LncRNA00067110通过靶向Cabyr调控B16-F10细胞增殖、凋亡和黑色素生成

长非编码RNA(lncRNA)是一类转录长度大于200个核苷酸的非编码RNA。现已证明,多个lncRNA是潜在的癌症治疗靶点。LncRNA00067110是从小鼠黑色素瘤B16-F10细胞和正常黑色素细胞转录物组图谱中发现的差异表达基因。为研究lncRNA00067110是否调控B16-F10细胞的增殖、凋亡和黑色素生成,本文通过LncTar预测和双荧光酶活性验证了钙结合酪氨酸磷酸化调节蛋白(Cabyr) 和lncRNA00067110存在靶向关系。通过构建lncRNA00067110的过表达载体,转染B16-F10细胞,经过对B16-F10细胞的转录图谱分析,并对细胞增殖、凋亡和黑色素生成的表型以及相关基因表达变化进行了检测。结果显示,lncRNA00067110靶向Cabyr,在过表达lncRNA00067110的B16细胞中,17个基因呈差异表达。其中,Cabyr的表达被上调,细胞增殖相关基因MEK/ERK/MNK/CREB和黑色素生成相关基因TYR家族成员及CREB的mRNA和蛋白质水平显著被下调,凋亡相关基因AKT和Bcl-2的mRNA水平和蛋白质丰度被上调。进一步通过细胞增殖和凋亡的表型的变化验证了lncRNA00067110的功能。结果提示,lncRNA00067110通过靶向Cabyr,调控相关基因表达,从而抑制B16-F10细胞的增殖和黑色素生成,并诱导黑色素瘤细胞的凋亡,可能成为治疗和抑制黑色素瘤的新的靶点。     

转染人核糖核酸酶抑制因子基因对B16黑色瘤细胞及肿瘤转移的影响(英)

人核糖核酸酶抑制因子(human ribonuclease inhibitor, RI)是一种细胞质中分子质量为50 ku的酸性糖蛋白.RI能抑制核糖核酸酶A（RNase A）的活性, RNase A与血管生成因子（angiogenin,Ang）的氨基酸有着高度保守的同源序列.Ang是RNase A超家族的一员,RI通过与RNase A和Ang的紧密结合而抑制其活性.血管生成及新血管的形成, 是肿瘤发生和转移的必要条件.所以抗血管生成将是一种很有希望的对抑制肿瘤生长和转移的有效方法.实验显示RI能有效地抑制肿瘤诱导血管的生成.RI由含有许多亮氨酸重复序列的多肽组成.含有这样重复序列的100多种蛋白质显示了广泛的功能,包括细胞周期调节,DNA修复,对细胞外基质相互作用以及抑制酶活性等.RI被认为是胚胎发育,创伤愈合及肿瘤发生中新血管形成的一种调节因子.RI定位于染色体的11p15.5,与ras基因邻近,在肿瘤病人中经常存在染色体11p15.5部位的变异和异常.RI可能与细胞的生长和分化有关, 因此,RI 可能还具有尚未知的生物学作用.为了进一步了解RI的潜在功能以及探讨RI与肿瘤浸润、转移的关系, 将人的核糖核酸酶抑制因子基因的cDNA通过逆转录包装细胞PA317,并转染到B16小鼠黑色瘤细胞中, 用转染空载体和未转染的B16细胞作为对照.通过PCR, RT-PCR, 蛋白质免疫印迹, 免疫荧光分析鉴定,获得稳定表达人核糖核酸酶抑制因子的细胞株.结果显示, 转染的RI基因在体外能显著地抑制细胞增殖和细胞迁移,增加了细胞的粘附以及改善细胞的恶性形态,B16,B16 pLNCX,B16 pLNCX-RI 3种细胞的倍增时间分别为(24.98±0.16) h, (25.62±0.28) h, (32.64±1.11) h.与对照组相比,转RI的细胞粘附率增加17.8%和19.5%而迁移降低了61.4%和60%.转RI的细胞比对照组细胞较平展,核仁和分裂相较少,胞质嗜碱性减弱,提示细胞增殖活性降低和恶性表型的改善. 将3种B16细胞静脉注射到C57BL/6小鼠中, 结果表明, 转染RI基因的实验组显著地抑制了肿瘤的转移, 与两个对照组相比,荷瘤小鼠有更长的存活时间, 少得多的转移节结, 更低的肿瘤血管密度和肺重量.结果显示,RI的表达可能与黑色瘤的转移有关, 提示RI能显著地抑制肿瘤的转移,可能由于其与抑制血管作用,增加细胞粘附,降低细胞迁移及增殖有关.     

LncRNA-177922靶向MAPK15调节B16-F10黑色素瘤细胞黑色素生成、增殖、迁移和自噬

黑色素瘤是一种预后较差的侵袭性癌症。了解黑色素瘤的分子机制和诊断标志物对黑色素瘤的防治极为重要。LncRNAs在肿瘤的发生发展中发挥重要作用。与正常黑色素细胞相比,LncRNA-177922在B16-F10黑色素瘤中高表达。丝裂源活化蛋白激酶15 (mitogen-activated protein kinase 15, MAPK15) 的缺失影响肿瘤的发生和进展。本研究中,LncRNA-177922在B16-F10细胞中过表达,结果显示,黑色素生成、增殖、迁移相关基因的mRNA和蛋白质水平显著上调(P< 0. 05),自噬相关基因的mRNA水平和蛋白质丰度下调(P< 0. 05),PI3K/AKT/mTOR通路被激活。同时,进一步验证了细胞迁移、增殖和自噬的表型。结果提示,LncRNA-177922靶向MAPK15通过交叉点细胞外调节蛋白激酶 (extracellular regulated protein kinases, ERK)参与调控黑色素生成、增殖、迁移、自噬等B16-F10细胞的生物学特性,可能是一个新的治疗靶点和诊断标志物。     

花青素单一成分矢车菊-3-O-葡萄糖苷抑制B16-F10细胞增殖和迁移的机理北大核心CSCD

本研究旨在探讨花青素单一成分矢车菊-3-O-葡萄糖苷(cyanidin-3-O-glucoside, Cy-3-glu)对小鼠黑色素瘤细胞B16-F10增殖和迁移的影响,并阐明相关机理。用不同浓度Cy-3-glu处理B16-F10细胞,采用CCK-8法检测细胞存活率,划痕法检测细胞迁移情况,流式细胞仪检测细胞周期,real-time PCR检测细胞周期相关基因的m RNA表达水平,Western blot检测p-AKT、E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)和波形蛋白(vimentin)的相对表达水平,并用小动物活体成像仪检测B16-F10细胞在C57BL/6J小鼠体内的生长和迁移情况。结果显示,Cy-3-glu能明显抑制体外B16-F10细胞的生长(P <0.001)和迁移(P <0.01),使细胞周期阻滞在S期。Cy-3-glu作用后p-AKT (P <0.05)、N-cadherin和vimentin (P <0.001)表达水平显著下降,E-cadherin表达水平升高(P <0.05)。饲喂Cy-3-glu饲料的肿瘤模型C57BL/6J小鼠肿瘤大小和重量显著减小(P <0.05),肿瘤的转移水平也明显降低。以上结果提示,Cy-3-glu通过抑制PI3K/AKT信号、调节细胞黏附和迁移信号使B16-F10细胞周期停滞在S期,最终抑制其在体内和体外的增殖和迁移。     

花青素单一成分矢车菊-3-O-葡萄糖苷抑制B16-F10细胞增殖和迁移的机理

本研究旨在探讨花青素单一成分矢车菊-3-O-葡萄糖苷(cyanidin-3-O-glucoside, Cy-3-glu)对小鼠黑色素瘤细胞B16-F10增殖和迁移的影响,并阐明相关机理。用不同浓度Cy-3-glu处理B16-F10细胞,采用CCK-8法检测细胞存活率,划痕法检测细胞迁移情况,流式细胞仪检测细胞周期,real-time PCR检测细胞周期相关基因的m RNA表达水平,Western blot检测p-AKT、E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)和波形蛋白(vimentin)的相对表达水平,并用小动物活体成像仪检测B16-F10细胞在C57BL/6J小鼠体内的生长和迁移情况。结果显示,Cy-3-glu能明显抑制体外B16-F10细胞的生长(P 0.001)和迁移(P 0.01),使细胞周期阻滞在S期。Cy-3-glu作用后p-AKT (P 0.05)、N-cadherin和vimentin (P 0.001)表达水平显著下降,E-cadherin表达水平升高(P 0.05)。饲喂Cy-3-glu饲料的肿瘤模型C57BL/6J小鼠肿瘤大小和重量显著减小(P 0.05),肿瘤的转移水平也明显降低。以上结果提示,Cy-3-glu通过抑制PI3K/AKT信号、调节细胞黏附和迁移信号使B16-F10细胞周期停滞在S期,最终抑制其在体内和体外的增殖和迁移。     

核糖核酸酶抑制因子对血管生成素功能的调控

血管生成素（angiogenin,ANG）能有效促进血管生成和肿瘤细胞增殖,在肿瘤发生发展中起重要作用.其主要分子机制是通过核转位和激活PI3K/AKT/mTOR信号通路,刺激rRNA转录和核糖体生成.ANG也被发现在肌萎缩侧索硬化症（ALS）和帕金森病（PD）患者中存在基因编码区的功能突变,表明其在运动神经元生理方面发挥作用,其缺陷是神经退行性疾病的一个危险因素.核糖核酸酶抑制因子（ribonuclease inhibitor,RI）是胞内酸性蛋白质,由460个氨基酸残基组成,分子质量约为50 kD,当其与核糖核酸酶A（RNaseA）结合形成复合物后,可抑制RNaseA 的90％以上活性,从而有效调节细胞内RNA水平. ANG具有低核糖核酸酶活性, 是RNase超家族一员,与RNase A有着高度保守的同源顺序. 序列、结构和酶学等分析表明,RI也能够与ANG紧密结合,且得到体外实验的证明. 研究发现,RI具抑癌基因功能;RI与ANG在细胞内共定位;Co IP和GST pull down证实其相互作用,获取了RI与ANG在体内结合的直接证据;RI与AKT磷酸化表达负相关.在膀胱癌细胞及临床标本中证实了RI与 ANG和PI3K/AKT通路分子表达的相关性及与肿瘤细胞生长与转移的关系．在细胞和动物模型研究表明,RI调节ANG活性的功能及其分子机制,即RI通过结合ANG而封锁其核转位和调控PI3K/AKT/mTOR信号通路及其相关通路交互应答（cross talk）的能力,从而抑制肿瘤生长及转移. RI是一个有希望的抗肿瘤蛋白新药和血管生成抑制剂,可望成为基因治疗的靶基因.     

重组天花粉蛋白与-16联合用药抑制黑色素瘤细胞增殖及成瘤

获得人源天花粉蛋白并研究其与YH-16联合用药对小鼠黑色素瘤细胞B16-F10增殖及成瘤的作用。以大肠杆菌BL21重组表达天花粉蛋白r TCS并以亲和层析法纯化r TCS;以MTT法检测r TCS、YH-16、r TCS+YH-16对B16-F10细胞生长的影响;体内实验检测r TCS、r TCS+YH-16对瘤体生长的作用。结果表明,获得了电泳纯重组天花粉蛋白r TCS;r TCS可显著抑制B16-F10细胞增殖,并且抑制率显著高于YH-16组,但是r TCS与YH-16联合处理组并未比r TCS组有显著差异;r TCS与YH-16联合用药明显抑制B16-F10细胞在小鼠体内成瘤。重组天花粉蛋白r TCS联合YH-16用药对黑色素瘤细胞B16-F10的增殖及成瘤有显著抑制作用。联合策略将为治疗黑色素瘤患者提供新的契机。     

异位（过）表达PGRN抑制IL-1β引起的髓核细胞损伤

炎症因子IL-1β是引起髓核细胞功能异常的关键因素之一。颗粒体蛋白原（progranulin,PGRN）是一种多功能生长因子,在组织修复、炎症反应等过程中发挥重要作用,但其在髓核细胞中的作用尚不清楚。本研究以IL-1β诱导的髓核细胞炎性损伤模型为研究对象,探讨PGRN对IL 1β诱导的髓核细胞损伤的保护作用及其机制。基因转染结合MTT方法证明,与IL-1β处理的细胞比较,过表达PGRN可逆转IL-1β引起的原代培养的髓核细胞生长抑制,促进细胞增殖。TUNEL技术和流式细胞分析显示,PGRN抑制IL-1β诱导的髓核细胞凋亡。Western印迹和RT-qPCR方法揭示,与IL-1β处理的细胞相比,过表达PGRN显著上调聚蛋白聚糖（aggrecan）和II型胶原（collagen type II）的蛋白质表达,但下调基质金属蛋白酶-13（MMP-13）、分解素-金属蛋白酶ADAMTS-5的表达,同时抑制IL-1β诱导的炎性因子IL-6、IL-8和TNF-α的表达,说明PGRN可缓解IL-1β引起的炎性反应,并减少细胞外基质（ECM）相关蛋白质的降解。此外,过表达PGRN还可降低p65、p-IkB a和β-catenin的蛋白质表达水平,提示PGRN可抑制IL-1β下游TNF-α介导的NF-κB信号途径及β-catenin途径。总之,上述结果提示,过表达PGRN可通过抑制IL-1β诱导的炎性反应、髓核细胞凋亡及基质代谢紊乱,缓解IL-1β诱导的髓核细胞损伤;PGRN的这种抗炎、抗基质降解作用可能与PGRN参与调控NF-κB和β-catenin信号途径有关。     

长链非编码RNA Linc00673过表达对胃癌MGC-803细胞增殖与凋亡的影响*

目的: 探讨长链非编码RNA Linc00673过表达对胃癌细胞增殖和凋亡的影响及其机制。方法: 将重组慢病毒表达质粒pLVX-Linc00673和对照空载体质粒pLVX-NC在293T细胞中进行慢病毒包装与扩增,将重组慢病毒转染胃癌细胞MGC-803建立稳定过表达 Linc00673的细胞系,实时荧光定量PCR方法检测Linc00673基因的表达; MTT实验和克隆形成实验观察细胞的生长增殖;流式细胞术检测细胞周期和细胞凋亡;qPCR检测细胞周期相关调控基因表达;免疫印迹法检测PI3K/Akt信号通路关键分子及肿瘤增殖相关蛋白的表达。结果: Linc00673在胃癌细胞系MGC-803、BGC-823和AGS中的表达量显著高于正常胃粘膜细胞GES-1(P＜0.05)。建立了稳定过表达Linc00673的MGC-803细胞系,Linc00673的表达量比对照空载体组高200倍。Linc00673过表达促进MGC-803细胞增殖和克隆形成(P＜0.05),抑制细胞凋亡并影响细胞周期G1→S期进程(P＜0.01);Linc00673过表达可影响MGC-803细胞周期调节基因CCNG2、p19和CDK1的表达;免疫印迹结果显示,Linc00673过表达不仅促进PI3K/Akt信号通路关键分子pAKT及其下游靶点NF-κB和Bcl-2蛋白的表达,而且上调肿瘤相关因子β-catenin和EZH2蛋白的表达。结论: Linc00673过表达可能通过PI3K/Akt信号通路促进MGC-803细胞增殖、抑制凋亡。     

纤黏连蛋白重组多肽CH50抑制黑色素瘤MMP-2和MMP-9表达、激活的研究

目的 研究纤黏连蛋白重组多肽CH50对黑色素瘤B16细胞侵袭能力的影响,探讨CH50多肽抑制肿瘤生长、侵袭的机制。方法 体外培养小鼠黑色素瘤B16细胞、小鼠腿部皮下注射B16细胞建立肿瘤动物模型。采用明胶电泳法检测B16细胞和黑色素瘤组织中基质金属蛋白酶MMP-2和MMP-9的表达和激活;以CH50多肽体外处理B16细胞或体内表达CH50,观察CH50下调MMPs表达以及对肿瘤细胞侵袭能力的抑制作用。结果 B16细胞在体外培养条件下主要表达MMP-2,而在肿瘤微环境中则同时表达MMP-2和MMP-9。肿瘤组织中MMPs的表达明显高于体外培养B16细胞。CH50多肽对体外培养B16细胞的MMPs表达和激活无明显抑制作用,但处理后的B16细胞进入体内后表达MMPs的能力受到明显抑制。体内转染表达的CHSO多肽亦可明显抑制肿瘤表达MMPs、并抑制肿瘤侵袭能力。结论 纤黏连蛋白重组多肽CHSO可以抑制肿瘤微环境中基质金属蛋白酶MMP-2和MMP-9的表达和激活,从而抑制黑色素瘤生长及侵袭能力。     

Up-regulating ribonuclease inhibitor inhibited epithelial-to-mesenchymal transition and metastasis in murine melanoma cells

Human ribonuclease inhibitor (RI) is a cytoplasmic acidic protein. RI is constructed almost entirely of leucine rich repeats, which might be involved in unknown biological effects except inhibiting RNase A and angiogenin activities. We previously reported that up-regulating RI inhibited the growth and metastasis of melanoma cells. Epithelial-mesenchymal transition (EMT) is a critical event of cancer cells that triggers invasion and metastasis. However, the role of RI in the EMT process remains unknown. Here we hypothesize that RI might inhibit melanoma invasion and metastasis by regulating EMT. We found that over-expression of RI induced up-regulation of E-cadherin, accompanied with decreased expressions of proteins associated with EMT such as N-cadherin, Snail, Slug, Vimentin and Twist both in vitro and in vivo. Furthermore, RI restrained matrix metalloproteinase MMP-2 and MMP-9 secretions in B16 and B16-F10 melanoma cells. In addition, we also found that up-regulation of RI inhibited cell proliferation, migration and invasion as well as changed cell morphology, adhesion and rearranged cytoskeleton in vitro. Finally, the effects of RI on phenotype and invasiveness translated into suppressing metastasis by the experimental metastasis models of melanoma with lighter lung weight, a fewer metastasis nodules and a lower incidence rate, with respect to the control groups. Taken together, our data highlight, for the first time, that RI plays a novel role in inhibiting development and progression of murine melanoma cells through regulating EMT. These results suggest that RI could be a therapeutic target protein for melanoma and may be of biological importance.     

Inhibition by tyroserleutide (YSL) on the invasion and adhesion of the mouse melanoma cell

Tyroserleutide (YSL) is an active, low-molecular-weight polypeptide, comprised of three amino acids, that has shown antitumor effects on human hepatocarcinoma BEL-7402 in vitro and in vivo. In this study, we evaluated the inhibition of YSL on invasion and adhesion of the mouse B16-F10 melanoma cell line by injecting B16-F10 cells into the tail veins of C57BL/6 mice to establish an experimental lung metastasis model. YSL inhibited B16-F10 cell metastasis to lung, reducing the number and area of metastasis lesions. When we treated B16-F10 cells with YSL (0.01, 0.1, 1, 10, or 100 microg/mL) in vitro, we found that YSL inhibited the proliferation of B16-F10 cells with a 28.11% rate of inhibition. YSL significantly decreased the adhesiveness of B16-F10 cells to Matrigel with a 29.15% inhibition rate; YSL also significantly inhibited the invasion of B16-F10 cells, producing an inhibition of 35.31%. By analyses with Western blot and real-time RT-PCR, we found that YSL markedly inhibited the expression of ICAM-1 in B16-F10 cells. These data suggest that YSL inhibits the growth, invasion, and adhesion of B16-F10 cells.     

A novel role of ribonuclease inhibitor in regulation of epithelial-to-mesenchymal transition and ILK signaling pathway in bladder cancer cells

Human ribonuclease inhibitor (RI) is a cytoplasmic acidic protein possibly involved in biological functions other than the inhibition of RNase A and angiogenin activities. We have previously shown that RI can inhibit growth and metastasis in some cancer cells. Epithelial-mesenchymal transition (EMT) is regarded as the beginning of invasion and metastasis and has been implicated in the metastasis of bladder cancer. We therefore postulate that RI regulates EMT of bladder cancer cells. We find that the over-expression of RI induces the up-regulation of E-cadherin, accompanied with the decreased expression of proteins associated with EMT, such as N-cadherin, Snail, Slug, vimentin and Twist and of matrix metalloprotein-2 (MMP-2), MMP-9 and Cyclin-D1, both in vitro and in vivo. The up-regulation of RI inhibits cell proliferation, migration and invasion, alters cell morphology and adhesion and leads to the rearrangement of the cytoskeleton in vitro. We also demonstrate that the up-regulation of RI can decrease the expression of integrin-linked kinase (ILK), a central component of signaling cascades controlling an array of biological processes. The over-expression of RI reduces the phosphorylation of the ILK downstream signaling targets p-Akt and p-GSK3β in T24 cells. We further find that bladder cancer with a high-metastasis capability shows higher vimentin, Snail, Slug and Twist and lower E-cadherin and RI expression in human clinical specimens. Finally, we provide evidence that the up-regulation of RI inhibits tumorigenesis and metastasis of bladder cancer in vivo. Thus, RI might play a novel role in the development of bladder cancer through regulating EMT and the ILK signaling pathway.     

Correlation between post-replication repair and metastatic potential in B16 mouse melanoma cell lines

The rate of postreplication repair of the B16-F1 and the B16-F10 variant clones was compared to the parent B16CL4 mouse melanoma cells in an attempt to correlate the postreplication repair efficiency with the metastatic potential of these melanoma cells. The rate of postreplication repair of the B16-F10 subline was 47% higher than that of the parent B16CL4 mouse melanoma cells and 20% higher than that of the B16-F1 cells. This higher rate of postreplication repair in the B16-F10 cells correlates with its higher metastatic potential. It was also of interest to notice that the rate of postreplication repair of the B16-F1 and the B16-F10 cells are comparable to their rate of replicon joining in non-irradiated cells, in contrast to the parent B16CL4 cells whose rate of post-replication repair was significantly lower than its rate of replicon joining.     

Characterization of mouse melanoma cell lines by their mortal malignancy using an experimental metastatic model

We characterized the metastatic ability and mortality of four different mouse melanoma cell lines, B16-F0, -F1, -F10 and -BL6. B16-F0 is the parent cell line. B16-F1 was obtained by a one-time selective procedure and B16-F10 by a ten-time selective procedure using Fidler's method. B16-BL6 derived from B16-F10 has much more invasive activity than B16-F10. To investigate the difference in mortal malignancy among B16-F0, -F1, -F10 and -BL6, we examined the survival time of syngeneic C57BL/6Cr mice intravenously inoculated with these cells. As a control, we used the C57BL/6J-embryo mouse fibroblast-like semi-normal cell line. The ability to form lung metastatic nodules in mice gradually increased in the order: B16-F0, -F1, and -F10 (=-BL6). C57BL/6J-embryo cell (1 x 10(5)/mouse)-inoculated mice survived for over 46 days. B16-F0, -F1, -F10 and -BL6 (1 x 10(5)/mouse)-inoculated mice survived 31.4+/-4.4 (7), 25.7+/-2.8 (7), 23.6+/-1.5 (7) and 25.3+/-2.3 (7) days [mean+/-S.D. (number of mice)], respectively. According to the Mann-Whitney test, the B16-F0 inoculated group versus -F1 inoculated group (P<0.05), -F0 inoculated group versus -BL6 inoculated group (P<0.05), and -F0 inoculated group versus -F10 inoculated group (P<0.01) were significantly different, but the B16-F1 group versus -F10 group, -F1 group versus -BL6 group, and -F10 group versus -BL6 group were not. These results suggest that mortal malignancy is not necessarily correlated with lung-colonizing potential and even only one-time selected B16-F0 mouse melanoma cells are useful as an experimental metastatic model in vivo.     

T-cadherin suppresses the cell proliferation of mouse melanoma B16F10 and tumor angiogenesis in the model of the chorioallantoic membrane

The influence of T-cadherin on the pigmentation and proliferation of mouse melanoma B16F10 cells in vitro and on the growth and neovascularization of tumor cell masses formed by the B16F10 cells in a model of the chorioallantoic membrane of a chicken embryo is studied. It is found that the proliferative activity of the cells decreases in the cell culture of mouse melanoma upon the overexpression of T-cadherin in comparison with the cells in the control. It is shown in experiments in vitro that the B16F10 cells with the overexpression of T-cadherin are rarely settling are to the chorioallantoic membrane than the control cells. In addition, it is found that the control cells of mouse melanoma form tumors with area more 0.1 mm² more often than the cells with the overexpression of T-cadherin and the amount of the vessels growing to tumor cell masses formed by the cells with the overexpression of T-cadherin is significantly lower than the same index for the cells in the control. Thus, the overexpression of T-cadherin in the B16F10 cells suppresses the proliferation of these cells in vitro and the growth of the tumor masses formed by melanoma cells on the chorioallantoic membrane and their neovascularization in vivo.     

PMA 及乏氧诱导VEGF 上调的细胞模型构建及用于RNAi 研究

目的:探讨佛波酯(PMA)与乏氧诱导对小鼠黑色素瘤细胞B16-F10中血管内皮细胞生长因子(VEGF)表达量的影响,构建适合RNA干扰(RNAi)的体外细胞模型。方法:通过酶联免疫吸附试验(ELISA法)在蛋白质水平上检测细胞分泌的VEGF量,并用激光共聚焦显微镜观察小干扰RNA(siRNA)转染的细胞胞吞及细胞形态。结果:1 M PMA处理细胞2 h能明显上调B16-F10细胞中VEGF蛋白的合成及分泌,与常规培养相比,细胞可增加50%的VEGF水平。再经乏氧诱导48 h,稳定释放到培养液里的VEGF浓度大幅提高200%,范围在55-65 pg/mL/h。结论:经PMA和乏氧诱导后,B16-F10细胞稳定的VEGF分泌量与一定时间内分泌的稳定性均表明其适合作为RNAi的体外细胞模型。初步的RNAi结果表明,TKO/siRNA纳米粒与壳聚糖/siRNA纳米粒对于VEGF的沉默效率达40%。     

Biochemical analysis of metastasis-related Ax actin in B16 mouse melanoma cells after chemical reversional modulation and of tumor progression-related A' actin in the ontogeny of human malignant melanoma

To examine the correlation between tumor metastasis and Ax actin in mouse melanoma and between tumor progression and A'.actin in human melanoma and further to investigate whether or not it is a generally existing principle, we studied the effects of reversion agents, which distinctly decrease metastatic ability of melanoma cells, on the appearance of Ax actin. Will an induced decrease in metastasis of established highly metastatic B16-F10 mouse melanoma cells cause the appearance of Ax actin? We also examined the appearance of A' actin in eight human benign pigment cell tumors and nine human malignant melanoma tissues or cells in relation to tumor progression. In vitro treatment of B16-F10 cells with each of these agents suppressed metastatic ability of the cells injected intravenously into syngenic mice; however, none of the treated cells represented Ax actin in vitro. These results suggest that the appearance of Ax actin may be a result of long-term tumor cell progression leading to changes in gene level, but because the treatments with these agents were only carried out over a short period, they could not effect changes in gene level; thus, Ax actin appearance remained unchanged. Appearance of A' actin was detected only in human benign pigment cell tumors such as nevus cell nevi, but not in malignant melanomas, which were also formed in a long period of tumor progression in vivo. These results suggest that A' actin is a clinically useful marker to determine the prognosis and level of tumor progression of human pigment cell tumors.     

Identification of metastasis-promoting sequences in the mouse laminin alpha 1 chain.

Laminin-1, a major basement membrane matrix glycoprotein, enhances adhesion, migration, and metastasis of tumor cells. We have screened 208 overlapping synthetic peptides covering the short and long arms of mouse laminin alpha1 chain for their adhesion activity with B16-F10 mouse melanoma cells. Cell adhesion activity was determined using various amounts of peptides coated on plastic dishes and by measuring cell adhesion on peptide-conjugated Sepharose beads. Nineteen peptides showed B16-F10 cell adhesion activity. Three peptides, designated A-13, -24, and -208, showed the strongest attachment activity in the plate assay, whereas 4 peptides, A-13, -51, -99, and -112, demonstrated the strongest cell adhesion when conjugated to beads. The 19 peptides were tested in vivo for their effect on experimental pulmonary metastasis by B16-F10 cells. Four peptides, A-13, -51, -64, and -119, significantly enhanced metastasis, with A-13 showing the strongest dramatic enhancement. The four metastasis-promoting peptides also stimulated migration of B16-F10 cells in the Boyden chamber assay in vitro with A-13 being the most potent stimulator. In addition, the 4 peptides inhibited laminin-induced cell attachment and migration, which indicates that these four sequences are possible functional B16-F10 cell binding sites in laminin-1. All the four sequences are located on the globular domains of the short arm. Other peptides, including strong adhesion-active peptides, A-24, -99, -112, and a scrambled A-13 peptide, did not stimulate either migration or metastasis. Thus, laminin-1 has multiple active sites in the globular domains of the short arm which promote migration and metastasis of B16-F10 cells.     

Proopiomelanocortin gene delivery induces apoptosis in melanoma through NADPH oxidase 4-mediated ROS generation

Hypoxia in the tumor microenvironment triggers differential signaling pathways for tumor survival. In this study, we characterize the involvement of hypoxia and reactive oxygen species (ROS) generation in the antineoplastic mechanism of proopiomelanocortin (POMC) gene delivery in a mouse B16-F10 melanoma model in vivo and in vitro. Histological analysis revealed increased TUNEL-positive cells and enhanced hypoxic activities in melanoma treated with adenovirus encoding POMC (Ad-POMC) but not control vector. Because the apoptotic cells were detected mainly in regions distant from blood vessels, it was hypothesized that POMC therapy might render melanoma cells vulnerable to hypoxic insult. Using a hypoxic chamber or cobalt chloride (CoCl₂), we showed that POMC gene delivery elicited apoptosis and caspase-3 activation in cultured B16-F10 cells only under hypoxic conditions. The apoptosis induced by POMC gene delivery was associated with elevated ROS generation in vitro and in vivo. Blocking ROS generation using the antioxidant N-acetyl-l-cysteine abolished the apoptosis and caspase-3 activities induced by POMC gene delivery and hypoxia. We further showed that POMC-derived melanocortins, including α-MSH, β-MSH, and ACTH, but not γ-MSH, contributed to POMC-induced apoptosis and ROS generation during hypoxia. To elucidate the source of ROS generation, application of the NADPH oxidase inhibitor diphenyleneiodonium attenuated α-MSH-induced apoptosis and ROS generation, implicating the proapoptotic role of NADPH oxidase in POMC action. Of the NADPH oxidase isoforms, only Nox4 was expressed in B16-F10 cells, and Nox4 was also elevated in Ad-POMC-treated melanoma tissues. Silencing Nox4 gene expression with Nox4 siRNA suppressed the stimulatory effect of α-MSH-induced ROS generation and cell apoptosis during hypoxia. In summary, we demonstrate that POMC gene delivery suppressed melanoma growth by inducing apoptosis, which was at least partly dependent on Nox4 upregulation.