TRPC通道参与U-87细胞缺氧诱导的VEGF表达的研究
摘要
研究背景
恶性多形性神经胶质细胞瘤(Glioblastoma multiforme,GBM)是最常见的恶性脑部肿瘤。GBM患者通常预后很差,是癌症相关死亡的主要原因。这一类型肿瘤呈现高度血管依赖性。通常,神经胶质瘤恶性程度越高,则血管化程度越高。许多生长因子可以调节肿瘤血管生成,包括:成纤维细胞生长因子(Fibroblast growth factors,FGF),血管内皮生长因子(Vascular endothelia growthfactors,VEGF),内皮生长因子(Endothelial growth factors,EGF)和转化生长因子(Transforming growth factors,TGF-β)等。其中,VEGF是最重要的实体瘤血管生成因子。VEGF可促进血管内皮细胞增殖,导致血管生成。缺氧是GBM的显著特征,同时也是一个已知的可诱导VEGF表达的重要因素。人们发现,GBM坏死区周围存在高度缺氧的区域(pseudopalisading region),该区细胞表达高水平的VEGF。
VEGF表达的调节是复杂的,涉及到多个不同的水平,包括:VEGF转录增加,VEGF mRNA的稳定,VEGF蛋白的分泌和扩散。在大多数细胞,缺氧诱导因子1α(Hypoxia inducible factor,HIF-1α)是调节VEGF表达的关键转录因子。缺氧情况下,HIF-1α蛋白增加且转录活性升高。它与HIF-1β形成异源二聚体,与VEGF启动子区结合,介导VEGF表达。
通常,转录因子直接调控下游靶基因表达。然而,离子通道可能在更上游的水平发挥调节基因表达的作用。嗜铬细胞瘤细胞系(PC12)是一个公认的可兴奋的氧感受细胞;缺氧可以抑制氧敏感的钾离子通道(Kv1.2),细胞因此产生去极化;Kv1.2的失活过程密切耦联于HIF-1靶基因酪氨酸羟化酶(Tyrosinehydroxylase)的表达。此外,非选择性阳离子通道(non-selective cation channels)也参与缺氧信号转导通路。严重缺氧联合葡萄糖剥夺(prolonged oxygen andglucose deprivation)激活TRPM7-ROS正反馈通路,介导钙超载事件,引起神经元坏死。中等程度缺氧(1%)条件下,Yamaji等发现非选择性阳离子通道参与缺氧诱导的内皮细胞糖酵解甘油醛-3-磷酸脱氢酶(GAPDH)表达。缺氧暴露时,非选择性阳离子通道被激活,通透钙离子导致星形胶质细胞胞内钙浓度升高,而钙是重要的基因表达的调节者。
非选择性离子通道包括很多成员:TRP通道(transient receptor potentialchannels),钙激活的非选择性通道(calcium activated non-selective channels),超极化激活的阳离子通道(hyperpolarization activated cation currents),酸感受离子通道(acid-sensitive cationic channels,ASIC)等等。其中TRP是近年来的研究热点。TRP离子通道超家族至少包括TRPC(TRP-canonical),TRPV(TRP-vanilloid)和TRPM(TRP-melastatin)亚家族。尽管以前的实验通过通道阻断剂La~(3+)或者Gd~(3+)证实非选择性阳离子通道参与细胞的缺氧反应性。但是这些通道阻断剂是非特异性的,可能对整个非选择性阳离子通道的成员都有作用(阻断或激活),因此无法确定参与缺氧反应的具体分子。本研究主要关注TRPC离子通道。TRPC通道是一个重要的TRP通道成员,可以感受多样性的外界刺激。人类细胞表达六个TRPC亚型(TRPC1-7),除了TRPC2(在人类是假基因)。通过联合使用药理学以及细胞分子生物学方法,我们确认了一个特异性的TRPC亚型,TRPC1,参与缺氧诱导的U-87细胞VEGF表达。
目的
本研究联合使用多种细胞生物学和分子生物学方法:MTT,hoechst染色,real-time RT-PCR,western blot,免疫荧光技术,RNA干扰,过表达,钙浓度测定方法等,探讨TRPC在缺氧诱导的U-87细胞VEGF表达中的作用。
方法
1.通过MTT、Hoechst、western blot和real-time RT-PCR方法,分析低氧(1%O_2)不同时间点细胞凋亡、增殖、缺氧诱导因子1α(HIF-1α)以及VEGF表达情况,以建立神经胶质细胞瘤细胞系U-87体外缺氧诱导VEGF表达的模型。
2.通过real-time RT-PCR,观察TRPC通道阻断剂SKF96365对缺氧诱导的VEGF表达的影响,以确定TRPC通道是否参与缺氧诱导的VEGF表达。
3.采用RT-PCR,检测常氧情况下不同TRPC通道亚型在U-87细胞上的表达情况。
4.使用激光共聚焦显微镜,观察加入TRPC通道激动剂OAG以及阻断剂2-APB时细胞内Ca~(2+)的动态变化,以确定U-87细胞上的TRPC通道是否是功能性的。
5.使用real-time RT-PCR,观察并分析缺氧后TRPC通道各亚型的表达情况,以确定下一步需要使用的分子生物学策略:过表达或RNA干扰。
6.通过RNAi下调TRPC1表达。分别用real-time RT-PCR和ELISA检测VEGFmRNA和蛋白水平的变化,观察下调TRPC1对缺氧诱导的VEGF上调的影响。
7.通过瞬时转染野生型TRPC3或TRPC5质粒增加TRPC3或TRPC5表达。观察TRPC3和TRPC5对缺氧诱导的VEGF表达的影响。
8.通过免疫荧光技术,使用激光共聚焦显微镜观察缺氧是否引起TRPC1胞内转位变化。
结果
1.Hoechst结果显示:缺氧(1%O_2)24h或48h不导致细胞凋亡;缺氧24h仅轻微减少了细胞增殖(下降3.23%),而缺氧48h细胞出现明显的增殖抑制(减少22.35%,P＜0.001);缺氧(1%O_2)诱导HIF-1α增加;缺氧10h显著诱导VEGF表达(升高4.24倍,P＜0.01)。
2.TRPC通道阻断剂SKF96365可明显抑制缺氧U-87细胞VEGF表达。25μMSKF96365抑制了VEGF mRNA水平的上调(P＜0.01),且增加SKF96365的浓度进一步抑制VEGF表达。
3.常氧条件下,PCR产物经琼脂糖凝胶电泳分析,检测到四个单一条带,大小为168bp,112bp,119bp,159bp(与预期的产物大小一致),分别对应于TRPC1,TRPC3,TRPC4和TRPC5亚型。
4.TRPC通道激动剂OAG可增加胞内Ca~(2+)浓度,而TRPC通道阻断剂2-APB可抑制激动剂诱导的胞内Ca~(2+)浓度增加。
5.缺氧后,U-87细胞TRPC3和TRPC4亚型表达分别减少67.7%,68.2%(P＜0.05)。此外,高敏感的荧光定量PCR无法检测到缺氧U-87细胞TRPC5亚型转录本。另一方面,常氧和缺氧胶质瘤细胞TRPC1的表达保持稳定,并不发生明显减少。
6.化学合成的siRNA显示出高度的细胞传输效率(超过90%);三个TRPC1特异性的siRNAs:siRNA-1,siRNA-2和siRNA-3分别减少TRPC1 mRNA到64%,39%和36%(P＜0.01);分别减少TRPC1蛋白到阴性对照组的48%,29%和33%(P＜0.01)。siRNA-2和siRNA-3显著抑制了缺氧诱导的VEGF基因上调(P＜0.01)。同时培养基中分泌的VEGF也发生类似的变化(P＜0.01)。
7.通过瞬时过表达TRPC3或TRPC5增加了缺氧情况下U-87细胞TRPC3或TRPC5的表达水平。但过表达这些TRPC野生型质粒DNA既不促进缺氧U-87细胞VEGF表达,也不减弱VEGF表达(P＞0.05)。
8.常氧条件下,TRPC1通道分布于胞膜及胞浆。然而缺氧后,胞浆区TRPC1荧光明显减少,而细胞膜出现强烈的荧光信号。
结论
1.1%O_2是人体内实体肿瘤低氧环境的氧气浓度。体外的缺氧实验证实该氧气浓度作用24h不能诱导显著的细胞凋亡且仅轻微抑制细胞增殖,但延长缺氧暴露时间至48h可导致细胞增殖抑制。说明1%O_2作用小于24h可能是合适的体外诱导U-87细胞VEGF表达的模型。进一步,缺氧(1%O_2)可增加HIF-1α蛋白水平,并显著诱导VEGF上调。
2.TRPC通道阻断剂可抑制缺氧U-87细胞VEGF表达,说明TRPC通道可能参与缺氧诱导的VEGF表达。
3.常氧条件下,U-87细胞共表达四个TRPC通道亚型,分别为:TRPC1,TRPC3,TRPC4和TRPC5。
4.对TRPC通道激动剂和阻断剂的反应性,说明U-87细胞上TRPC通道是功能性的。
5.缺氧后TRPC3,4,5亚型表达量显著减少,而TRPC1表达保持不变。说明相对于其他TRPC亚型,TRPC1可能在胶质瘤细胞缺氧信号通路中扮演不同的角色。并提示下一步针对缺氧抑制表达的TRPC亚型,可采用过表达方法;而针对TRPC1,则采用RNA干扰技术。
6.通过RNAi下调TRPC1表达可抑制缺氧诱导的VEGF基因表达,此外,分泌型VEGF也受到抑制。说明TRPC1参与缺氧诱导的VEGF表达。
7.过表达可显著增加TRPC3或TRPC5亚型表达。但过表达这些TRPC野生型质粒DNA既不促进缺氧U-87细胞VEGF表达,也不减弱VEGF表达。说明TRPC3或TRPC5不参与缺氧诱导的VEGF上调过程。
8.缺氧诱导TRPC1细胞膜转位,说明缺氧条件下,TRPC1可能通过转位而被激活,参与缺氧诱导的VEGF表达。
Background
Glioblastoma multiforme(GBM) is the most common malignant brain tumor. The patients of GBM usually have a bad prognosis,representing a leading cause of cancer-related death.Usually,the growth of these tumors is highly angiogenesis-dependent,while higher grade malignant astrocytoma show higher degree of vascularity.Many growth factors can regulate angiogenesis including: Fibroblast growth factors(FGF),Vascular endothelia growth factors(VEGF), Endothelial growth factors(EGF) and Transforming growth factors(TGF-β).Of these factors,VEGF is a most potent angiogenic factor implicated in solid tumor angiogenesis.VEGF can promote vascular endothelial cell(VEC) proliferation, leading to Angiogenesis.Hypoxia is an important characteristic of glioblastomas, and a well-known stimulus for inducing VEGF expression.It has been found that in GBM,pseudopalisading cells in areas adjacent to areas of necrosis are severely hypoxic and express high levels of VEGF.
Regulation of VEGF is complex and occurs at many different levels,including induction of VEGF transcription,stabilization of VEGF mRNA,and secretion and diffusion of mature protein.In most cells,Hypoxia inducible factor(HIF-1α) is the key transcription factor accounting for hypoxia-induced VEGF expression.Under hypoxic condition,protein levels and transcription activity of HIF-1αincrease. HIF-1αtranslocates into cell nucleus due to hypoxia and form heterodimer with HIF-1β.Then,HIF-1 binds to a specific consensus sequence,5'-RCGTG-3',which is found within the HRE in the VEGF promoter and induces VEGF gene expression.
Usually,transcription factors directly regulate expression of downstream target gene.Meanwhile,ion channels also play an important role in regulating gene expression.Pheochromocytoma(PC12),an generally accepted excitable oxygen-sensing cell line,depolarized during hypoxia as the result of inhibition of hypoxia sensitive potassium ehannel(Kvl.2),which coupled to expression of HIF-1 target gene for tyrosine hydroxylase,in addition,non-selective cation channels are also found to be involved in hypoxia signal pathway.Severe hypoxia combined with glucose deprivation caused neuronal death by Ca2+ overload via TRPM7-ROS positive feedback pathway.Under moderate hypoxia(1%O2),non-selective cation channels have been reported to participate in the regulation of gene expression in endothelial cells.Ca2+ entry via non-selective cation channels raised[Ca~(2+)]_i in astrocytes exposed to hypoxia,while Ca~(2+) was an important regulator for gene expression.
Non-selective cation channels were formed by transient receptor potential(TRP) channels,calcium activated non-selective channels,hyperpolarization activated cation currents,acid-sensitive cationic channels(ASIC),etc.Recently,TRP channels become hot spot in this research filed.TRP channels have at least three subfamilies: TRPC(TRP-canonical),TRPV(TRP-vanilloid) and TRPM(TRP-melastatin). Although previous reports showed that non-selective cation channels participate in hypoxic response using channel blockers such as La~(3+) or Gd~(3+),these blockers are not specific and can not be used to identify specific member molecule.We focus on TRPC channels which can sense diverse stimulus in local environments.Human express six isoforms(TRPC1-7) except TRPC2(a pseudogene for human).By combined use of pharmacological and molecular biology methods,we identify that a specific molecule of TRPC channels,TRPC1,is involved in hypoxia-induced VEGF expression in U-87 MG cells.
Objective
By combined use of several cell biology and molecular biology methods:MTT, hoechst staining,real-time RT-PCR,western blot,immunofluorescence technique, RNA interference,overexpression,calcium concentration assay,etc,Effect of TRPC channels on hypoxia-induced VEGF expression in U-87 MG cells is investigated.
Methods
1.To establish a model of hypoxia-induced VEGF expression in vitro in U-87 MG cells,cell apoptosis,proliferation,expression of HIF-1αand VEGF was examined in cells treated with hypoxia(1%O2) for different time.Cell apoptosis,proliferation, expression of HIF-1αand VEGF was examined using Hoechst,MTT,western blot and real-time RT-PCR.
2.U-87 MG cells were exposed to normoxia or hypoxia for 10h in the presence of 0, 25 or 40μM SKF96365.Total RNA was extracted and real-time RT-PCR was performed to determine whether TRPC channels participate in VEGF up-regulation in hypoxic U-87 MG cells.
3.Total RNA was isolated from U-87 MG cells in normoxia.The cDNAs were amplified for 35 cycles and PCR products(5μl) were subjected to 2%agarose gel electrophoresis to analyze expression profile of TRPC in normoxic U-87 cells.
4.Confocal microscopy was used to observe changes of calcium concentration when agonist or antagonist of TRPC channels were added.
5.U-87 MG cells were incubated in normoxia or hypoxia for 16h.Then,TRPCs mRNA levels were determined by real-time RT-PCR.
6.TRPC1 was down-regulated by RNA interference.Real-time RT-PCR and ELISA assay were used to determine whether knockdown of TRPC1 affected VEGF expression in mRNA level and protein level induced by hypoxia.
7.TRPC3 and TRPC5 were overexpressed by transiently transfecting corresponding plasmids.After overexpression,real-time RT-PCR was used to evaluate effects of TRPC3 and TRPC5 on expression of VEGF.
8.By immunofluorescence technique,location of TRPC1 was observed after hypoxic exposure in U-87 MG cells.
Results
1.Results from Hoechst staining show that hypoxia(1%O2) for 24h or 48h do not cause cell apoptosis;Hypoxia for 24h slightly inhibits cell proliferation(decrease by 3.23%) and for 48h inhibits cell proliferation by 22.35%(P<0.001).Hypoxia(1%O2) for 2h and 4h increase expression of HIF-1α.Also,VEGF was substantially induced by hypoxia for 10h(P<0.01).
2.The up-regulation of VEGF mRNA during hypoxia was inhibited by 25μM SKF96365(P<0.01) and further suppressed by a increased dose of SKF96365.
3.Agarose gel electrophoresis of the product from RT-PCR detected four single bands of 168bp,112bp,119bp,159bp as expected size,corresponding to TRPC1, TRPC3,TRPC4,TRPC5 in normoxic U-87 MG cells.
4.OAG(agonist of TRPC) can increase calcium concentration of U-87 cells,while 2-APB(antagonist of TRPC) can attenuate OAG-induced calcium concentration rise.
5.Under hypoxia stress,TRPC3 and TRPC4 expression decreased by 67.7%,68.2% (P<0.05),respectively.The mRNA of TRPC5 can not be detected after hypoxia; On the other hand,No change was found in TRPC1 mRNA level between normoxia and hypoxia.
6.siRNAs delivery efficiency was over 90%.Real-time RT-PCR showed the mRNA of TRPC1 in U-87 cells transfected with three different siRNAs against TRPC1,si-1, si-2 and si-3,were reduced to 63%,39%and 36%,respectively,of that found in cultures transfected with the NC siRNA(P<0.01).Furthermore,si-1,si-2 and si-3 reduced the amount of TRPC1 protein to 48%,29%and 33%,respectively.Hypoxia enhanced VEGF gene expression in NC siRNA-transfected U-87 cells.However, transfection with either si-2 or si-3 greatly inhibited up-regulation of VEGF gene expression by hypoxia(P<0.01).Moreover,the protein levels of secreted VEGF in medium were in accordance with mRNA results.
7.Transient transfection of TRPC3 or TRPC5 increase expression of these genes under hypoxic conditions.However,overexpression of TRPC3 or TRPC5 neither promote VEGF expression in hypoxic U-87 MG cells,nor prevent VEGF expression (P>0.05).
8.Under normoxic conditions,TRPC1 protein located in cytoplasm and cell membrane.After hypoxia,TRPC1 mainly located in cell membrane,rather than cytoplasm.
Conclusion
1.Hypoxic conditions(1%O2) exists in microenvironment of most solid tumors. Experiments in vitro demonstrate that hypoxic conditions(1%O2) can induce expression of HIF-1αand VEGF.
2.TRPC channels participate in hypoxia-induced VEGF expression in U-87 MG cells.
3.Under normoxic conditions,U-87 MG cells express four TRPC isoforms:TRPC1, TRPC3,TRPC4 and TRPC5
4.Effective response of TRPC channels to agonist and antagonist demonstrates TRPC in U-87 MG cells is functional channels.
5.Hypoxia has different effects on TRPCs gene expression,and imply TRPC1 and other TRPCs might play different roles in hypoxia signal pathway.
6.Knockdown of TRPC1 largely suppressed hypoxia-induced VEGF gene expression.Moreover,secreted VEGF protein was also inhibited.These data suggest TRPC1 is critical for hypoxia-induced VEGF expression and therefore it's expression is stable during hypoxia.
7.Overexpression of TRPC3 or TRPC5 neither promote VEGF expression in hypoxic U-87 MG cells,nor prevent VEGF expression.These results suggest that these TRPCs do not contribute to VEGF up-regulation induced by hypoxia in U-87 MG cells
8.Hypoxia can induce translocation of TRPC1 into cell membrane,which imply TRPC1 was activated by translocation and play a role in VEGF expression induced by hypoxia.
恶性多形性神经胶质细胞瘤(Glioblastoma multiforme,GBM)是最常见的恶性脑部肿瘤。GBM患者通常预后很差,是癌症相关死亡的主要原因。这一类型肿瘤呈现高度血管依赖性。通常,神经胶质瘤恶性程度越高,则血管化程度越高。许多生长因子可以调节肿瘤血管生成,包括:成纤维细胞生长因子(Fibroblast growth factors,FGF),血管内皮生长因子(Vascular endothelia growthfactors,VEGF),内皮生长因子(Endothelial growth factors,EGF)和转化生长因子(Transforming growth factors,TGF-β)等。其中,VEGF是最重要的实体瘤血管生成因子。VEGF可促进血管内皮细胞增殖,导致血管生成。缺氧是GBM的显著特征,同时也是一个已知的可诱导VEGF表达的重要因素。人们发现,GBM坏死区周围存在高度缺氧的区域(pseudopalisading region),该区细胞表达高水平的VEGF。
VEGF表达的调节是复杂的,涉及到多个不同的水平,包括:VEGF转录增加,VEGF mRNA的稳定,VEGF蛋白的分泌和扩散。在大多数细胞,缺氧诱导因子1α(Hypoxia inducible factor,HIF-1α)是调节VEGF表达的关键转录因子。缺氧情况下,HIF-1α蛋白增加且转录活性升高。它与HIF-1β形成异源二聚体,与VEGF启动子区结合,介导VEGF表达。
通常,转录因子直接调控下游靶基因表达。然而,离子通道可能在更上游的水平发挥调节基因表达的作用。嗜铬细胞瘤细胞系(PC12)是一个公认的可兴奋的氧感受细胞;缺氧可以抑制氧敏感的钾离子通道(Kv1.2),细胞因此产生去极化;Kv1.2的失活过程密切耦联于HIF-1靶基因酪氨酸羟化酶(Tyrosinehydroxylase)的表达。此外,非选择性阳离子通道(non-selective cation channels)也参与缺氧信号转导通路。严重缺氧联合葡萄糖剥夺(prolonged oxygen andglucose deprivation)激活TRPM7-ROS正反馈通路,介导钙超载事件,引起神经元坏死。中等程度缺氧(1%)条件下,Yamaji等发现非选择性阳离子通道参与缺氧诱导的内皮细胞糖酵解甘油醛-3-磷酸脱氢酶(GAPDH)表达。缺氧暴露时,非选择性阳离子通道被激活,通透钙离子导致星形胶质细胞胞内钙浓度升高,而钙是重要的基因表达的调节者。
非选择性离子通道包括很多成员:TRP通道(transient receptor potentialchannels),钙激活的非选择性通道(calcium activated non-selective channels),超极化激活的阳离子通道(hyperpolarization activated cation currents),酸感受离子通道(acid-sensitive cationic channels,ASIC)等等。其中TRP是近年来的研究热点。TRP离子通道超家族至少包括TRPC(TRP-canonical),TRPV(TRP-vanilloid)和TRPM(TRP-melastatin)亚家族。尽管以前的实验通过通道阻断剂La~(3+)或者Gd~(3+)证实非选择性阳离子通道参与细胞的缺氧反应性。但是这些通道阻断剂是非特异性的,可能对整个非选择性阳离子通道的成员都有作用(阻断或激活),因此无法确定参与缺氧反应的具体分子。本研究主要关注TRPC离子通道。TRPC通道是一个重要的TRP通道成员,可以感受多样性的外界刺激。人类细胞表达六个TRPC亚型(TRPC1-7),除了TRPC2(在人类是假基因)。通过联合使用药理学以及细胞分子生物学方法,我们确认了一个特异性的TRPC亚型,TRPC1,参与缺氧诱导的U-87细胞VEGF表达。
目的
本研究联合使用多种细胞生物学和分子生物学方法:MTT,hoechst染色,real-time RT-PCR,western blot,免疫荧光技术,RNA干扰,过表达,钙浓度测定方法等,探讨TRPC在缺氧诱导的U-87细胞VEGF表达中的作用。
方法
1.通过MTT、Hoechst、western blot和real-time RT-PCR方法,分析低氧(1%O_2)不同时间点细胞凋亡、增殖、缺氧诱导因子1α(HIF-1α)以及VEGF表达情况,以建立神经胶质细胞瘤细胞系U-87体外缺氧诱导VEGF表达的模型。
2.通过real-time RT-PCR,观察TRPC通道阻断剂SKF96365对缺氧诱导的VEGF表达的影响,以确定TRPC通道是否参与缺氧诱导的VEGF表达。
3.采用RT-PCR,检测常氧情况下不同TRPC通道亚型在U-87细胞上的表达情况。
4.使用激光共聚焦显微镜,观察加入TRPC通道激动剂OAG以及阻断剂2-APB时细胞内Ca~(2+)的动态变化,以确定U-87细胞上的TRPC通道是否是功能性的。
5.使用real-time RT-PCR,观察并分析缺氧后TRPC通道各亚型的表达情况,以确定下一步需要使用的分子生物学策略:过表达或RNA干扰。
6.通过RNAi下调TRPC1表达。分别用real-time RT-PCR和ELISA检测VEGFmRNA和蛋白水平的变化,观察下调TRPC1对缺氧诱导的VEGF上调的影响。
7.通过瞬时转染野生型TRPC3或TRPC5质粒增加TRPC3或TRPC5表达。观察TRPC3和TRPC5对缺氧诱导的VEGF表达的影响。
8.通过免疫荧光技术,使用激光共聚焦显微镜观察缺氧是否引起TRPC1胞内转位变化。
结果
1.Hoechst结果显示:缺氧(1%O_2)24h或48h不导致细胞凋亡;缺氧24h仅轻微减少了细胞增殖(下降3.23%),而缺氧48h细胞出现明显的增殖抑制(减少22.35%,P＜0.001);缺氧(1%O_2)诱导HIF-1α增加;缺氧10h显著诱导VEGF表达(升高4.24倍,P＜0.01)。
2.TRPC通道阻断剂SKF96365可明显抑制缺氧U-87细胞VEGF表达。25μMSKF96365抑制了VEGF mRNA水平的上调(P＜0.01),且增加SKF96365的浓度进一步抑制VEGF表达。
3.常氧条件下,PCR产物经琼脂糖凝胶电泳分析,检测到四个单一条带,大小为168bp,112bp,119bp,159bp(与预期的产物大小一致),分别对应于TRPC1,TRPC3,TRPC4和TRPC5亚型。
4.TRPC通道激动剂OAG可增加胞内Ca~(2+)浓度,而TRPC通道阻断剂2-APB可抑制激动剂诱导的胞内Ca~(2+)浓度增加。
5.缺氧后,U-87细胞TRPC3和TRPC4亚型表达分别减少67.7%,68.2%(P＜0.05)。此外,高敏感的荧光定量PCR无法检测到缺氧U-87细胞TRPC5亚型转录本。另一方面,常氧和缺氧胶质瘤细胞TRPC1的表达保持稳定,并不发生明显减少。
6.化学合成的siRNA显示出高度的细胞传输效率(超过90%);三个TRPC1特异性的siRNAs:siRNA-1,siRNA-2和siRNA-3分别减少TRPC1 mRNA到64%,39%和36%(P＜0.01);分别减少TRPC1蛋白到阴性对照组的48%,29%和33%(P＜0.01)。siRNA-2和siRNA-3显著抑制了缺氧诱导的VEGF基因上调(P＜0.01)。同时培养基中分泌的VEGF也发生类似的变化(P＜0.01)。
7.通过瞬时过表达TRPC3或TRPC5增加了缺氧情况下U-87细胞TRPC3或TRPC5的表达水平。但过表达这些TRPC野生型质粒DNA既不促进缺氧U-87细胞VEGF表达,也不减弱VEGF表达(P＞0.05)。
8.常氧条件下,TRPC1通道分布于胞膜及胞浆。然而缺氧后,胞浆区TRPC1荧光明显减少,而细胞膜出现强烈的荧光信号。
结论
1.1%O_2是人体内实体肿瘤低氧环境的氧气浓度。体外的缺氧实验证实该氧气浓度作用24h不能诱导显著的细胞凋亡且仅轻微抑制细胞增殖,但延长缺氧暴露时间至48h可导致细胞增殖抑制。说明1%O_2作用小于24h可能是合适的体外诱导U-87细胞VEGF表达的模型。进一步,缺氧(1%O_2)可增加HIF-1α蛋白水平,并显著诱导VEGF上调。
2.TRPC通道阻断剂可抑制缺氧U-87细胞VEGF表达,说明TRPC通道可能参与缺氧诱导的VEGF表达。
3.常氧条件下,U-87细胞共表达四个TRPC通道亚型,分别为:TRPC1,TRPC3,TRPC4和TRPC5。
4.对TRPC通道激动剂和阻断剂的反应性,说明U-87细胞上TRPC通道是功能性的。
5.缺氧后TRPC3,4,5亚型表达量显著减少,而TRPC1表达保持不变。说明相对于其他TRPC亚型,TRPC1可能在胶质瘤细胞缺氧信号通路中扮演不同的角色。并提示下一步针对缺氧抑制表达的TRPC亚型,可采用过表达方法;而针对TRPC1,则采用RNA干扰技术。
6.通过RNAi下调TRPC1表达可抑制缺氧诱导的VEGF基因表达,此外,分泌型VEGF也受到抑制。说明TRPC1参与缺氧诱导的VEGF表达。
7.过表达可显著增加TRPC3或TRPC5亚型表达。但过表达这些TRPC野生型质粒DNA既不促进缺氧U-87细胞VEGF表达,也不减弱VEGF表达。说明TRPC3或TRPC5不参与缺氧诱导的VEGF上调过程。
8.缺氧诱导TRPC1细胞膜转位,说明缺氧条件下,TRPC1可能通过转位而被激活,参与缺氧诱导的VEGF表达。
Background
Glioblastoma multiforme(GBM) is the most common malignant brain tumor. The patients of GBM usually have a bad prognosis,representing a leading cause of cancer-related death.Usually,the growth of these tumors is highly angiogenesis-dependent,while higher grade malignant astrocytoma show higher degree of vascularity.Many growth factors can regulate angiogenesis including: Fibroblast growth factors(FGF),Vascular endothelia growth factors(VEGF), Endothelial growth factors(EGF) and Transforming growth factors(TGF-β).Of these factors,VEGF is a most potent angiogenic factor implicated in solid tumor angiogenesis.VEGF can promote vascular endothelial cell(VEC) proliferation, leading to Angiogenesis.Hypoxia is an important characteristic of glioblastomas, and a well-known stimulus for inducing VEGF expression.It has been found that in GBM,pseudopalisading cells in areas adjacent to areas of necrosis are severely hypoxic and express high levels of VEGF.
Regulation of VEGF is complex and occurs at many different levels,including induction of VEGF transcription,stabilization of VEGF mRNA,and secretion and diffusion of mature protein.In most cells,Hypoxia inducible factor(HIF-1α) is the key transcription factor accounting for hypoxia-induced VEGF expression.Under hypoxic condition,protein levels and transcription activity of HIF-1αincrease. HIF-1αtranslocates into cell nucleus due to hypoxia and form heterodimer with HIF-1β.Then,HIF-1 binds to a specific consensus sequence,5'-RCGTG-3',which is found within the HRE in the VEGF promoter and induces VEGF gene expression.
Usually,transcription factors directly regulate expression of downstream target gene.Meanwhile,ion channels also play an important role in regulating gene expression.Pheochromocytoma(PC12),an generally accepted excitable oxygen-sensing cell line,depolarized during hypoxia as the result of inhibition of hypoxia sensitive potassium ehannel(Kvl.2),which coupled to expression of HIF-1 target gene for tyrosine hydroxylase,in addition,non-selective cation channels are also found to be involved in hypoxia signal pathway.Severe hypoxia combined with glucose deprivation caused neuronal death by Ca2+ overload via TRPM7-ROS positive feedback pathway.Under moderate hypoxia(1%O2),non-selective cation channels have been reported to participate in the regulation of gene expression in endothelial cells.Ca2+ entry via non-selective cation channels raised[Ca~(2+)]_i in astrocytes exposed to hypoxia,while Ca~(2+) was an important regulator for gene expression.
Non-selective cation channels were formed by transient receptor potential(TRP) channels,calcium activated non-selective channels,hyperpolarization activated cation currents,acid-sensitive cationic channels(ASIC),etc.Recently,TRP channels become hot spot in this research filed.TRP channels have at least three subfamilies: TRPC(TRP-canonical),TRPV(TRP-vanilloid) and TRPM(TRP-melastatin). Although previous reports showed that non-selective cation channels participate in hypoxic response using channel blockers such as La~(3+) or Gd~(3+),these blockers are not specific and can not be used to identify specific member molecule.We focus on TRPC channels which can sense diverse stimulus in local environments.Human express six isoforms(TRPC1-7) except TRPC2(a pseudogene for human).By combined use of pharmacological and molecular biology methods,we identify that a specific molecule of TRPC channels,TRPC1,is involved in hypoxia-induced VEGF expression in U-87 MG cells.
Objective
By combined use of several cell biology and molecular biology methods:MTT, hoechst staining,real-time RT-PCR,western blot,immunofluorescence technique, RNA interference,overexpression,calcium concentration assay,etc,Effect of TRPC channels on hypoxia-induced VEGF expression in U-87 MG cells is investigated.
Methods
1.To establish a model of hypoxia-induced VEGF expression in vitro in U-87 MG cells,cell apoptosis,proliferation,expression of HIF-1αand VEGF was examined in cells treated with hypoxia(1%O2) for different time.Cell apoptosis,proliferation, expression of HIF-1αand VEGF was examined using Hoechst,MTT,western blot and real-time RT-PCR.
2.U-87 MG cells were exposed to normoxia or hypoxia for 10h in the presence of 0, 25 or 40μM SKF96365.Total RNA was extracted and real-time RT-PCR was performed to determine whether TRPC channels participate in VEGF up-regulation in hypoxic U-87 MG cells.
3.Total RNA was isolated from U-87 MG cells in normoxia.The cDNAs were amplified for 35 cycles and PCR products(5μl) were subjected to 2%agarose gel electrophoresis to analyze expression profile of TRPC in normoxic U-87 cells.
4.Confocal microscopy was used to observe changes of calcium concentration when agonist or antagonist of TRPC channels were added.
5.U-87 MG cells were incubated in normoxia or hypoxia for 16h.Then,TRPCs mRNA levels were determined by real-time RT-PCR.
6.TRPC1 was down-regulated by RNA interference.Real-time RT-PCR and ELISA assay were used to determine whether knockdown of TRPC1 affected VEGF expression in mRNA level and protein level induced by hypoxia.
7.TRPC3 and TRPC5 were overexpressed by transiently transfecting corresponding plasmids.After overexpression,real-time RT-PCR was used to evaluate effects of TRPC3 and TRPC5 on expression of VEGF.
8.By immunofluorescence technique,location of TRPC1 was observed after hypoxic exposure in U-87 MG cells.
Results
1.Results from Hoechst staining show that hypoxia(1%O2) for 24h or 48h do not cause cell apoptosis;Hypoxia for 24h slightly inhibits cell proliferation(decrease by 3.23%) and for 48h inhibits cell proliferation by 22.35%(P<0.001).Hypoxia(1%O2) for 2h and 4h increase expression of HIF-1α.Also,VEGF was substantially induced by hypoxia for 10h(P<0.01).
2.The up-regulation of VEGF mRNA during hypoxia was inhibited by 25μM SKF96365(P<0.01) and further suppressed by a increased dose of SKF96365.
3.Agarose gel electrophoresis of the product from RT-PCR detected four single bands of 168bp,112bp,119bp,159bp as expected size,corresponding to TRPC1, TRPC3,TRPC4,TRPC5 in normoxic U-87 MG cells.
4.OAG(agonist of TRPC) can increase calcium concentration of U-87 cells,while 2-APB(antagonist of TRPC) can attenuate OAG-induced calcium concentration rise.
5.Under hypoxia stress,TRPC3 and TRPC4 expression decreased by 67.7%,68.2% (P<0.05),respectively.The mRNA of TRPC5 can not be detected after hypoxia; On the other hand,No change was found in TRPC1 mRNA level between normoxia and hypoxia.
6.siRNAs delivery efficiency was over 90%.Real-time RT-PCR showed the mRNA of TRPC1 in U-87 cells transfected with three different siRNAs against TRPC1,si-1, si-2 and si-3,were reduced to 63%,39%and 36%,respectively,of that found in cultures transfected with the NC siRNA(P<0.01).Furthermore,si-1,si-2 and si-3 reduced the amount of TRPC1 protein to 48%,29%and 33%,respectively.Hypoxia enhanced VEGF gene expression in NC siRNA-transfected U-87 cells.However, transfection with either si-2 or si-3 greatly inhibited up-regulation of VEGF gene expression by hypoxia(P<0.01).Moreover,the protein levels of secreted VEGF in medium were in accordance with mRNA results.
7.Transient transfection of TRPC3 or TRPC5 increase expression of these genes under hypoxic conditions.However,overexpression of TRPC3 or TRPC5 neither promote VEGF expression in hypoxic U-87 MG cells,nor prevent VEGF expression (P>0.05).
8.Under normoxic conditions,TRPC1 protein located in cytoplasm and cell membrane.After hypoxia,TRPC1 mainly located in cell membrane,rather than cytoplasm.
Conclusion
1.Hypoxic conditions(1%O2) exists in microenvironment of most solid tumors. Experiments in vitro demonstrate that hypoxic conditions(1%O2) can induce expression of HIF-1αand VEGF.
2.TRPC channels participate in hypoxia-induced VEGF expression in U-87 MG cells.
3.Under normoxic conditions,U-87 MG cells express four TRPC isoforms:TRPC1, TRPC3,TRPC4 and TRPC5
4.Effective response of TRPC channels to agonist and antagonist demonstrates TRPC in U-87 MG cells is functional channels.
5.Hypoxia has different effects on TRPCs gene expression,and imply TRPC1 and other TRPCs might play different roles in hypoxia signal pathway.
6.Knockdown of TRPC1 largely suppressed hypoxia-induced VEGF gene expression.Moreover,secreted VEGF protein was also inhibited.These data suggest TRPC1 is critical for hypoxia-induced VEGF expression and therefore it's expression is stable during hypoxia.
7.Overexpression of TRPC3 or TRPC5 neither promote VEGF expression in hypoxic U-87 MG cells,nor prevent VEGF expression.These results suggest that these TRPCs do not contribute to VEGF up-regulation induced by hypoxia in U-87 MG cells
8.Hypoxia can induce translocation of TRPC1 into cell membrane,which imply TRPC1 was activated by translocation and play a role in VEGF expression induced by hypoxia.
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