliferation and Metastasis of Non-Small Cell Lung Cancer Cells

Hypoxia in non-small cell lung cancer (NSCLC) affects cancer progression, metastasis and metabolism. We previously showed that FAM13A was induced by hypoxia in NSCLC but the biological function of this gene has not been fully elucidated. This study aimed to investigate the role of hypoxia-induced FAM13A in NSCLC progression and metastasis. Lentiviral shRNAs were used for FAM13A gene silencing in NSCLC cell lines (A549, CORL-105). MTS assay, cell tracking VPD540 dye, wound healing assay, invasion assay, BrdU assay and APC Annexin V staining assays were performed to examine cell proliferation ability, migration, invasion and apoptosis rate in NSCLC cells. The results of VPD540 dye and MTS assays showed a significant reduction in cell proliferation after FAM13A knockdown in A549 cells cultured under normal and hypoxia (1% O₂) conditions (p < 0.05), while the effect of FAM13A downregulation on CORL-105 cells was observed after 96 h exposition to hypoxia. Moreover, FAM13A inhibition induced S phase cell cycle arrest in A549 cells under hypoxia conditions. Silencing of FAM13A significantly suppressed migration of A549 and CORL-105 cells in both oxygen conditions, especially after 72 and 96 h (p < 0.001 in normoxia, p < 0.01 after hypoxia). It was showed that FAM13A reduction resulted in disruption of the F-actin cytoskeleton altering A549 cell migration. Cell invasion rates were significantly decreased in A549 FAM13A depleted cells compared to controls (p < 0.05), mostly under hypoxia. FAM13A silencing had no effect on apoptosis induction in NSCLC cells. In the present study, we found that FAM13A silencing has a negative effect on proliferation, migration and invasion activity in NSCLC cells in normal and hypoxic conditions. Our data demonstrated that FAM13A depleted post-hypoxic cells have a decreased cell proliferation ability and metastatic potential, which indicates FAM13A as a potential therapeutic target in lung cancer.     

吴茱萸碱对人结肠癌lovo细胞生长的抑制作用

目的探讨吴茱萸碱(Evodiamine,Evo)在体内外对人结肠癌lovo细胞生长的抑制作用及其机制。方法采用MTT法检测Evo对人结肠癌lovo细胞、人乳腺癌MDA-MB-231细胞以及人肺癌A549细胞生长的影响;流式细胞术分析Evo对lovo细胞细胞周期和细胞凋亡的影响;采用Evo治疗lovo细胞荷瘤裸鼠,并绘制肿瘤生长曲线;Westernblot法检测Evo对lovo细胞和肿瘤组织中Bcl-2及procaspase-3表达的影响。结果 Evo能抑制lovo细胞生长(P<0.01),促进MDA-MB-231细胞增殖(P<0.01),对A549细胞无明显作用(P>0.05);可将lovo细胞阻滞于S期(P<0.01),并呈时间依赖性诱导其凋亡(P<0.01);能抑制lovo细胞荷瘤裸鼠肿瘤生长(P<0.05);Westernblot检测结果显示,Evo在体内外均可降低lovo细胞Bcl-2及procas-pase-3的表达。结论 Evo可通过抑制Bcl-2表达,激活caspase-3,诱导凋亡,从而抑制lovo细胞的生长。