乏氧诱导因子-1α与食管鳞癌血管生成和化疗疗效的关系及其机制
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摘要
近年来研究发现乏氧是实体瘤中一种常见现象，是实体肿瘤微环境的基本特征之一。乏氧作为一种应激源，能激活肿瘤细胞内的一系列基因对乏氧作出应答反应，使肿瘤细胞内很多基因转录活性发生改变，其基因产物引起乏氧肿瘤细胞的一系列生物学行为改变。研究表明，在乏氧环境中为了保持能量供应，肿瘤细胞促血管生成能力极大提高，使肿瘤细胞更具有侵袭性，容易发生远处转移。而且乏氧会导致肿瘤细胞对放化疗的抗拒性增加，从而导致治疗失败。而在此过程中，乏氧诱导因子1(hypoxia inducible factor-1，HIF-1)起关键作用。但这种复杂的机制目前尚不清楚。因此HIF-1作为乏氧环境中起关键作用的转录因子，已成为近年来的研究热点。
HIF-1是存在于哺乳动物和人体内一种转录因子，它是一个属于bHLH-PAS蛋白族的转录因子，由HIF-1α和HIF-1β两个亚单位组成。HIF-1的稳定和活性是由HIF-1α决定的，且HIF-1α是专一受O_2调节的亚基。由于在乏氧诱导肿瘤基因表达的信息通路上，HIF-1α起着中枢纽带作用。因此，研究HIF-1α与肿瘤生长、浸润、转移和治疗方面的关系有重要的意义。
食管癌作为我国高发肿瘤，是否也存在乏氧耐受的分子机制目前尚不十分清楚。关于HIF-1α在食管癌中的表达研究较少，其与食管癌血管生成及化疗耐药的关系目前未见系统的研究报道。特别是其是否参与食管癌化疗耐药及其机制的研究，目前未见报道。
为了探讨HIF-1α在食管癌组织中的表达及HIF-1α与食管癌血管生成的关系，本文采用半定量聚合酶链反应技术及免疫组化技术检测食管鳞癌组织中HIF-1α在mRNA水平及蛋白水平的表达，采用免疫组化技术检测食管鳞癌组织中VEGF、CD34的表达，观察了HIF-1α在食管癌中的表达情况及其与临床病理特征、VEGF和微血管密度的关系。为了探讨HIF-1α是否通过上调VEGF的表达而促进食管癌血管生成的机制，本文以食管鳞癌细胞株EC9706为研究对象，通过化学性乏氧诱导剂氯化钴(cobaltchloride，COCl_2)建立乏氧模型，体外模拟肿瘤乏氧微环境。采用半定量RT-PCR、Western-blot、免疫细胞化学技术检测乏氧诱导的HIF-1α、VEGF表达情况。进一步利用RNA干扰技术(RNA intefercnce，RNAi)观察小干扰RNA(small interference RNA，siRNA)转染EC9706细胞后乏氧诱导的HIF-1α和VEGF的表达的改变，初步探讨了HIF-1α在乏氧条件下对食管癌血管生成的调控作用及其机制以及以HIF-1α为靶点抗食管癌血管生成治疗的可能性。
为探讨HIF-1α是否与食管癌化疗耐药有关及其参与耐药的机制，采用免疫组化技术检测食管鳞癌组织中HIF-1α的表达，并与临床化疗疗效相结合，探讨HIF-1α与食管癌化疗疗效的关系。从体外方面，以EC9706为研究对象，通过化学性乏氧诱导剂建立乏氧模型，采用MTS、半定量RT-PCR、免疫细胞化学、RNA干扰技术进一步从多药耐药、凋亡、细胞周期等方面，探讨了HIF-1α参与食管癌细胞乏氧条件下对不同机制的化疗药物产生耐药的机制。并进一步体外探讨以HIF-1α为新的靶点，应用RNA干扰技术逆转食管癌耐药的新途径。为以此为突破口进一步提高食管癌的化疗水平提供理论依据。
第一部分乏氧诱导因子-1α在食管鳞癌组织中的表达及乏氧调节
方法
1．应用半定量RT-PCR及免疫组化技术对46例食管鳞癌组织及20例正常食管黏膜组织进行HIF-1αmRNA及蛋白水平的检测。
2．应用化学乏氧法COCl_2(COCl_2加入培养液的终浓度为75μmol/l)体外模拟肿瘤乏氧微环境。
3．应用半定量RT-PCR、免疫细胞化学、Western-blot检测乏氧环境下食管鳞癌细胞EC9706中HIF-1αmRNA及蛋白的表达情况。
4．统计学处理：所有实验数据应用SPSS11.0软件进行分析，统计学数据用均数±标准差((?)±s)表示，采用χ~2检验、t检验、单因素方差分析、相关性检验。以α=0.05作为检验水准。
结果
1．食管鳞癌组织及正常食管黏膜组织中均可见HIF-1αmRNA的表达，食管鳞癌组织中HIF-1αmRNA表达水平(范围：0.32～1.62；平均1.17±0.25)显著高于正常食管黏膜组织(范围：0.18～0.58；平均0.35±0.12)(P＜0.05)。而且癌组织中HIF-1αmRNA表达强弱不一，46例癌组织中高表达者(大于1.17)为24例，高表达率为52.17％。
2．免疫组化法显示：食管鳞癌组织中HIF-1α蛋白的表达位于胞浆/核中，表达阳性率为41.31％(19/46)；而在正常食管黏膜组织中呈阴性表达。
3．食管鳞癌组织中HIF-1αmRNA表达水平与HIF-1α蛋白表达阳性率之间有显著正相关(P＜0.05)。
4．食管鳞癌组织中HIF-1αmRNA表达水平与肿瘤组织学分级、淋巴结转移、浸润深度、临床分期均无相关性(P＞0.05)。
5．食管鳞癌组织中HIF-1α蛋白的表达与淋巴结转移、浸润深度有显著相关性(P＜0.05)。
6．免疫细胞化学法结果：常氧培养EC9706细胞HIF-1α蛋白表达阴性，化学乏氧法培养8h，可见细胞浆/核呈强棕黄色染色。
7．Western-blot结果显示：化学乏氧法4h即可诱导食管鳞癌EC9706细胞HIF-1α蛋白的表达，8h处于稳定表达状态。乏氧8h再氧合4h后HIF-1α蛋白表达显著减弱。
8．而乏氧前后HIF-1αmRNA表达水平无明显变化(P＞0.05)。
第二部分乏氧诱导因子-1α与食管癌血管生成的关系及其机制
方法
1．血管生成抗原指标检测VEGF、CD34。应用免疫组化技术对上述46例食管鳞癌组织及20例正常食管黏膜组织标本进行VEGF、CD34的检测。微血管密度(MVD)以CD34特异性血管内皮染色强度计算。
2．应用化学乏氧法CoCl_2(CoCl_2加入培养液的终浓度为75μmol/l)体外模拟肿瘤乏氧微环境。
3．利用RNA干扰技术沉默HIF-1α基因的表达。应用半定量RT-PCR检测HIF-1α基因沉默效果。
4．应用Western-blot和免疫细胞化学技术检测HIF-1α基因沉默后乏氧诱导HIF-1α蛋白表达变化。
5．采用半定量RT-PCR、免疫细胞化学技术检测常氧培养细胞、乏氧培养细胞及RNA干扰后乏氧培养细胞中VEGF表达的变化。
6．统计学处理：所有实验数据应用SPSS11.0软件进行分析，统计学数据用均数±标准差((?)±s)表示，采用χ~2检验、t检验、单因素方差分析。以α=0.05作为检验水准。
结果
1．VEGF阳性表达位于细胞浆内，食管鳞癌组织中阳性表达率为58.70％，显著高于正常食管黏膜组织的4.34％(P＜0.05)。
2．以组织中微血管内皮细胞CD34免疫组化染色评估MVD，癌组织中MVD范围在10～101之间，平均46.12±7.64。
3．食管鳞癌组织中VEGF的表达与食管癌浸润深度、淋巴结转移显著相关(P＜0.05)。MVD与食管癌浸润深度、淋巴结转移及临床分期显著相关(P＜0.05)。
4．食管鳞癌组织中HIF-1αmRNA表达水平与VEGF表达及MVD均无相关性(P＞0.05)，而HIF-1α蛋白的表达与VEGF表达及MVD均呈显著正相关(P＜0.05)。
5．针对HIF-1α基因的siRNA干扰技术，有效沉默HIF-1α基因的表达及乏氧诱导的HIF-1α蛋白表达。
6．EC9706细胞乏氧培养后VEGF在mRNA水平和蛋白水平均表达增加(P＜0.05)。针对HIF-1α基因的siRNA干扰技术，有效抑制乏氧诱导VEGF表达增加(P＜0.05)。
第三部分食管鳞癌中HIF-1α的表达与食管癌化疗耐药的关系及其机制
方法
1．48例中晚期食管鳞癌患者采用顺铂／紫杉醇方案化疗，评定疗效。
2．应用免疫组化技术检测上述病例的石蜡包埋标本中HIF-1α蛋白的表达，探讨其与化疗疗效的关系。
3．利用RNA干扰技术沉默HIF-1α基因的表达。
4．采用MTS比色法检测常氧培养组和乏氧培养组EC9706细胞在顺铂／紫杉醇作用下细胞的抑制率。及RNA干扰后乏氧培养细胞在顺铂／紫杉醇作用下细胞的抑制率。
5．采用RT-PCR、免疫细胞化学技术分别检测常氧和乏氧环境下EC9706细胞中的MRP1、Bcl-2、Bax的表达。及RNA干扰后乏氧培养细胞中的MRP1、Bcl-2、Bax表达变化。
6．应用原位末端标记TUNEL法，Annexin V／PI双标记法检测RNA干扰前后紫杉醇对乏氧培养细胞EC9706细胞凋亡的影响
7．用流式细胞仪检测RNA干扰前后乏氧条件下EC9706细胞的细胞周期。
8．统计学处理：所有实验数据应用SPSS11.0软件进行分析，统计学数据用均数±标准差((?)±s)表示，采用χ~2检验、t检验、单因素方差分析。以α=0.05作为检验水准。
结果
1．48例食管鳞癌标本，21例为HIF-1α阳性表达，阳性表达率为43.75％。HIF-1α阳性表达者中无一例CR，5例达PR，化疗有效率为23.81％；而27例HIF-1α阴性表达者，化疗有效者19例，其中CR8例，PR11例，化疗有效率为70.37％。两组比较有显著性差异(P＜0.05)。
2．(1)以终浓度为10μg／ml、5μg／ml、2μg／ml的顺铂分别作用于常氧及乏氧培养细胞24h后，每一药物浓度常氧培养组细胞抑制率均显著高于乏氧培养组(P＜0.05)。(2)以终浓度为100μg／ml、50μg／ml、20μg／ml的紫杉醇分别作用于常氧及乏氧培养细胞24h后，每一药物浓度常氧培养组细胞抑制率均显著高于乏氧培养组(P＜0.05)。
3．(1)以终浓度为10μg／ml、5μg／ml、2μg／ml的顺铂分别作用于乏氧条件下未转染、转染control siRNA和转染HIF-1αsiRNA培养细胞，相同药物浓度RNA干扰组抑制率显著高于未转染组及转染control siRNA组(P＜0.05)。(2)以终浓度为100μg／ml、50μg／ml、20μg／ml紫杉醇分别作用于乏氧条件下未转染、转染control siRNA和转染HIF-1αsiRNA培养细胞，同一药物浓度RNA干扰组抑制率显著高于未转染组及转染control siRNA组(P＜0.05)。
4．乏氧后EC9706细胞MRP1在mRNA水平和蛋白水表达均增加(P＜0.05)。同样乏氧条件，转染HIF-1αsiRNA后EC9706细胞较未转染及转染controlsiRNA组细胞相比，MRP1无论是mRNA水平或蛋白水平均表达减少(P＜0.05)。
5．TUNEL法检测结果显示：50μg／ml紫杉醇作用于相同乏氧条件下EC9706细胞24h后，转染HIF-1αsiRNA组出现大量凋亡细胞，凋亡细胞核被染成棕黄色，阳性率达38.63％，同未转染组的阳性率12.54％、转染control siRNA组的13.69％相比，差异均有显著性(P＜0.05)。未转染组与转染阴性组间差异无统计学意义。(P＞0.05)。
6．流式细胞仪Annexin V FITC／PI测定细胞凋亡率结果：50μg／ml紫杉醇作用于相同乏氧条件下EC9706细胞24h后，转染HIF-1αsiRNA组可显著引起细胞凋亡，凋亡率达(36.57±3.36)％，与未转染组(13.54±2.61)％、转染controlsiRNA组(15.61±2.42)％相比，差异均有统计学意义(P＜0.05)。而未转染组与转染control siRNA组差异无统计学意义(P＞0.05)。
7．乏氧后EC9706细胞，Bcl-2在蛋白水平和mRNA水平表达无显著变化(P＞0.05)。相同乏氧条件，转染HIF-1αsiRNA后EC9706细胞较未转染及转染control siRNA组细胞相比，Bcl-2在蛋白水平和mRNA水平表达均无显著性差异(P＞0.05)。
8．乏氧后EC9706细胞，Bax在蛋白水平和mRNA水平表达均显著降低(P＜0.05)。相同乏氧条件，RNA干扰组Bax在蛋白水平和mRNA水平较未转染组及转染control siRNA组表达均显著增强(P＜0.05)。而未转染组与转染controlsiRNA组差异无统计学意义(P＞0.05)。
9．流式细胞仪检测细胞周期结果显示：常氧培养条件下，EC9706细胞G_1期、S期所占比例分别为(69.05±1.30)％、(23.72±1.30)％，乏氧培养8h组G_1期、S期所占比例分别为(78.29±2.70)％、(11.94±2.70)％，与常氧培养组相比乏氧培养组G_1期显著增加(P＜0.05)，S期显著减少(P＜0.05)。RNA干扰组(转染HIF-1αsiRNA)乏氧8h后G_1期、S期细胞所占比例分别为(70.48±1.91)％、(21.86±1.90)％。转染control siRNA组乏氧8h后G_1期、S期所占比例分别为(77.86±2.31)％、(12.07±1.90)％。相同的乏氧条件下转染HIF-1αsiRNA与未转染组／转染control siRNA组相比，S期显著增加(P＜0.05)、G_1期细胞减少(P＜0.05)。
结论
1．食管鳞癌组织中HIF-1αmRNA水平显著高于正常食管黏膜组织。而食管鳞癌组织中HIF-1α在mRNA表达水平的高低与食管癌临床病理特征、VEGF的表达及MVD无相关性。HIF-1αmRNA水平与蛋白表达阳性率之间有相关性。
2．食管鳞癌组织中HIF-1α在蛋白表达水平与淋巴结转移情况、侵润深度均相关。而且食管鳞癌中HIF-1α在蛋白表达水平与VEGF的表达及MVD呈显著正相关。而正常食管粘膜组织中HIF-1α蛋白表达阴性。
3．食管鳞癌组织中HIF-1α在蛋白表达水平与化疗疗效有关，表达阳性者化疗有效率低。提示HIF-1α蛋白表达情况可作为预测食管癌化疗疗效的又一新的指标。
4．体外化学乏氧法可诱导食管癌细胞HIF-1α蛋白表达，而对RNA水平表达无影响。提示，乏氧主要在转录后水平上影响食管癌细胞中HIF-1α的表达而达到调节其靶基因的目的。HIF-1α在蛋白水平的表达对食管癌的临床治疗具有重要的指导意义。
5．针对HIF-1α的RNA干扰技术能有效沉默HIF-1α的基因表达。
6．乏氧通过诱导HIF-1α蛋白表达上调VEGF表达引起新生血管形成。通过RNA干扰技术沉默HIF-1α基因表达后，乏氧诱导的VEGF表达可被显著抑制，提示以HIF-1α为靶点抗血管生成治疗的可能性。
7．乏氧培养EC9706细胞对顺铂、紫杉醇这两种作用机制不同的药物均产生耐药。通过RNA干扰技术沉默HIF-1α基因表达后，可以逆转乏氧条件下的细胞耐药。
8．乏氧通过诱导HIF-1α蛋白表达上调食管鳞癌细胞中多药耐药相关基因(MRP1)的表达可能是乏氧条件下化疗耐药的机制之一。
9．乏氧条件下HIF-1α可能通过抑制促凋亡基因Bax的表达而发挥抗凋亡作用从而参与食管癌化疗耐药。
10．HIF-1α参与的乏氧所导致EC9706细胞周期停滞可能是乏氧环境下细胞耐药的又一机制。通过RNA干扰技术沉默HIF-1α基因表达后，乏氧导致的细胞周期抑制被解除，从而逆转乏氧条件下的细胞耐药。
11．乏氧环境下HIF-1α蛋白的过表达通过上调VEGF的表达导致食管鳞癌细胞促血管生成能力提高以及对不同机制的化疗药物耐药，因此HIF-1α可望成为新的靶点从而提高食管癌的治疗水平。
创新点：
1．首次系统地探讨了食管鳞癌组织中HIF-1α在mRNA水平及蛋白水平的表达。其蛋白水平的表达有望成为预测食管癌化疗疗效及临床预后的新的指标。
2．利用RNAi技术及化学乏氧法，进一步探讨了乏氧环境中食管鳞癌细胞中VEGF在mRNA水平及蛋白水平的表达，从分子水平上明确了乏氧环境中HIF-1α促食管鳞癌血管生成的机制。
3．首次系统地从分子水平上探讨了乏氧环境中食管鳞癌细胞耐药的复杂机制：乏氧环境中HIF-1α蛋白表达增加，从而上调MRP1表达；乏氧环境中HIF-1α通过抑制促凋亡基因Bax的表达而使乏氧细胞抗凋亡能力增加；以及乏氧环境中HIF-1α抑制细胞周期中的G_1期向S期转换，使乏氧细胞停滞在G_1期。
In recent years, a mounting body of evidence has demonstrated that a hypoxic microenvironment is common phenomena of many tumor types. Hypoxia, as a stimulating factor, can activate a series of genes responsing to hypoxia. It changes the transcription activity of many genes which induce many biological changes. Strong evidence has accumulated that hypoxia plays a pivotal role in tumor angiogenesis, progression and acquired treatment resistance. In the process, hypoxia-inducible factor 1 (HIF-1) plays a vital role in response to hypoxia. But the mechanism which may be compromised are not fully understood. Transcription factor HIF-1, as a key determinant in hypoxic microenvironment, has become one of the most popular subjects in recent.
HIF-1, as Transcrettion factor, widespreadly presents in human and mammalian cells under hypoxia. It is a member of the basic helix-loop-helix Per, AhR, and Sim (bHLH-PAS) family. HIF-1 is composed of two subunits of HIF-1αand HIF-1β. The activity and stability of HIF-1, is decided by HIF-1α, a subnnit regulated by O_2. HIF-1αplays a crucial role in tumor cell adaptation to the hypoxic microenvironment through transcriptional regulation of its target genes. Therefore, it is of great importance to study the effects of HIF-1αin tumor growth, invasion, metastasis and therapy.
Esophageal squamous cell carcinoma(ESCC) is one of the leading cancers in China. Is there the molecular mechanism of hypoxia response in it? At present, studies on the expression of HIF-1αare limited. The detail study about relationship between HIF-1αand angiogenesis, chemotherapy response in ESCC are seldom seen. Especially studies on relationship between HIF-1αand chemotherapy resistance and its mechanism in ESCC are not found.
In order to study the relationship between HIF-1αand angiogenesis in ESCC, expression of HIF-1αon mRNA and protein levels in ESCC tissue was investigated respectively by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. The expressions of VEGF and CD34 were detected using immunohistochemistry. The relationship between HIF-1αand clinical pathological factors,VEGF, and microvessel density (MVD) was analyzed. In order to investigate whether HIF-1αinduces angiogenesis through regulating the expression of VEGF, Cobalt chloride (COC12) was used to establish hypoxia model with EC9706 cell line to mimic hypoxic microenvironment of tumors, and RT-PCR, immunocytochemistry and Western-blot were used to observe the expression of HIF-1αand VEGF. In addition, RNA interference(RNAi) was used to silence HIF-1αgene expression. VEGF expression after silencing HIF-1αgene was observed in EC9706 cells under hypoxia. The regulation effect of HIF-1αon angiogenesis of ESCC under hypoxia and its mechanism were studied, and evaluate whether HIF-1αcould be used as a novel, tumor-specific target for anti-angiogenesis therapy in ESCC.
In order to investigate the relationship between HIF-1αand chemotherapeutic resistance and its mechanism in ESCC, immunohistochemistry and 3-(4,5-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) methods were used. Semi-quantative RT-PCR, immunocytochemistry, RNA interference were used to study the mechanism of chemotherapeutic resistance induced by HIF-1αunder hypoxia from aspects of multidrug resistance, apoptosis and cell cycle. In addition, RNA interference was used to investigate whether it could reverse the chemotherapeutic resistance of ESCC in vitro. All these studies would offer a theorectical foundation for enhancing therapeutic level by targeting HIF-1αas a breaking point in ESCC.
PartⅠ: HIF-1αexpression in ESCC and its mechanism induced by hypoxia
Methods
1. The expression of HIF-1αin 46 cases of ESCC tissue and 20 cases of normal mucosa of esophagus was measured on mRNA and protein level by RT-PCR and immunohistochemistry.
2. To establish a hypoxia model of EC9706 cells by a chemical hypoxia inducer (Cobalt chloride, COCl_2).
3. The expression of HIF-1αin EC9706 cells under hypoxia on mRNA and protein level was measured by RT-PCR immunocytochemistry and Western-blot.
4. Statistical analysis: The SPSS11.0 Statistical Package was used for all analyses. Values were expressed as means±SD. Data were analyzed by t test, ANOVA and x~2 test. The standard of significant level wasα=0.05.
Results:
1. The mRNA expressions of HIF-1αwas detected in 48 cases of ESCC and 20 cases of normal mucosal tissue of esophagus. But the level in tumor tissue(range:0.32～1.62; mean: 1.17±0.25)was significantly higher than that in corresponding normal tissue(range: 0.18～0.58; mean 0.35±0. 12)(P＜0.05). High level HIF-1αmRNA (more than 1.17) was detected in 24 out of 46 tumor cases, The high expression rate was 52.17%.
2. HIF-1αimmunoreactivity was recognized in both cytoplasm and nuclei. The positive expression rate of HIF-1αwas 41.31%(19/46) in tumor samples. And it was negative expression in normal tissue.
3. The mRNA expression level of HIF-1αwas positively correlated with its protein expression(P＜0.05).
4. The mRNA expression level of HIF-1αwas not associated with histological grades, lympha node metastasis, infutration depth, orclinical stage(P＞0.05).
5. HIF-1αprotein expression was positively correlated with infiltration depth and lympha node metastasis(P＜0.05).
6. Results of immunocytochemistry: HIF-1αimmunoreactivity of EC9706 cells in normoxia was negative, but strongly stained in plasmid/nuclear of EC9706 cells induced by chemical hypoxic condition.
7. Results ofWestem-blot: HIF-1αprotein expression detected in EC9706 cells under hypoxia condition for 4h and it was stable expressied till 8h hypoxia. HIF-1αprotein expression significantly decreased when EC9706 cells were reoxygenated for 4h after being under hypoxia for 8h.
8. Whereas HIF-la mRNA expression did not change before and after hypoxia
PartⅡ: Relationship between HIF-1 alpha and angiogenesis and its
mechanism in ESCC
Methods
1. The angiogenenic profile was assessed using VEGF and CD34. The expression of VEGF and CD34 in above 46 cases of ESCC tissue and 20 cases of normal mucosa of esophagus were measured by immunohistochemistry. MVD was calculated according to immunohistochemical staining of the adhesion molecule CD34 of the endothelial cells.
2. A hypoxia model of EC9706 cells was established by a chemical hypoxia inducer (Cobalt chloride, COCl_2).
3. RNA-interference targeting HIF-1αwas used to silence the expression of HIF-1αin human EC9706 cell line. The effect of HIF-1αgene silencing was measured by RT-PCR.
4. The expression level of HIF-1αinduced by hypoxia after RNAi were measured by Western-blot and immuncytochemistry.
5. The expression of VEGF in EC9706 cells, which were under normaxia or under hypoxia or after RNAi under hypoxia, was measured respectively by immunocytochemistry and Semi-quantitative RT-PCR.
6. Statistical analysis: The SPSS11.0 Statistical Package was used for all analyses. Values were expressed as means±SD. Data were analyzed by t test, ANOVA and x~2 test. The standard of significant level wasα＝0.05.
Results:
1. VEGF expression was detected in cytoplasm. The positive rate was 58.70%, which was significantly higher than that (4.34%) in normal mucosa (P＜0.05).
2. MVD in the ESCC tissue ranged from 10 to 101, median 46.12±7.64.
3. The expression of VEGF in ESCC was closely related to tumor infiltration depth (P＜0.05) and lymph node metastasis(P＜0.05). MVD was closely related to tumor infiltration depth (P＜0.05), lymph node metastasis(P＜0.05), and clinical stage(P＜0.05).
4. The mRNA expression level of HIF-1αwas not associated with VEGF expression or MVD(P＞0.05). Whereas HIF-1αprotein expression was positively correlated with VEGF expression(P＜0.05) and MVD(P＜0.05).
5. HIF-1αmRNA expression was obviously knocted down by small interferencing RNA(siRNA). In addition, HIF-1αprotein expression induced by hypoxia in EC9706 cells was inhibited effectively by RNAi (P＜0.05).
6. The expression of VEGF on mRNA and protein level was significantly increased under hypoxia condition(P＜0.05). The expression of VEGF on mRNA and protein level under hypoxia was greatly down-regulated by small interferencing RNA(siRNA) targeting HIF-1α(P＜0.05).
PartⅢ: Relationship between HIF-1αand chemotherapy resistance and its mechanism in ESCC
Methods
1. Platixal in combination with cisplatin was used in 48 patients with ESCC at advanced stage.The chemotherapy response was evaluated.
2. HIF-1αprotein in the specimens from the 48 patients was measured by immunohistochemistry, and the relation of HIF-1αand chemotherapy response was analyzed.
3. RNA-interference targeting HIF-1αwas used to silence the expression of HIF-1αin human EC9706 cell line.
4. Growth inhibition rates of EC9706 cells treated with cisplatian/platixal were measured by MTS colorimetric assay under normoxic or hypoxic condition after RNA interference under hypoxia.
5. The expressions of MRP1, Bcl-2 and Bax were measured respectively by RT-PCR, immunocytochemistry in EC9706 cells under normia, hypoxia and after RNA interference under hypoxia.
6. The apoptosis rate of EC9706 cells treated by platixal under hypoxia before and after RNAi was studied by TUNEL assay and Annexin V/PI double staining, respectively.
8. Cell cycles of EC9706 cells before and after RNAi under hypoxia were analyzed with flow cytometry.
10. Statistical analysis: The SPSS Statistical Package was used for all analyses. Association between the variables were tested by analysis of variance and x~2 test, t test. The standard of significant level wasα=0.05.
Results:
1. Of 48 specimens 21 (43.75%) had positive expression of HIF-1αprotein. Among 21 cases with positive expression of HIF-1α, there was none complete response case and 5 cases of partial responses, and the response rate was 23.81%; whereas in 27 eases with negative expression of HIF-1α, there were 8 cases of complete responses and 11 cases of partial responses, and the response rate was 70.37%. There was significant difference between two groups (P＜0.05).
2. After being treated by cisplatin (10μg/ml, 5μg/ml, 2μg/ml), the growth inhibition rates of EC9706 cells under normoxia were significant higher than those under hypoxia(P＜0.05). After being treated by platixal (100μg/ml, 50μg/ml, 20μg/ml) the growth inhibition rates of EC9706 cells under normoxia were significantly higher than those under hypoxia (P＜0.05) .
3. In the untransfected group, transfected with control siRNA group and transfected with HIF-1αsiRNA group treatedby cisplatin (10μg/ml, 5μg/ml, 2μg/ml) under hypoxia, respectively, the growth inhibition rates of EC9706 cells transfected with HIF-1αsiRNA were significantly higher than those in the untransfected group and transfected with control siRNA group (P＜0.05). In the untransfected group, transfected with control siRNA group and transfected with HIF-1αsiRNA group treated with platixal (100μg/ml, 50μg/ml, 20μ/ml) under hypoxia respectively, the growth inhibition rates of cells transfected with HIF-1αsiRNA were significantly higher than those in the untransfected group and transfected with control siRNA group (P＜0.05).
4. Expression of MRP1 on mRNA and protein level was significantly increased in EC9706 cells after hypoxia (P＜0.05). MRP1 expression on mRNA and protein level in cells transfected with HIF-1αsiRNA was significantly lower than those in untransfected group and transfected with control siRNA group under hypoxia (P<0.05).
5. TUNEL detection revealed that after being treated with platixal (50μg/ml)under hypoxia, the apoptosis rate in HIF-1αsiRNA group(38.63%) was significantly higher than that in untransfected group (12.54%), and the transfected with control siRNA group (13.69%) (P＜0.05).
6. Flow cytometer(Annexin V FITC/PI) revealed that after being treated with platixal (50μg/ml) under hypoxia, the apoptosis rate in the transfected with HIF-1αsiRNA group (36.57±3.36)% was significantly higher than that in untransfected group (13.54±2.61)%, and the transfected with control siRNA group (15.61±2.42)% (P＜0.05).
7. There was not significant difference of Bcl-2 expression on mRNA and protein level between normoxia group and hypoxia group (P＞0.05), as well as between the transfected with HIF-1αsiRNA group, the transfected with control siRNA group and the untransfected group (P＜0.05) .
8. The expression of Bax on mRNA and protein level was significantly decreased in EC9706 cells after hypoxia(P＜0.05). Bax expression on mRNA and protein level in cells transfected with HIF-1αsiRNA was significantly higher than those in untransfected group and transfected with control siRNA group under hypoxia(P＜0.05).
9. The results of flow cytometry showed that the cells in G_1-phase, S-phase were (69.05±1.30)% and (23.72±1.30)%, respectively during normoxia. The cells in G_1-phase, S-phase was (78.29±2.70)% and (11.94±2.70)%, respectively during hypoxia. An increase of G_1-phase and a decrease of S-phase was observed in EC9706 cells in response to hypoxia(P＜0.05); the cells in G_1-phase, S-phase of cells in HIF-1αsiRNA transfection group was (70.48±1.91)% and (21.86±1.90)%, respectively after hypoxia for 8h, whereas the cells in G_1-phase, S-phase of cells in transfected with control siRNA group was (77.86±2.31)% and (12.07±1.90)% respectively, under the same hypoxia. Compared with untransfected group and transfected with control siRNA group, the cells in G_1-phase decreased and the cells in S-phase increased significantly(P＜0.05).
Conclusions
1. The mRNA expression of HIF-1αin ESCC tissue is Significantly higher than that in corresponding normal mucosa. The level of HIF-1αmRNA expression is not correlated with clinicopathological features, VEGF expression or MVD.
2. There is positive correlation between the level of HIF-1αmRNA expression and the positive rate of HIF-1αprotein expression. HIF-1αprotein expression in ESCC tissue is correlated with lymph node metastasis and invasion depth. HIF-1αprotein expression is correlated with VEGF expression and MVD, which indicats that HIF-1αprotein expression in ESCC tissue is correlated with angiogenesis.
3. The expression of HIF-1αprotein was associated with chemotherapy response. The patients with positive expression of HIF-1αprotein have lower chemotherapeutic efficacy, suggesting that HIF-1αprotein can be used as a new indicator to predict chemotherapy response.
4. Chemical hypoxia inducer (Cobalt chloride, COCl_2) establishs a hypoxia model in vitro to mimic hypoxia environment in tumor, and overexpression of HIF-1αprotein in EC9706 cells can be induced, while the expression of HIF-1αmRNA does not change after hypoxia. It indicates that hypoxia mainly affects the expression HIF-1αon protein level. Therefore, it is more important pratice to measure HIF-1αexpression on protein in the treatment of ESCC.
5. RNA interference targeting HIF-1αmakes a specificity of HIF-1αsilencing.
6. The overexpression of VEGF regulated by HIF-1αcould be attenuated markedly by HIF-1αsiRNA treatment. Therefore, to silence the expression of HIF-1αmay inhibit the angiogenesis, which offers an avenue of anti-angiogenesis gene therapy for ESCC.
7. EC9706 cells under hypoxia is resistant to cisplatin and taxol, the two different chemotherapeutic drugs. Whereas RNAi silencing HIF-1αgene can reverse the resistance to the two drugs.
8. HIF-1αcan upregulate the expression of MRP1, which may involve in the chemotherapeutic resistance of EC9706 cells under hypoxia.
9. Hypoxia-mediated down-regulation of Bax in EC9706 cells occurs via HIF-1α-dependent mechanism and contributes to chemoresistance.
10. HIF-1αis involved in the cell cycle arrest in EC9706 cells in response to hypoxia which may be another therapeutic resistance mechanism under hypoxia.
11. The overexpression of HIF-1αinduces angiogenesis and resistance to varied chemotherapeutic drugs. Therefore, HIF-1αcan be used as a novel, tumor-specific target for anticancer therapy in ESCC. This would offer one new way and a theoretic foundation to target therapy for ESCC.
Original points:
1. The expression of HIF-1αon mRNA and protein level in ESCC was systematically studied. The results indicated that HIF-1αprotein expression can be regarded as a new index to predict chemotherapeutic response in ESCC.
2. RNAi and chemical mimic hypoxia were used to further explore the expression of VEGF on mRNA and protein level. The results indicated that HIF-1αprotein was involved in angiogenesis through upregulating VEGF expression.
3. The complex mechanism of cell resistance to chemotherapeutic drugs under hypoxia in ESCC was explored systematically. The results indicated that HIF-1αprotein was involved in chemoresistance under hypoxia by by inducing cell cycle arrest in G_1 phrase and up-regulation the expression of MRP1 and down-regulation the expression of Bax which play a role in apoptosis.
HIF-1是存在于哺乳动物和人体内一种转录因子，它是一个属于bHLH-PAS蛋白族的转录因子，由HIF-1α和HIF-1β两个亚单位组成。HIF-1的稳定和活性是由HIF-1α决定的，且HIF-1α是专一受O_2调节的亚基。由于在乏氧诱导肿瘤基因表达的信息通路上，HIF-1α起着中枢纽带作用。因此，研究HIF-1α与肿瘤生长、浸润、转移和治疗方面的关系有重要的意义。
食管癌作为我国高发肿瘤，是否也存在乏氧耐受的分子机制目前尚不十分清楚。关于HIF-1α在食管癌中的表达研究较少，其与食管癌血管生成及化疗耐药的关系目前未见系统的研究报道。特别是其是否参与食管癌化疗耐药及其机制的研究，目前未见报道。
为了探讨HIF-1α在食管癌组织中的表达及HIF-1α与食管癌血管生成的关系，本文采用半定量聚合酶链反应技术及免疫组化技术检测食管鳞癌组织中HIF-1α在mRNA水平及蛋白水平的表达，采用免疫组化技术检测食管鳞癌组织中VEGF、CD34的表达，观察了HIF-1α在食管癌中的表达情况及其与临床病理特征、VEGF和微血管密度的关系。为了探讨HIF-1α是否通过上调VEGF的表达而促进食管癌血管生成的机制，本文以食管鳞癌细胞株EC9706为研究对象，通过化学性乏氧诱导剂氯化钴(cobaltchloride，COCl_2)建立乏氧模型，体外模拟肿瘤乏氧微环境。采用半定量RT-PCR、Western-blot、免疫细胞化学技术检测乏氧诱导的HIF-1α、VEGF表达情况。进一步利用RNA干扰技术(RNA intefercnce，RNAi)观察小干扰RNA(small interference RNA，siRNA)转染EC9706细胞后乏氧诱导的HIF-1α和VEGF的表达的改变，初步探讨了HIF-1α在乏氧条件下对食管癌血管生成的调控作用及其机制以及以HIF-1α为靶点抗食管癌血管生成治疗的可能性。
为探讨HIF-1α是否与食管癌化疗耐药有关及其参与耐药的机制，采用免疫组化技术检测食管鳞癌组织中HIF-1α的表达，并与临床化疗疗效相结合，探讨HIF-1α与食管癌化疗疗效的关系。从体外方面，以EC9706为研究对象，通过化学性乏氧诱导剂建立乏氧模型，采用MTS、半定量RT-PCR、免疫细胞化学、RNA干扰技术进一步从多药耐药、凋亡、细胞周期等方面，探讨了HIF-1α参与食管癌细胞乏氧条件下对不同机制的化疗药物产生耐药的机制。并进一步体外探讨以HIF-1α为新的靶点，应用RNA干扰技术逆转食管癌耐药的新途径。为以此为突破口进一步提高食管癌的化疗水平提供理论依据。
第一部分乏氧诱导因子-1α在食管鳞癌组织中的表达及乏氧调节
方法
1．应用半定量RT-PCR及免疫组化技术对46例食管鳞癌组织及20例正常食管黏膜组织进行HIF-1αmRNA及蛋白水平的检测。
2．应用化学乏氧法COCl_2(COCl_2加入培养液的终浓度为75μmol/l)体外模拟肿瘤乏氧微环境。
3．应用半定量RT-PCR、免疫细胞化学、Western-blot检测乏氧环境下食管鳞癌细胞EC9706中HIF-1αmRNA及蛋白的表达情况。
4．统计学处理：所有实验数据应用SPSS11.0软件进行分析，统计学数据用均数±标准差((?)±s)表示，采用χ~2检验、t检验、单因素方差分析、相关性检验。以α=0.05作为检验水准。
结果
1．食管鳞癌组织及正常食管黏膜组织中均可见HIF-1αmRNA的表达，食管鳞癌组织中HIF-1αmRNA表达水平(范围：0.32～1.62；平均1.17±0.25)显著高于正常食管黏膜组织(范围：0.18～0.58；平均0.35±0.12)(P＜0.05)。而且癌组织中HIF-1αmRNA表达强弱不一，46例癌组织中高表达者(大于1.17)为24例，高表达率为52.17％。
2．免疫组化法显示：食管鳞癌组织中HIF-1α蛋白的表达位于胞浆/核中，表达阳性率为41.31％(19/46)；而在正常食管黏膜组织中呈阴性表达。
3．食管鳞癌组织中HIF-1αmRNA表达水平与HIF-1α蛋白表达阳性率之间有显著正相关(P＜0.05)。
4．食管鳞癌组织中HIF-1αmRNA表达水平与肿瘤组织学分级、淋巴结转移、浸润深度、临床分期均无相关性(P＞0.05)。
5．食管鳞癌组织中HIF-1α蛋白的表达与淋巴结转移、浸润深度有显著相关性(P＜0.05)。
6．免疫细胞化学法结果：常氧培养EC9706细胞HIF-1α蛋白表达阴性，化学乏氧法培养8h，可见细胞浆/核呈强棕黄色染色。
7．Western-blot结果显示：化学乏氧法4h即可诱导食管鳞癌EC9706细胞HIF-1α蛋白的表达，8h处于稳定表达状态。乏氧8h再氧合4h后HIF-1α蛋白表达显著减弱。
8．而乏氧前后HIF-1αmRNA表达水平无明显变化(P＞0.05)。
第二部分乏氧诱导因子-1α与食管癌血管生成的关系及其机制
方法
1．血管生成抗原指标检测VEGF、CD34。应用免疫组化技术对上述46例食管鳞癌组织及20例正常食管黏膜组织标本进行VEGF、CD34的检测。微血管密度(MVD)以CD34特异性血管内皮染色强度计算。
2．应用化学乏氧法CoCl_2(CoCl_2加入培养液的终浓度为75μmol/l)体外模拟肿瘤乏氧微环境。
3．利用RNA干扰技术沉默HIF-1α基因的表达。应用半定量RT-PCR检测HIF-1α基因沉默效果。
4．应用Western-blot和免疫细胞化学技术检测HIF-1α基因沉默后乏氧诱导HIF-1α蛋白表达变化。
5．采用半定量RT-PCR、免疫细胞化学技术检测常氧培养细胞、乏氧培养细胞及RNA干扰后乏氧培养细胞中VEGF表达的变化。
6．统计学处理：所有实验数据应用SPSS11.0软件进行分析，统计学数据用均数±标准差((?)±s)表示，采用χ~2检验、t检验、单因素方差分析。以α=0.05作为检验水准。
结果
1．VEGF阳性表达位于细胞浆内，食管鳞癌组织中阳性表达率为58.70％，显著高于正常食管黏膜组织的4.34％(P＜0.05)。
2．以组织中微血管内皮细胞CD34免疫组化染色评估MVD，癌组织中MVD范围在10～101之间，平均46.12±7.64。
3．食管鳞癌组织中VEGF的表达与食管癌浸润深度、淋巴结转移显著相关(P＜0.05)。MVD与食管癌浸润深度、淋巴结转移及临床分期显著相关(P＜0.05)。
4．食管鳞癌组织中HIF-1αmRNA表达水平与VEGF表达及MVD均无相关性(P＞0.05)，而HIF-1α蛋白的表达与VEGF表达及MVD均呈显著正相关(P＜0.05)。
5．针对HIF-1α基因的siRNA干扰技术，有效沉默HIF-1α基因的表达及乏氧诱导的HIF-1α蛋白表达。
6．EC9706细胞乏氧培养后VEGF在mRNA水平和蛋白水平均表达增加(P＜0.05)。针对HIF-1α基因的siRNA干扰技术，有效抑制乏氧诱导VEGF表达增加(P＜0.05)。
第三部分食管鳞癌中HIF-1α的表达与食管癌化疗耐药的关系及其机制
方法
1．48例中晚期食管鳞癌患者采用顺铂／紫杉醇方案化疗，评定疗效。
2．应用免疫组化技术检测上述病例的石蜡包埋标本中HIF-1α蛋白的表达，探讨其与化疗疗效的关系。
3．利用RNA干扰技术沉默HIF-1α基因的表达。
4．采用MTS比色法检测常氧培养组和乏氧培养组EC9706细胞在顺铂／紫杉醇作用下细胞的抑制率。及RNA干扰后乏氧培养细胞在顺铂／紫杉醇作用下细胞的抑制率。
5．采用RT-PCR、免疫细胞化学技术分别检测常氧和乏氧环境下EC9706细胞中的MRP1、Bcl-2、Bax的表达。及RNA干扰后乏氧培养细胞中的MRP1、Bcl-2、Bax表达变化。
6．应用原位末端标记TUNEL法，Annexin V／PI双标记法检测RNA干扰前后紫杉醇对乏氧培养细胞EC9706细胞凋亡的影响
7．用流式细胞仪检测RNA干扰前后乏氧条件下EC9706细胞的细胞周期。
8．统计学处理：所有实验数据应用SPSS11.0软件进行分析，统计学数据用均数±标准差((?)±s)表示，采用χ~2检验、t检验、单因素方差分析。以α=0.05作为检验水准。
结果
1．48例食管鳞癌标本，21例为HIF-1α阳性表达，阳性表达率为43.75％。HIF-1α阳性表达者中无一例CR，5例达PR，化疗有效率为23.81％；而27例HIF-1α阴性表达者，化疗有效者19例，其中CR8例，PR11例，化疗有效率为70.37％。两组比较有显著性差异(P＜0.05)。
2．(1)以终浓度为10μg／ml、5μg／ml、2μg／ml的顺铂分别作用于常氧及乏氧培养细胞24h后，每一药物浓度常氧培养组细胞抑制率均显著高于乏氧培养组(P＜0.05)。(2)以终浓度为100μg／ml、50μg／ml、20μg／ml的紫杉醇分别作用于常氧及乏氧培养细胞24h后，每一药物浓度常氧培养组细胞抑制率均显著高于乏氧培养组(P＜0.05)。
3．(1)以终浓度为10μg／ml、5μg／ml、2μg／ml的顺铂分别作用于乏氧条件下未转染、转染control siRNA和转染HIF-1αsiRNA培养细胞，相同药物浓度RNA干扰组抑制率显著高于未转染组及转染control siRNA组(P＜0.05)。(2)以终浓度为100μg／ml、50μg／ml、20μg／ml紫杉醇分别作用于乏氧条件下未转染、转染control siRNA和转染HIF-1αsiRNA培养细胞，同一药物浓度RNA干扰组抑制率显著高于未转染组及转染control siRNA组(P＜0.05)。
4．乏氧后EC9706细胞MRP1在mRNA水平和蛋白水表达均增加(P＜0.05)。同样乏氧条件，转染HIF-1αsiRNA后EC9706细胞较未转染及转染controlsiRNA组细胞相比，MRP1无论是mRNA水平或蛋白水平均表达减少(P＜0.05)。
5．TUNEL法检测结果显示：50μg／ml紫杉醇作用于相同乏氧条件下EC9706细胞24h后，转染HIF-1αsiRNA组出现大量凋亡细胞，凋亡细胞核被染成棕黄色，阳性率达38.63％，同未转染组的阳性率12.54％、转染control siRNA组的13.69％相比，差异均有显著性(P＜0.05)。未转染组与转染阴性组间差异无统计学意义。(P＞0.05)。
6．流式细胞仪Annexin V FITC／PI测定细胞凋亡率结果：50μg／ml紫杉醇作用于相同乏氧条件下EC9706细胞24h后，转染HIF-1αsiRNA组可显著引起细胞凋亡，凋亡率达(36.57±3.36)％，与未转染组(13.54±2.61)％、转染controlsiRNA组(15.61±2.42)％相比，差异均有统计学意义(P＜0.05)。而未转染组与转染control siRNA组差异无统计学意义(P＞0.05)。
7．乏氧后EC9706细胞，Bcl-2在蛋白水平和mRNA水平表达无显著变化(P＞0.05)。相同乏氧条件，转染HIF-1αsiRNA后EC9706细胞较未转染及转染control siRNA组细胞相比，Bcl-2在蛋白水平和mRNA水平表达均无显著性差异(P＞0.05)。
8．乏氧后EC9706细胞，Bax在蛋白水平和mRNA水平表达均显著降低(P＜0.05)。相同乏氧条件，RNA干扰组Bax在蛋白水平和mRNA水平较未转染组及转染control siRNA组表达均显著增强(P＜0.05)。而未转染组与转染controlsiRNA组差异无统计学意义(P＞0.05)。
9．流式细胞仪检测细胞周期结果显示：常氧培养条件下，EC9706细胞G_1期、S期所占比例分别为(69.05±1.30)％、(23.72±1.30)％，乏氧培养8h组G_1期、S期所占比例分别为(78.29±2.70)％、(11.94±2.70)％，与常氧培养组相比乏氧培养组G_1期显著增加(P＜0.05)，S期显著减少(P＜0.05)。RNA干扰组(转染HIF-1αsiRNA)乏氧8h后G_1期、S期细胞所占比例分别为(70.48±1.91)％、(21.86±1.90)％。转染control siRNA组乏氧8h后G_1期、S期所占比例分别为(77.86±2.31)％、(12.07±1.90)％。相同的乏氧条件下转染HIF-1αsiRNA与未转染组／转染control siRNA组相比，S期显著增加(P＜0.05)、G_1期细胞减少(P＜0.05)。
结论
1．食管鳞癌组织中HIF-1αmRNA水平显著高于正常食管黏膜组织。而食管鳞癌组织中HIF-1α在mRNA表达水平的高低与食管癌临床病理特征、VEGF的表达及MVD无相关性。HIF-1αmRNA水平与蛋白表达阳性率之间有相关性。
2．食管鳞癌组织中HIF-1α在蛋白表达水平与淋巴结转移情况、侵润深度均相关。而且食管鳞癌中HIF-1α在蛋白表达水平与VEGF的表达及MVD呈显著正相关。而正常食管粘膜组织中HIF-1α蛋白表达阴性。
3．食管鳞癌组织中HIF-1α在蛋白表达水平与化疗疗效有关，表达阳性者化疗有效率低。提示HIF-1α蛋白表达情况可作为预测食管癌化疗疗效的又一新的指标。
4．体外化学乏氧法可诱导食管癌细胞HIF-1α蛋白表达，而对RNA水平表达无影响。提示，乏氧主要在转录后水平上影响食管癌细胞中HIF-1α的表达而达到调节其靶基因的目的。HIF-1α在蛋白水平的表达对食管癌的临床治疗具有重要的指导意义。
5．针对HIF-1α的RNA干扰技术能有效沉默HIF-1α的基因表达。
6．乏氧通过诱导HIF-1α蛋白表达上调VEGF表达引起新生血管形成。通过RNA干扰技术沉默HIF-1α基因表达后，乏氧诱导的VEGF表达可被显著抑制，提示以HIF-1α为靶点抗血管生成治疗的可能性。
7．乏氧培养EC9706细胞对顺铂、紫杉醇这两种作用机制不同的药物均产生耐药。通过RNA干扰技术沉默HIF-1α基因表达后，可以逆转乏氧条件下的细胞耐药。
8．乏氧通过诱导HIF-1α蛋白表达上调食管鳞癌细胞中多药耐药相关基因(MRP1)的表达可能是乏氧条件下化疗耐药的机制之一。
9．乏氧条件下HIF-1α可能通过抑制促凋亡基因Bax的表达而发挥抗凋亡作用从而参与食管癌化疗耐药。
10．HIF-1α参与的乏氧所导致EC9706细胞周期停滞可能是乏氧环境下细胞耐药的又一机制。通过RNA干扰技术沉默HIF-1α基因表达后，乏氧导致的细胞周期抑制被解除，从而逆转乏氧条件下的细胞耐药。
11．乏氧环境下HIF-1α蛋白的过表达通过上调VEGF的表达导致食管鳞癌细胞促血管生成能力提高以及对不同机制的化疗药物耐药，因此HIF-1α可望成为新的靶点从而提高食管癌的治疗水平。
创新点：
1．首次系统地探讨了食管鳞癌组织中HIF-1α在mRNA水平及蛋白水平的表达。其蛋白水平的表达有望成为预测食管癌化疗疗效及临床预后的新的指标。
2．利用RNAi技术及化学乏氧法，进一步探讨了乏氧环境中食管鳞癌细胞中VEGF在mRNA水平及蛋白水平的表达，从分子水平上明确了乏氧环境中HIF-1α促食管鳞癌血管生成的机制。
3．首次系统地从分子水平上探讨了乏氧环境中食管鳞癌细胞耐药的复杂机制：乏氧环境中HIF-1α蛋白表达增加，从而上调MRP1表达；乏氧环境中HIF-1α通过抑制促凋亡基因Bax的表达而使乏氧细胞抗凋亡能力增加；以及乏氧环境中HIF-1α抑制细胞周期中的G_1期向S期转换，使乏氧细胞停滞在G_1期。
In recent years, a mounting body of evidence has demonstrated that a hypoxic microenvironment is common phenomena of many tumor types. Hypoxia, as a stimulating factor, can activate a series of genes responsing to hypoxia. It changes the transcription activity of many genes which induce many biological changes. Strong evidence has accumulated that hypoxia plays a pivotal role in tumor angiogenesis, progression and acquired treatment resistance. In the process, hypoxia-inducible factor 1 (HIF-1) plays a vital role in response to hypoxia. But the mechanism which may be compromised are not fully understood. Transcription factor HIF-1, as a key determinant in hypoxic microenvironment, has become one of the most popular subjects in recent.
HIF-1, as Transcrettion factor, widespreadly presents in human and mammalian cells under hypoxia. It is a member of the basic helix-loop-helix Per, AhR, and Sim (bHLH-PAS) family. HIF-1 is composed of two subunits of HIF-1αand HIF-1β. The activity and stability of HIF-1, is decided by HIF-1α, a subnnit regulated by O_2. HIF-1αplays a crucial role in tumor cell adaptation to the hypoxic microenvironment through transcriptional regulation of its target genes. Therefore, it is of great importance to study the effects of HIF-1αin tumor growth, invasion, metastasis and therapy.
Esophageal squamous cell carcinoma(ESCC) is one of the leading cancers in China. Is there the molecular mechanism of hypoxia response in it? At present, studies on the expression of HIF-1αare limited. The detail study about relationship between HIF-1αand angiogenesis, chemotherapy response in ESCC are seldom seen. Especially studies on relationship between HIF-1αand chemotherapy resistance and its mechanism in ESCC are not found.
In order to study the relationship between HIF-1αand angiogenesis in ESCC, expression of HIF-1αon mRNA and protein levels in ESCC tissue was investigated respectively by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. The expressions of VEGF and CD34 were detected using immunohistochemistry. The relationship between HIF-1αand clinical pathological factors,VEGF, and microvessel density (MVD) was analyzed. In order to investigate whether HIF-1αinduces angiogenesis through regulating the expression of VEGF, Cobalt chloride (COC12) was used to establish hypoxia model with EC9706 cell line to mimic hypoxic microenvironment of tumors, and RT-PCR, immunocytochemistry and Western-blot were used to observe the expression of HIF-1αand VEGF. In addition, RNA interference(RNAi) was used to silence HIF-1αgene expression. VEGF expression after silencing HIF-1αgene was observed in EC9706 cells under hypoxia. The regulation effect of HIF-1αon angiogenesis of ESCC under hypoxia and its mechanism were studied, and evaluate whether HIF-1αcould be used as a novel, tumor-specific target for anti-angiogenesis therapy in ESCC.
In order to investigate the relationship between HIF-1αand chemotherapeutic resistance and its mechanism in ESCC, immunohistochemistry and 3-(4,5-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) methods were used. Semi-quantative RT-PCR, immunocytochemistry, RNA interference were used to study the mechanism of chemotherapeutic resistance induced by HIF-1αunder hypoxia from aspects of multidrug resistance, apoptosis and cell cycle. In addition, RNA interference was used to investigate whether it could reverse the chemotherapeutic resistance of ESCC in vitro. All these studies would offer a theorectical foundation for enhancing therapeutic level by targeting HIF-1αas a breaking point in ESCC.
PartⅠ: HIF-1αexpression in ESCC and its mechanism induced by hypoxia
Methods
1. The expression of HIF-1αin 46 cases of ESCC tissue and 20 cases of normal mucosa of esophagus was measured on mRNA and protein level by RT-PCR and immunohistochemistry.
2. To establish a hypoxia model of EC9706 cells by a chemical hypoxia inducer (Cobalt chloride, COCl_2).
3. The expression of HIF-1αin EC9706 cells under hypoxia on mRNA and protein level was measured by RT-PCR immunocytochemistry and Western-blot.
4. Statistical analysis: The SPSS11.0 Statistical Package was used for all analyses. Values were expressed as means±SD. Data were analyzed by t test, ANOVA and x~2 test. The standard of significant level wasα=0.05.
Results:
1. The mRNA expressions of HIF-1αwas detected in 48 cases of ESCC and 20 cases of normal mucosal tissue of esophagus. But the level in tumor tissue(range:0.32～1.62; mean: 1.17±0.25)was significantly higher than that in corresponding normal tissue(range: 0.18～0.58; mean 0.35±0. 12)(P＜0.05). High level HIF-1αmRNA (more than 1.17) was detected in 24 out of 46 tumor cases, The high expression rate was 52.17%.
2. HIF-1αimmunoreactivity was recognized in both cytoplasm and nuclei. The positive expression rate of HIF-1αwas 41.31%(19/46) in tumor samples. And it was negative expression in normal tissue.
3. The mRNA expression level of HIF-1αwas positively correlated with its protein expression(P＜0.05).
4. The mRNA expression level of HIF-1αwas not associated with histological grades, lympha node metastasis, infutration depth, orclinical stage(P＞0.05).
5. HIF-1αprotein expression was positively correlated with infiltration depth and lympha node metastasis(P＜0.05).
6. Results of immunocytochemistry: HIF-1αimmunoreactivity of EC9706 cells in normoxia was negative, but strongly stained in plasmid/nuclear of EC9706 cells induced by chemical hypoxic condition.
7. Results ofWestem-blot: HIF-1αprotein expression detected in EC9706 cells under hypoxia condition for 4h and it was stable expressied till 8h hypoxia. HIF-1αprotein expression significantly decreased when EC9706 cells were reoxygenated for 4h after being under hypoxia for 8h.
8. Whereas HIF-la mRNA expression did not change before and after hypoxia
PartⅡ: Relationship between HIF-1 alpha and angiogenesis and its
mechanism in ESCC
Methods
1. The angiogenenic profile was assessed using VEGF and CD34. The expression of VEGF and CD34 in above 46 cases of ESCC tissue and 20 cases of normal mucosa of esophagus were measured by immunohistochemistry. MVD was calculated according to immunohistochemical staining of the adhesion molecule CD34 of the endothelial cells.
2. A hypoxia model of EC9706 cells was established by a chemical hypoxia inducer (Cobalt chloride, COCl_2).
3. RNA-interference targeting HIF-1αwas used to silence the expression of HIF-1αin human EC9706 cell line. The effect of HIF-1αgene silencing was measured by RT-PCR.
4. The expression level of HIF-1αinduced by hypoxia after RNAi were measured by Western-blot and immuncytochemistry.
5. The expression of VEGF in EC9706 cells, which were under normaxia or under hypoxia or after RNAi under hypoxia, was measured respectively by immunocytochemistry and Semi-quantitative RT-PCR.
6. Statistical analysis: The SPSS11.0 Statistical Package was used for all analyses. Values were expressed as means±SD. Data were analyzed by t test, ANOVA and x~2 test. The standard of significant level wasα＝0.05.
Results:
1. VEGF expression was detected in cytoplasm. The positive rate was 58.70%, which was significantly higher than that (4.34%) in normal mucosa (P＜0.05).
2. MVD in the ESCC tissue ranged from 10 to 101, median 46.12±7.64.
3. The expression of VEGF in ESCC was closely related to tumor infiltration depth (P＜0.05) and lymph node metastasis(P＜0.05). MVD was closely related to tumor infiltration depth (P＜0.05), lymph node metastasis(P＜0.05), and clinical stage(P＜0.05).
4. The mRNA expression level of HIF-1αwas not associated with VEGF expression or MVD(P＞0.05). Whereas HIF-1αprotein expression was positively correlated with VEGF expression(P＜0.05) and MVD(P＜0.05).
5. HIF-1αmRNA expression was obviously knocted down by small interferencing RNA(siRNA). In addition, HIF-1αprotein expression induced by hypoxia in EC9706 cells was inhibited effectively by RNAi (P＜0.05).
6. The expression of VEGF on mRNA and protein level was significantly increased under hypoxia condition(P＜0.05). The expression of VEGF on mRNA and protein level under hypoxia was greatly down-regulated by small interferencing RNA(siRNA) targeting HIF-1α(P＜0.05).
PartⅢ: Relationship between HIF-1αand chemotherapy resistance and its mechanism in ESCC
Methods
1. Platixal in combination with cisplatin was used in 48 patients with ESCC at advanced stage.The chemotherapy response was evaluated.
2. HIF-1αprotein in the specimens from the 48 patients was measured by immunohistochemistry, and the relation of HIF-1αand chemotherapy response was analyzed.
3. RNA-interference targeting HIF-1αwas used to silence the expression of HIF-1αin human EC9706 cell line.
4. Growth inhibition rates of EC9706 cells treated with cisplatian/platixal were measured by MTS colorimetric assay under normoxic or hypoxic condition after RNA interference under hypoxia.
5. The expressions of MRP1, Bcl-2 and Bax were measured respectively by RT-PCR, immunocytochemistry in EC9706 cells under normia, hypoxia and after RNA interference under hypoxia.
6. The apoptosis rate of EC9706 cells treated by platixal under hypoxia before and after RNAi was studied by TUNEL assay and Annexin V/PI double staining, respectively.
8. Cell cycles of EC9706 cells before and after RNAi under hypoxia were analyzed with flow cytometry.
10. Statistical analysis: The SPSS Statistical Package was used for all analyses. Association between the variables were tested by analysis of variance and x~2 test, t test. The standard of significant level wasα=0.05.
Results:
1. Of 48 specimens 21 (43.75%) had positive expression of HIF-1αprotein. Among 21 cases with positive expression of HIF-1α, there was none complete response case and 5 cases of partial responses, and the response rate was 23.81%; whereas in 27 eases with negative expression of HIF-1α, there were 8 cases of complete responses and 11 cases of partial responses, and the response rate was 70.37%. There was significant difference between two groups (P＜0.05).
2. After being treated by cisplatin (10μg/ml, 5μg/ml, 2μg/ml), the growth inhibition rates of EC9706 cells under normoxia were significant higher than those under hypoxia(P＜0.05). After being treated by platixal (100μg/ml, 50μg/ml, 20μg/ml) the growth inhibition rates of EC9706 cells under normoxia were significantly higher than those under hypoxia (P＜0.05) .
3. In the untransfected group, transfected with control siRNA group and transfected with HIF-1αsiRNA group treatedby cisplatin (10μg/ml, 5μg/ml, 2μg/ml) under hypoxia, respectively, the growth inhibition rates of EC9706 cells transfected with HIF-1αsiRNA were significantly higher than those in the untransfected group and transfected with control siRNA group (P＜0.05). In the untransfected group, transfected with control siRNA group and transfected with HIF-1αsiRNA group treated with platixal (100μg/ml, 50μg/ml, 20μ/ml) under hypoxia respectively, the growth inhibition rates of cells transfected with HIF-1αsiRNA were significantly higher than those in the untransfected group and transfected with control siRNA group (P＜0.05).
4. Expression of MRP1 on mRNA and protein level was significantly increased in EC9706 cells after hypoxia (P＜0.05). MRP1 expression on mRNA and protein level in cells transfected with HIF-1αsiRNA was significantly lower than those in untransfected group and transfected with control siRNA group under hypoxia (P<0.05).
5. TUNEL detection revealed that after being treated with platixal (50μg/ml)under hypoxia, the apoptosis rate in HIF-1αsiRNA group(38.63%) was significantly higher than that in untransfected group (12.54%), and the transfected with control siRNA group (13.69%) (P＜0.05).
6. Flow cytometer(Annexin V FITC/PI) revealed that after being treated with platixal (50μg/ml) under hypoxia, the apoptosis rate in the transfected with HIF-1αsiRNA group (36.57±3.36)% was significantly higher than that in untransfected group (13.54±2.61)%, and the transfected with control siRNA group (15.61±2.42)% (P＜0.05).
7. There was not significant difference of Bcl-2 expression on mRNA and protein level between normoxia group and hypoxia group (P＞0.05), as well as between the transfected with HIF-1αsiRNA group, the transfected with control siRNA group and the untransfected group (P＜0.05) .
8. The expression of Bax on mRNA and protein level was significantly decreased in EC9706 cells after hypoxia(P＜0.05). Bax expression on mRNA and protein level in cells transfected with HIF-1αsiRNA was significantly higher than those in untransfected group and transfected with control siRNA group under hypoxia(P＜0.05).
9. The results of flow cytometry showed that the cells in G_1-phase, S-phase were (69.05±1.30)% and (23.72±1.30)%, respectively during normoxia. The cells in G_1-phase, S-phase was (78.29±2.70)% and (11.94±2.70)%, respectively during hypoxia. An increase of G_1-phase and a decrease of S-phase was observed in EC9706 cells in response to hypoxia(P＜0.05); the cells in G_1-phase, S-phase of cells in HIF-1αsiRNA transfection group was (70.48±1.91)% and (21.86±1.90)%, respectively after hypoxia for 8h, whereas the cells in G_1-phase, S-phase of cells in transfected with control siRNA group was (77.86±2.31)% and (12.07±1.90)% respectively, under the same hypoxia. Compared with untransfected group and transfected with control siRNA group, the cells in G_1-phase decreased and the cells in S-phase increased significantly(P＜0.05).
Conclusions
1. The mRNA expression of HIF-1αin ESCC tissue is Significantly higher than that in corresponding normal mucosa. The level of HIF-1αmRNA expression is not correlated with clinicopathological features, VEGF expression or MVD.
2. There is positive correlation between the level of HIF-1αmRNA expression and the positive rate of HIF-1αprotein expression. HIF-1αprotein expression in ESCC tissue is correlated with lymph node metastasis and invasion depth. HIF-1αprotein expression is correlated with VEGF expression and MVD, which indicats that HIF-1αprotein expression in ESCC tissue is correlated with angiogenesis.
3. The expression of HIF-1αprotein was associated with chemotherapy response. The patients with positive expression of HIF-1αprotein have lower chemotherapeutic efficacy, suggesting that HIF-1αprotein can be used as a new indicator to predict chemotherapy response.
4. Chemical hypoxia inducer (Cobalt chloride, COCl_2) establishs a hypoxia model in vitro to mimic hypoxia environment in tumor, and overexpression of HIF-1αprotein in EC9706 cells can be induced, while the expression of HIF-1αmRNA does not change after hypoxia. It indicates that hypoxia mainly affects the expression HIF-1αon protein level. Therefore, it is more important pratice to measure HIF-1αexpression on protein in the treatment of ESCC.
5. RNA interference targeting HIF-1αmakes a specificity of HIF-1αsilencing.
6. The overexpression of VEGF regulated by HIF-1αcould be attenuated markedly by HIF-1αsiRNA treatment. Therefore, to silence the expression of HIF-1αmay inhibit the angiogenesis, which offers an avenue of anti-angiogenesis gene therapy for ESCC.
7. EC9706 cells under hypoxia is resistant to cisplatin and taxol, the two different chemotherapeutic drugs. Whereas RNAi silencing HIF-1αgene can reverse the resistance to the two drugs.
8. HIF-1αcan upregulate the expression of MRP1, which may involve in the chemotherapeutic resistance of EC9706 cells under hypoxia.
9. Hypoxia-mediated down-regulation of Bax in EC9706 cells occurs via HIF-1α-dependent mechanism and contributes to chemoresistance.
10. HIF-1αis involved in the cell cycle arrest in EC9706 cells in response to hypoxia which may be another therapeutic resistance mechanism under hypoxia.
11. The overexpression of HIF-1αinduces angiogenesis and resistance to varied chemotherapeutic drugs. Therefore, HIF-1αcan be used as a novel, tumor-specific target for anticancer therapy in ESCC. This would offer one new way and a theoretic foundation to target therapy for ESCC.
Original points:
1. The expression of HIF-1αon mRNA and protein level in ESCC was systematically studied. The results indicated that HIF-1αprotein expression can be regarded as a new index to predict chemotherapeutic response in ESCC.
2. RNAi and chemical mimic hypoxia were used to further explore the expression of VEGF on mRNA and protein level. The results indicated that HIF-1αprotein was involved in angiogenesis through upregulating VEGF expression.
3. The complex mechanism of cell resistance to chemotherapeutic drugs under hypoxia in ESCC was explored systematically. The results indicated that HIF-1αprotein was involved in chemoresistance under hypoxia by by inducing cell cycle arrest in G_1 phrase and up-regulation the expression of MRP1 and down-regulation the expression of Bax which play a role in apoptosis.
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