LRIG1基因抑制胶质瘤细胞生长的机制

目的 探讨富含亮氨酸重复序列免疫球蛋白样蛋白1(LRIG1)基因与表皮生长因子受体(EGFR)在人脑胶质瘤细胞中的相互作用及其对肿瘤细胞生长影响的机制.方法 用免疫共沉淀法检测人脑胶质瘤细胞系GL15中LRIG1与EGFR的相瓦作用.构建针对LRIG1基因的干扰片段及非特异性的对照片段分别转染GL15细胞系,G418(600 mg/L)筛选出稳定株,Western blot法检测LRIG1表达的改变及其对EGFR表达的影响;嚷唑蓝(MTT)比色法检测LRIG1表达下调对胶质瘤细胞增殖的影响.结果 免疫共沉淀法表明胶质瘤细胞系GL15中LRlG1与EGFR存在相互作用.成功构建了针对LRIG1基因的特异性shRNA表达载体,转染细胞后LRIG1基因表达下降54.7%;同时干扰组细胞EGFR蛋白水平与阴性对照组比较上升了57.1%.MTT结果显示干扰组细胞增殖率高于对照组.结论 LRIG1通过与EGFR结合发挥抑癌作用,干扰LRIG1表达可削弱这种作用.导致肿瘤细胞增殖加速.     

RNA干扰富含亮氨酸重复序列免疫球蛋白样蛋白2后对胶质瘤细胞生长的影响及其机制

目的 探讨RNA干扰富含亮氨酸重复序列免疫球蛋白样蛋白2(LRIG2)基因表达后对胶质瘤细胞生长的影响及其机制.方法 用携带U6启动子和LRIG2特异性短发夹RNA(shRNA)序列的质粒载体pGenesi12-LRIG2-shRNA(siRNA)及含非特异性shRNA编码序列的对照质粒pGenesi12-scramble-shRNA(scr)转染胶质瘤细胞系GL15、G418(600mg/L)筛选出稳定株,逆转录-聚合酶链反应(RT-PCR)和Western blot法检测LRIG2和表皮生长因子受体(EGFR)表达的改变.噻唑蓝(MTT)比色法检测稳定转染对照组和实验组细胞的增殖,流式细胞仪检测对照组和实验组细胞的细胞周期改变.结果 实验组细胞LRIG2 mRNA和蛋白水平与对照组比较分别下降了68.6%和62.3%,EGFR蛋白水平下降了32.6%.MTT结果显示实验组细胞增殖率低于对照组.细胞周期分析表明对照组G0/G1期为(26.72±2.10)%,实验组为(36.58±3.29)%(P<0.05),LRIG2表达下调后GL15细胞细胞周期部分阻滞在G0/G1期.结论 GL15细胞经RNA干扰基因表达后,LRIG2 mRNA和蛋白水平明显降低,同时EGFR蛋白水平下降,从而抑制细胞增殖和使细胞周期阻滞在G0/G1期.LRIG2可通过EGFR信号系统影响胶质瘤细胞的生长.     

过度表达LRIG1基因逆转人胶质瘤侵袭性的机制

目的　探讨LRIG1基因抑制恶性胶质瘤细胞侵袭、浸润的分子机制。方法　应用脂质体介导的基因转染技术将p3XFLAG CMV9 LRIG1质粒转染入人神经胶质瘤H4细胞系 ,通过逆转录 聚合酶链反应 (RT PCR)和Westernblot方法检测转染前、后LRIG1和表皮生长因子受体(EGFR)基因mRNA和蛋白表达水平 ,用Boyden小室体外侵袭试验观察H4细胞通过人工重组基底膜能力的变化。结果　转染p3XFLAG CMV 9 LRIG1质粒后 ,H4神经胶质瘤细胞的LRIG1mRNA和蛋白表达均较对照组明显增强 (P <0 .0 1) ;而EGFRmRNA和蛋白表达均较对照组明显减弱 (P <0 .0 1)。LRIG1质粒转染的H4细胞穿过人工重组基底膜的相对百分率为 (13 .3± 1.8) % ,明显低于未处理组和转染空载体组的穿膜细胞百分率 (2 4.7± 1.8) % ,(2 4.0± 1.5 ) % (P =0 .0 0 8和P =0 .0 10 )。结论　EGFR的过表达促进了胶质瘤的侵袭、浸润 ;LRIG1通过抑制EGFR逆转了恶性胶质瘤侵袭、浸润。     

表皮生长因子受体在前列腺癌雄激素非依赖型细胞系中表达的意义

目的探讨转化生长因子(TGF)-α和表皮生长因子(EGF)对前列腺癌雄激素非依赖型细胞系中表皮生长因子受体(EGFR)表达的调控作用.方法采用逆转录-多聚酶链式反应和免疫印迹法分别对TGF-α和EGF刺激前列腺癌雄激素非依赖型细胞系PC3、ARCaP后EGFR mRNA表达及其蛋白水平进行定量分析.结果EGF引起PC3、ARCaP的EGFR mRNA升高,分别为5.01±0.45和2.41±0.26,差异无显著性(P＞0.05);TGF-α引起各细胞系EGFR mRNA升高,分别为9.05±0.63和3.54±0.33,差异无显著性(P＞0.05).各细胞系EGFR蛋白水平TGF-α组明显高于EGF组(P＜0.05).结论TGF/EGF-EGFR通路在发生发展中起重要作用;TGFα-EGFR自分泌环在非依赖型前列腺癌中的作用强于EGF-EGFR自分泌环.     

TGFα和EGF对前列腺癌细胞系EGFR表达的调控作用

目的：探讨转化生长因子（TGF）α和表皮生长因子（EGF）对前列腺癌细胞系中表皮生长子受体（EGFR）表达的调控作用。方法：采用RT－PCR和Western印迹法分别对TGFα和EGF刺激前列腺癌细胞系LNCaP、PC3、ARCaP后EGFR mRNA表达及其蛋白水平进行定量分析。结果：EGF引起前列腺癌细胞系LNCaP、PC3、ARCaP的EGFR mRNA升高2－5倍,TGFα引起各细胞系EGFR mRNA升高2－10倍。TGFα使各细胞系EGFR不同程度升高;EGF处理的LNCaP EGFR蛋白水平略升高,PC3、ARCaP EGFR蛋白水平降低。结论：TGF／EGF-EGFR通路在前列腺癌发生发展中起重要作用;非依赖型前列腺癌中TGFα－EGFR自分泌环的作用可能强于EGF－EGFR自分泌环。     

RNA干扰富含亮氨酸重复序列免疫球蛋白样蛋白3表达对胶质瘤细胞周期和凋亡的影响

目的 探讨RNA干扰富含亮氨酸重复序列免疫球蛋白样蛋白3(LRIG3)基因表达后对胶质瘤细胞生长的影响及其机制.方法 设计两对含LRIG3特异性短发夹RNA(shRNA)序列的质粒及含非特异性shRNA的对照质粒,转染胶质瘤细胞系GL15、G418筛选出稳定株,逆转录-聚合酶链反应(RT-PCR)和Western blot法检测LRIG3表达的改变,流式细胞仪检测各组细胞的细胞周期变化,Annexin V-FITC/PI双标记分析细胞凋亡.结果 实验组细胞LRIG3 mRNA和蛋白水平与对照组比较分别下降了52.4%、63.8%和50.9%、67.4%.流式细胞仪分析显示,实验组的G2/M期细胞细胞百分率比对照组明显增加,细胞增殖指数显著增加(P<0.01);LRIG3基因表达下调后还具有显著的抗凋亡作用(P<0.05).结论 LRIG3特异性siRNA可明显抑制LRIG3基因的转录和表达,从而促进GL15细胞的增殖和使细胞周期阻滞在G2/M期并能抑制其凋亡.     

LRIG1在星形细胞瘤肿瘤发生中的作用

目的 观察表皮生长因子受体(EGFR)内源性抑制因子LRIG1在转录水平于星形细胞瘤中的表达及其与EGFR mRNA和肿瘤恶性程度的相关性,探讨其在肿瘤发生中的作用.方法 逆转录-聚合酶链反应(RT-PCR)法检测LRIG1和EGFR mRNA在35例Ⅰ～Ⅳ级星形细胞瘤组织中的表达水平.统计学分析各组间表达的差异性.结果 在多数肿瘤组织中LRIG1 mRNA低表达(27/35),EGFR mRNA存在过度表达(21/35),在18例低级别(Ⅰ～Ⅱ级)与17例高级别(Ⅲ～Ⅳ级)肿瘤中前者表达差异无统计学意义(P>0.05),后者表达差异有统计学意义(P<0.05).两者表达趋势无明显相关(r=0.187,P>0.05).结论 作为EGFR信号系统的负反馈调节因子,LRIG1在mRNA水平的表达下调提示了它和星形细胞瘤的发生有关,其与EGFR的表达趋势及肿瘤恶性度的非相关提示其可能作为胶质瘤发生的早期影响因素.     

AG1478对U87细胞增殖、细胞周期和凋亡的影响

目的 观察表皮生长因子受体抑制剂Tyrphostin AG1478对人脑胶质瘤U87细胞增殖、细胞周期和细胞凋亡的影响.方法 噻唑蓝(MTT)比色法检测不同浓度Tyrphostin AG1478作用于体外培养的人脑胶质瘤U87细胞24 h后的细胞生存率,流式细胞仪检测不同浓度Tyrphostin AG1478作用于体外培养的人脑胶质瘤U87细胞24 h后细胞周期分布和细胞凋亡.结果 5、10、15、20 μmol/L的Tyrphostin AG1478作用人脑胶质瘤U87细胞24 h后细胞生存率分别为(7 8.93±11.95)%、(46.42±4.12)%、(42.13±7.54)%和(37.48±4.69)%;0、10、20 μmol/L的Tyrphostin AG1478作用人脑胶质瘤U87细胞24 h后细胞凋亡率分别为(10.19±3.15)%、(32.02±1.60)%和(54.35±2.80)%;0、10、20 μmol/L的Tyrphostin AG1478作用人脑胶质瘤U87细胞24 h后细胞主要分布在G0～G1期,分别为(51.20±1.21)%、(78.61±1.57)%和(82.73±0.77)%,实验组与对照组比较差异均有统计学意义(P＜0.05).结论 Tyrphostin AG1478抑制体外人脑胶质瘤细胞增殖,阻滞细胞周期在G0～G1期,诱导细胞凋亡,均呈浓度依赖性.

Abstract：

Objective To investigate the effects of Tyrphostin AG1478 on glioma U87 cells proliferation, cells cycle and apoptosis. Methods U87 cells were cultured for 24 h in the medium which contained AG1478 with different concentrations (0, 5, 10, 15, 20 μmol/L). The methyl thiazolyl tetrazolium(MTT) assay was used to detect the survival rate, and the cells cycle and apoptosis of the cells were examined by using flow cytometry. Results The cells proliferation was obviously inhibited by AG1478 in a dose-dependent manner. The survival rate of the cells in 5, 10, 15, 20 μmol/L AG1478 groups was (78.93 ±11.95)%, (46.42 ±4. 12)%, (42. 13 ±7.54)% and (37.48 ±4.69)% respectively, which was all significantly higher than that in 0 μ mol/L AG1478 group (P ＜0. 05 ). The apoptotic rate in 10, 25μmol/L AG1478 groups was (32.02 ± 1.60)% and (54. 35 ± 2. 80)% respectively, significantly higher than that in 0 μmol/L AG1478 group ( P ＜ 0. 05 ). The cells cycle was obviously inhibited by AG1478 in a dose-dependent manner. The percentage of cells treated with 10 and 20 μmol/L AG1478 in Go-G1 phase was ( 78. 61 ± 1.57 ) % and ( 82. 73 ± 0. 77 ) % respectively, significantly higher than that in 0 μmol/L AG1478 group (P ＜ 0. 05 ). Conclusion The growth inhibition of glioma U87 cells caused by AG1478 may be associated with apoptotsis induction and the G0-G1 arrest. AG1478 was expected to become a new anti-tumor drug in human glioma.     

LRIG1基因表达下调对胶质瘤细胞周期及凋亡的调控作用

目的 探讨富含亮氨酸重复序列免疫球蛋白样蛋白1(LRIG1)基因表达下调后对胶质瘤细胞的周期及凋亡的影响及其机制.方法 将携带针对LRIG1基因特异性RNA干扰序列及非特异性shRNA编码序列的质粒载体分别转染GL15细胞株,用G418(600 mg/L)筛选出稳定株后,Western blot法检测LRIG1蛋白表达的改变,PI(0.05 g/L)与Rnasine(0.5 g/L)标记后流式细胞仪检测对照组与实验组细胞的周期差异,Annexin V-FTTC/PI双标后用流式细胞仪观察两组细胞凋亡的改变.结果 成功获得干扰LRIG1基因的GL15细胞稳定株,实验组较对照组LRIG1基因表达下降54.7%.流式细胞仪分析显示,实验组的G2/M期细胞百分率较对照组明显增高(P＜0.01),实验组细胞的凋亡比例明显低于对照组(P＜0.01).讨论抑制LRIG1表达后,GL15细胞的抗凋亡能力明显增强并能使细胞阻滞在G2/M期.     

LRIG基因家族在垂体腺瘤HP75细胞系中的表达下调

目的 观察LRIG基因家族成员及EGFR基因在垂体腺瘤HP75细胞系及正常垂体组织中的表达.方法 采用实时定量逆转录-聚合酶链反应(RT-PCR)测定LRIG基因家族及EG-FR基因在垂体腺瘤HP75细胞系及正常垂体组织中的表达.RT-PCR参数为60℃5 min,95℃5min,然后45个循环95℃15s和60℃30s.结果 LRIG基因家族及EGFR基因在HP75细胞系及正常垂体组织中均有不同水平的表达.LRIG基因家族在HP75细胞系中的表达水平低于正常垂体组织,EGFR在HP75细胞系中的表达高于正常垂体组织.HP75细胞系细胞中EGFR/LRIG1比值是正常垂体组织的13倍,EGFR/LRIG2比值是正常垂体组织的14倍,EGFR/LRIG3比值是正常垂体组织的11倍.结论 LRIG基因家族在垂体腺瘤HP75细胞系中的表达下调.     

Plasmin-induced smooth muscle cell proliferation requires epidermal growth factor activation through an extracellular pathway

BACKGROUND: Plasminogen activators are used routinely for thrombolysis. They lead to the generation of the protease, plasmin, which can induce smooth muscle cell proliferation and may thus promote further intimal hyperplasia in the thrombolysed vessel. We have shown recently that plasmin induces extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated cell proliferation. Plasmin can also activate metalloproteinases on the cell surface, which can release the tethered ligand heparin-binding epidermal growth factor (HB-EGF), which can in turn activate the epidermal growth factor receptor (EGFR). METHODS: Murine aortic smooth muscle cells were cultured in vitro. Assays of DNA synthesis and cell proliferation, EGFR phosphorylation, and ERK1/2 activation were examined in response to plasmin in the presence and absence of the plasmin inhibitors (epsilon-aminocaproic acid and aprotinin), matrix metalloproteinase (MMP) inhibitor GM6001, HB-EGF inhibitor CRM197, HB-EGF inhibitory antibodies, EGF inhibitory antibodies, and the EGFR inhibitor AG1478. RESULTS: Plasmin-induced smooth muscle cell DNA synthesis, which was blocked by EGFR and HB-EGF inhibition. Plasmin-induced time-dependent EGFR phosphorylation and ERK1/2 activation, which were inhibited by AG1478. This response was dependent on the proteolytic activity of plasmin since both plasmin inhibitors blocked the response. EGFR phosphorylation by plasmin was blocked by inhibition of MMP activity and the ligand HB-EGF. EGFR phosphorylation by EGF was not interrupted by inhibition of plasmin, MMPs, or HB-EGF. Direct blockade of the EGFR prevented activation by both plasmin and EGF. CONCLUSIONS: Plasmin can induce smooth muscle cell proliferation through activation of EGFR by an extracellular MMP-mediated, HB-EGF-dependent process.     

LRIG1基因对膀胱癌细胞侵袭力的影响

目的探讨抑癌基因LRIG1对膀胱癌细胞BIU87侵袭能力的影响。方法用脂质体介导的基因转染技术,将pLRIG1-GFP质粒转染人膀胱癌细胞BIU87,用G418筛选出抗性克隆。用Boyden小室观察转染前后细胞侵袭能力的变化,并用逆转录-聚合酶链式反应（RT—PCR）法和Western—blot检测转染前后LRIG1和表皮生长因子受体（EGFR）的mRNA和蛋白表达水平。结果转染pLRIG1-GFP后的BIU87细胞侵袭能力显著降低,LRIG1的mRNA和蛋白表达水平明显升高,而EGFR的mRNA和蛋白表达水平则显著降低（P〈0．01）。结论LRIG1对膀胱癌细胞BIU87的侵袭能力有显著的抑制作用。     

Calcium-sensing receptor activation stimulates parathyroid hormone-related protein secretion in prostate cancer cells: role of epidermal growth factor receptor transactivation

We have previously reported that high extracellular Ca2+ stimulates parathyroid hormone-related protein (PTHrP) release from human prostate and breast cancer cell lines as well as from H-500 rat Leydig cancer cells, an action mediated by the calcium-sensing receptor (CaR). Activating the CaR leads to phosphorylation of mitogen-activated protein kinases (MAPKs) that participate in PTHrP synthesis and secretion. Because the CaR is a G protein-coupled receptor (GPCR), it is likely to transactivate the epidermal growth factor receptor (EGFR) or the platelet-derived growth factor receptor (PDGFR). In this study, we hypothesized that activation of the CaR transactivates the EGFR or PDGFR, and examined whether transactivation affects PTHrP secretion in PC-3 human prostate cancer cells. Using Western analysis, we observed that an increase in extracellular Ca2+ resulted in delayed activation of extracellular signal-regulated kinase (ERK) in PC-3 cells. Pre-incubation with AG1478 (an EGFR kinase inhibitor) or an EGFR neutralizing antibody inhibited the high Ca2+ -induced phosphorylation of ERK1/2. GM6001, a pan matrix metalloproteinase (MMP) inhibitor, also partially suppressed the ERK activation, but AG1296 (a PDGFR kinase inhibitor) did not. High extracellular Ca2+ stimulates PTHrP release during a 6-h incubation (1.5- to 2.5- and 3- to 4-fold increases in 3.0 and 7.5 mM Ca2+, respectively). When cells were preincubated with AG1478, GM6001, or an antihuman heparin-binding EGF (HB-EGF) antibody, PTHrP secretion was significantly inhibited under basal as well as high Ca2+ conditions, while AG1296 had no effect on PTHrP secretion. Taken together, these findings indicate that activation of the CaR transactivates the EGFR, but not the PDGFR, leading to phosphorylation of ERK1/2 and resultant PTHrP secretion, although CaR-EGFR-ERK might not be the only signaling pathway for PTHrP secretion. This transactivation is most likely mediated by activation of MMP and cleavage of proheparin-binding EGF (proHB-EGF) to HB-EGF.     

大鼠星形胶质瘤细胞系C6细胞μ受体的表达

目的 鉴定大鼠星形胶质瘤细胞系C6细胞μ受体的表达.方法 星形胶质瘤细胞系C6细胞培养、消化、传代后,接种于50 ml培养瓶中,待细胞生长至融合率达80%～90%时,采用RT-PCR法测定μ受体mRNA的表达,免疫组化SP法测定μ受体蛋白的表达.将C6细胞接种于无菌的盖玻片上,待细胞生长至融合率达40%～50%时,孵育、去脂后,随机分为5组:A组加入PBS 20μl;B组加入μ受体选择性激动剂DAMGO 1 μmol/L 20 μl;C组加入吗啡1 μmol/L20μl;D组先加入κ受体选择性抑制剂nor-Binaltorphimine 1 μmol/L 20μl,10 min后再加入吗啡1 μmol/L 20μl;E组先加入纳洛酮lμmol/L 20μl,10 min后再加入吗啡1 μmol/L 20μl.采用FV500型激光扫描共聚焦显微镜测定给予激动剂前、后细胞内游离钙离子浓度([Ca2+]j).结果 C6细胞存在μ受体mRNA,且μ受体主要分布于细胞膜及细胞浆;A组、B组和E组[Ca2+]i无变化(P>0.05),C组和D组[ca2+]i呈一过性升高(P<0.05).结论 标准条件下培养的大鼠星形胶质细胞系C6细胞膜表面可能存在μ3型受体.     

内皮抑素质粒联合阿霉素对人脑胶质肉瘤GL15细胞株增殖和凋亡的影响

目的 探讨内皮抑素(ES)以及ES和阿霉素联合应用对人脑胶质肉瘤细胞株GL15增殖和凋亡的影响.方法 通过质粒转染的方法,运用MTT法和流式细胞仪检测了ES以及ES和阿霉素联合应用对GL15细胞株增殖和凋亡的影响.结果 MTT法检测表明阿霉素对转染pcDNA3.1 ES的GL15细胞的生长抑制作用明显增强,细胞增殖明显减慢,差异有统计学意义(P<0.05);流式细胞仪检测表明,转染pcDNA3.1 ES后,G0/G1期细胞明显增加(P<0.05),S期细胞明显减少(P<0.01),凋亡率明显上升(P<0.01);联合阿霉素治疗后,上述差异更加显著(P<0.01).结论 ES联合ADM可明显抑制人脑胶质肉瘤GL15细胞株的生长,二者联用可以产生协同抗瘤作用.