HIF-1α在肝细胞癌中的表达及反义HIF-1α寡核苷酸对HepG2细胞增殖的抑制作用
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摘要
第一部分HIF-1α、VEGF及MvD在肝细胞癌中的表达及临床意义
缺氧诱导因子-1(hypoxia inducible factor-1,HIF-1)作为一种重要的转录激活因子,能诱导多种缺氧反应性表达,可促进包括血管内皮生长因子(Vascular Endothelial Growth Fatcor,VEGF)在内的多种血管生长相关基因转录,促进肿瘤血管生成,并在促进肿瘤细胞增殖和转移、调节糖代谢、增加肿瘤耐药性等方面发挥重要作用,与肿瘤的发展与转移密切相关。其中,HIF-1α是HIF-1的活性亚基。为进一步探讨HIF-1α在肝细胞癌(HepatocellularCarcinoma,HCC)发生、发展和转移中的作用,本研究将在第一部分采用免疫组化方法观察人HCC及癌旁组织中HIF-1α、VEGF及微血管密度(microvesseldensity,MVD)的表达及其临床意义。
材料和方法
1、一般资料收集2005年12月—2006年12月浙江大学医学院附属第一医院手术切除肝脏标本55例,并经病理检查证实。其中HCC组织45例,并取距离肿瘤边缘1cm处组织为癌旁组织。正常肝脏组织10例。
2、主要试剂HIF-1α鼠抗人多克隆抗体,购自Lab Vision公司:VEGF鼠抗人多克隆抗体,购自长岛生物技术公司;CD34鼠抗人多克隆抗体,购自长岛生物技术公司;PV9000试剂盒,购自北京中山生物技术公司。
3、实验方法HIF-1α、VEGF、CD34采用免疫组化PV9000二步法,染色步骤按试剂盒说明书进行。
4、结果判断HIF-1α染色综合染色强度和阳性细胞占总细胞数的百分比进行半定量评分。染色强度:1分,染色弱但明显强于阴性对照;2分,染色清晰,中等程度;3分,染色程度强。阳性细胞数:2分,阳性细胞占11-50%;3分,阳性细胞占51-80%;4分,阳性细胞＞81%。上述两项评分相加,不论染色强度,阳性细胞≤10%为(-),3分为(+),4-5分为(++),6-7分为(+++),其中以(++)和(+++)为强阳性。VEGF判别标准为:随机选择5个视野,计算100个细胞/视野,共计500个,阳性细胞≦10%为阴性,阳性细胞＞10%为阳性。微血管密度(MVD)计数方法:在200倍高倍镜下随机选择4个不同视野计数,计算每例标本的单位视野平均血管数。
5、统计学处理数据统计在SPSS 13.0统计软件上进行。样本率的比较采用X~2检验;计量资料两样本均数比较t检验:多个样本均数比较采用单因素方差分析;参数间相关性采用Spearman等级相关分析。以P＜0.05为差异有显著性。
结果
1、HIF-1α、VEGF与MVD在HCC、癌旁及正常组织中的表达HIF-1α在HCC组织中广泛表达,与正常及癌旁组织比较,HIF-1α表达显著增高,表达阳性率和强阳性率分别为91.1%和68.9%。在癌旁组织中,表达阳性率和强阳性率分别为20.0%和6.7%。10例正常组织中,仅1例阳性。癌旁组织与正常组织比较,差异无显著性。VEGF在HCC中表达阳性率为53.3%;在癌旁组织中,表达阳性率为13.3%。而正常组织中均为阴性表达。与正常及癌旁组织比较,HCC组织中VEGF表达显著增高。与正常组织比较,癌旁组织中VEGF表达差异无显著性。HCC组织MVD值较癌旁、正常组织明显增高,为52.2±18.3;癌旁组织与正常组织的MVD值分别为23.2±9.8和16.5±9.7,差异无显著性。
2、HCC中HIF-1α、VEGF表达及MVD与临床病理的关系在HCC中,HIF-1α表达强阳性率在高分化、中分化和低分化HCC分别为16.7%、62.5%和100.0%,差异具显著性;在伴门脉癌栓形成和无癌栓形成HCC组织中,HIF-1α强阳性率分别为100.0%和60.0%;在瘤体直径＞5cm和≤5cm HCC组织中,HIF-1α强阳性率分别为86.3%和52.2%,组间差异均具统计学意义。而随远处转移的发生,HIF-1α表达阳性程度有增高趋势,但无统计学意义。VEGF表达随HCC组织分型减低呈增高趋势,但无统计学意义。在伴门脉癌栓形成和无癌栓形成HCC组织中,VEGF表达阳性率分别为90.0%和42.9%,差异具显著性。VEGF表达随远处转移发生、肿瘤体积增大而增高,但无统计学意义。在高分化HCC和低分化HCC组织中,MVD均值分别为40.5±14.8和61.0±17.2,差异具显著性。在伴发门脉癌栓形成、远处转移发生HCC组织中,MVD均值分别为71.3±12.7和64.1±12.2,与无门脉癌栓形成(46.8±16.0)和远处转移发生(49.7±18.5)HCC组织相比,组间差异具有统计学意义。MVD均值随肿瘤体积增大而增高,但无统计学意义。
3、HCC癌旁HIF-1α、VEGF表达及MVD与临床病理的关系在伴门脉癌栓形成和无癌栓形成的癌旁组织中,HIF-1α表达阳性率分别为50.0%和11.4%,差异具显著性。随远处转移发生、伴发肝硬化、HCC组织分型减低,表达阳性程度有增高趋势,但无统计学意义。VEGF表达随门脉癌栓形成、远处转移发生、HCC组织分型减低、伴发肝硬化呈增高趋势,但无统计学意义。在低分化HCC癌旁组织,MVD均值为28.5±10.6,与高分化(16.6±7.1)和中分化(21.6±8.4)HCC癌旁组织相比,差异具有统计学意义。在伴发门脉癌栓形成、远处转移发生及肝硬化的癌旁组织中,MVD均值分别为33.7±5.6、31.5±9.5和26.2±10.8,与无门脉癌栓形成(20.2±8.6)、无远处转移发生(21.4±8.7)及不伴肝硬化(20.1±7.6)癌旁组织相比,组间差异具有统计学意义。
4、HIF-1α与VEGF、MVD表达的关系在HCC中,HIF-1α阳性表达程度与VEGF表达、MVD均值有显著的正相关性(r=0.545,r=0.705)。HIF-1α表达(+)、(++)和(+++)癌组织中MVl)分别为36.5±7.9、53.8±14.3、75.3±9.1。VEGF阳性表达的HCC组织其MVD(64.1±15.9)明显高于VEGF阴性HCC组织(38.7±10.9),VEGF阳性表达程度与MvD呈正相关(r=0.710)。在HCC癌旁组织中,HIF-1α表达与VEGF表达、MVD值同样具有相关性(r=0.784,r=0.608)。HIF-1α阴性与HIF-1α阳性表达癌旁组织的MVD值分别为20.0±7.6和36.0±6.2。VEGF阳性表达的癌旁组织MVD值为38.5±6.0,明显高于VEGF阴性癌旁组织(20.9±7.9),VEGF阳性表达程度与MVD呈正相关(r=0.554)。
本研究采用免疫组化PV9000二步法检测肝组织中HIF-1α,VEGF和MVD的表达,结果表明:
1、HIF-1α,VEGF和MVD在HCC肿瘤组织中表达上调,与癌旁组织以及正常肝组织有显著性差异。提示上述各种因子可能在HCC发生发展过程中起重要作用。
2、HIF-1α,VEGF和MVD表达上调与HCC临床病理特征有一定的相关性。其中,HIF-1α表达与肿瘤组织分型、肿瘤体积、门脉癌栓形成密切相关;VEGF表达与门脉癌栓形成密切相关;MVD与肿瘤组织分型、肿瘤远处转移、门脉癌栓形成密切相关。
3、HIF-1α,VEGF和MVD在HCC肿瘤组织及癌旁组织中的表达具有相关性。
第二部分HIF-1αAS-ODN对肝癌细胞HepG2增殖的抑制作用
由于HIF-1α在肝癌发生、发展与转移中的关键作用,HIF-1α可能成为肝癌治疗的新靶向,在抑制肝癌血管生成、抑制肝癌细胞增殖和转移、降低肝癌细胞对缺氧的耐受性等环节中发挥作用。反义寡核苷酸(antisenseoligodeoxynucleotides,AS-ODN)是人工合成的单链小片段核苷酸,能在转录水平或翻译水平特异性地抑制靶基因的表达,以达到治疗效果。
为进一步研究HIF-1αAS-ODN对肝癌细胞生长的抑制作用,本研究将在第二部分实验中设计和合成若干条HIF-1αAS-ODN,以脂质体介导转染HepG2细胞,观察AS-ODN对肝癌细胞增殖的抑制作用,选择出细胞生长抑制效应最强的AS-ODN,并进一步验证该AS-ODN对肝癌细胞靶基因HIF-1αmRNA转录和翻译的抑制作用。
材料与方法
1、实验材料HIF-1αAS-ODN_(1-6),由北京军事医学科学院放射研究所设计、合成、修饰和纯化。HIF-1αRT-PCR引物,北京军事医学科学院放射研究所合成。PCR反应试剂盒,购于美国Promega公司;Trizol RNA提取试剂盒、购于Gibco BRL公司;HIF-1α抗体(一抗和二抗),购自stressgen公司;β-actin抗体(一抗和二抗),购自Santa CruzBiotechnology公司。
2、实验方法
2.1脂质体寡核苷酸转染肝癌细胞HepG2培养至对数生长期后,将HepG2细胞接种到96孔板(4×10~3细胞/孔/100μL)。培养至细胞贴壁且丰度达到40%～60%时,吸弃各孔中的培养液。按脂质体说明书进行AS-ODN的转染。用无血清无抗生素DMEM细胞培养液分别配制各序列及浓度寡核苷酸和阳离子脂质体溶解液,室温静置40分钟。振荡并混和寡核苷酸液和阳离子脂质体液成转染液,使脂质体充分包裹寡核苷酸。转染液混匀后滴加至细胞上,置于培养箱转染6小时。每个浓度寡核苷酸设置4个平行孔。吸弃各孔转染液,加入100μl含血清的DMEM培养液。封闭缺氧的条件下置于37℃、5%CO_2孵箱继续培养。
2.2 MTS检测法脂质体寡核苷酸转染96孔板后继续培养72小时后,每孔加入20μl MTS(5mg/ml),在微量振荡器上混匀后,置于37℃、5%CO_2孵箱继续培养90分钟后,用多功能酶标仪(Wallac美国)在492nm测定吸光值,并计算细胞增殖抑制率(Ip)。
2.3 RT-PCR法脂质体寡核苷酸转染后继续培养48小时,提取HepG2细胞RNA,取2μg RNA加入20μl逆转录体系中,42℃孵育1小时反转录合成cDNA,72℃,5分钟,灭活逆转录酶,中止反应。PCR反应体系:cDNA1μl,Taq酶0.5μl,PCR mix 15μl,20μmol/L内参照β-actin和HIF-1α上、下游引物各0.5μl,去离子水补充至20μl。PCR扩增条件:94℃预循环4分钟;94℃变性30秒,56℃退火30秒,72℃延伸30秒,30个循环;最后72℃延伸5分钟。产物进行2%琼脂糖电泳。利用ImageQuant5.2软件读取各条电泳带的信号值,计算抑制率Im。
2.4 Western blot检测脂质体寡核苷酸转染后继续培养48小时,提取HepG2细胞蛋白,改良Lowry法进行蛋白定量后将各组蛋白提取物调至相同浓度。取60μg/10μl蛋白与等体积2×SDS上样缓冲液混合均匀,煮沸5分钟充分变性。用8%的SDS-聚丙烯酰胺凝胶进行电泳分离。凝胶行半干转移至PVDF膜。用含1%脱脂奶粉的TBST缓冲液室温封闭1小时。室温结合β-actin和HIF-1α一抗1小时,浓度比例(1:400),用TBST洗涤;室温结合β-actin和HIF-1α二抗1小时,浓度比例(1:2000),用TBST洗涤后加ECL显色剂反应2分钟,X光胶片成像。
3、统计学处理数据统计在SPSS 13.0统计软件上进行。计量资料多个样本均数比较采用单因素方差分析,以P＜0.05为差异有显著性。
结果
1、HIF-1αAS-ODN细胞水平活性筛选脂质体法AS-ODN转染HepG2细胞72小时后,采用MTS检测法测定各序列对HepG2细胞增殖的抑制率。结果显示,与脂质体对照组相比,AS-ODN_(1-6)各序列均可以显著抑制HepG2细胞的增殖(p＜0.01),随剂量增大,抑制作用呈增强趋势,当浓度为0.8μmol/L时,AS-ODN_(1-6)的细胞增殖抑制率分别达57.2±2.4%、61.8±1.9%、57.4±2.6%、49.1±3.4%、53.3±3.1%和53.7±2.7%。尤其是AS-ODN_2当浓度为0.2μmol/L、0.4μmol/L和0.8μmol/L时,细胞增殖抑制率分别为37.8±2.0%、49.2±1.7%、61.8±1.9%;与其它反义链相比,AS-ODN_2抑制HepG2细胞增殖作用更明显,具有显著性差异(p＜0.05)。因此我们筛选出AS-ODN_2作为反义药物继续以下研究。
2、HIF-1αAS-ODN_2对HepG2细胞增殖的抑制作用在AS-ODN_2对细胞增殖抑制作用的实验中,设置0.2、0.4、0.8、1.0μmol/L四个剂量,结果显示,与S-ODN相比,AS-ODN_2可以显著抑制HepG2细胞的增殖(p＜0.01);随剂量增大,抑制作用呈增强趋势,当浓度达到1.0μmol/L时,细胞增殖抑制率达89.7±3.8%。
3、HIF-1αAS-ODN_2对HepG2细胞HIF-1αmRNA表达的抑制作用脂质体法AS-ODN_2转染HepG2细胞48小时后,采用RT-PCR法,对AS-ODN_2抑制HepG2细胞HIF-1αmRNA表达进行检测。RT-PCR产物分析结果显示,与S-ODN相比,AS-ODN_2可以明显抑制HIF-1αmRNA的表达,浓度为0.4μmol/L和0.8μmol/L时,抑制率分别为79.5%和78.2%。
4、HIF-1αAS-ODN_2对HepG2细胞HIF-1α蛋白表达的抑制作用脂质体法AS-ODN_2转染HepG2细胞48小时后,采用Western blot印迹检测HIF-1αAS-ODN_2对HIF-1α蛋白表达的抑制。免疫印迹结果显示,与S-ODN和细胞对照组相比,AS-ODN_2可以显著抑制相应蛋白的表达。该抑制作用随浓度增加有增强趋势,当浓度为0.8μmol/L时,HIF-1α蛋白表达基本受到抑制。
结论
本研究合成6条HIF-1α硫代AS-ODN,阳离子脂质体法转染HepG2细胞,观察其对HepG2细胞增殖、HIF-1αmRNA转录及翻译的抑制作用,结果表明:
1、HIF-1αAS-ODN_(1-6)各序列均具有不同程度抑制HepG2细胞增殖的作用,当浓度为0.8μmol/L时,AS-ODN_(1-6)的细胞增殖抑制率分别达57.2±2.4%、61.8±1.9%、57.4±2.6%、49.1±3.4%、53.3±3.1%和53.7±2.7%。
2、与其它反义链相比,HIF-1αAS-ODN_2抑制HepG2细胞增殖作用最明显,当浓度达到1.0μmol/L时,细胞增殖抑制率达89.7±3.8%。
3、HIF-1αAS-ODN_2可以明显抑制HepG2细胞HIF-1αmRNA和HIF-1α蛋白的表达,提示HIF-1αAS-ODN_2对肝癌HepG2细胞增殖的抑制作用,由通过阻断HIF-1αmRNA的转录及翻译而实现。
Part 1 Expression and significance of HIF-1α, VEGF and MVD in hepatocellular carcinoma
Introduction
Hepatocellular carcinoma(HCC)is one of the most common malignancies in China.Owing to the improvement of surgical technique and early diagnostic methods,the resection rate of HCC has increased. However,the postoperative relapse and metastatic rate remains high. Neovascularization may play important roles in the relapse and metastasis of HCC.Hypoxia-inducible factor 1(HIF-1)is a heterodimeric transcriptional factor composed of theαandβsubunits.HIF-1αis the O_2-regulated subunit that determines HIF-1 activity.A series of genes and proteins that may increase the survival of tumor cells under hypoxia conditions,including vascular endothelial growth factor(VEGF),are regulated by HIF-1α.Thus,HIF-1αmay play important roles in tumor progression and neovascularization.The objective of this clinical study is to investigate the expression of HIF-1α,VEGF and microvessel density (MVD)in HCC and to explore their correlation with clinical pathological features.
Materials and Methods
Patients
Forty-five patients with HCC who underwent hepatic resection between Dec 2005 and Dec 2006 are included in this study.Paraneoplastic liver tissue is taken from non-cancerous tissue 1cm away from the tumor margin. Ten samples of normal liver tissue were also included.All the sections are re-evaluated and the diagnosis are confirmed.
Immunohistochemistry
Formalin-fixed,paraffin-embedded tissue specimens are obtained. Serial 4μmol/L sections are prepared,and one is stained with H&E. HIF-1α,VEGF and CD34 were detected by PV9000 immunohistochemistry according to the manufacturer's instruction.
Expression of HIF-1αwas determined by assessing semiquantitatively the percentage of stained tumor cells and the staining intensity.The percentage of positive cells was rated as follows:2 points,11-50% positive cells;3 points,51-80%positive cells;4 points,＞81% positive cells.The staining intensity was rated as follows:1 points, weak intensity;2 points,moderate intensity;3 points,strong intensity; points for expression and percentage of positive cells were added.(-), regardless of intensity,≤10%of cells stained positive;(+),3 points; (++),4-5 points;(+++),6-7 points.The results for VEGF are classified as follows:(-),≤10%positive cells;(+),＞10%positive cells. Microvessel density,assessed by immunostaining for CD34,is determined by counting all vessels at a total magnification of×200.
Statistical analysis
All statistics were calculated through SPSS 13.0 software. Chi-square test,t test,One-way ANOVA and Spearman coefficient of correlation are used as appropriate.P＜0.05 was considered statistically significant.
Results
Expression of HIF-1α,VEGF and MVD in liver tissue
The expression of HIF-1αin HCC was significantly higher than that in paraneoplastic tissue and normal liver tissue.The positive rate and strong positive rate of HIF-1αin HCC were 91.1%and 68.9%respectively, whereas the positive rate and strong positive rate of HIF-1αin paraneoplastic tissue are 20.0%and 6.7%respectively,and only 1 mild positive staining was observed in normal liver tissue.
Compared with paraneoplastic tissue and normal liver tissue,the expression of VEGF in HCC is significantly higher.The positive rate of VEGF in HCC and paraneoplastic tissue is 53.3%and 13.3%respectively, whereas no positive staining observed in normal liver tissue.
Compared with paraneoplastic tissue and normal liver tissue,the MVD value in HCC is significantly higher.The MVD value in HCC, paraneoplastic tissue and normal liver tissue is 52.2±18.3,23.2±9.8 and 16.5±9.7 respectively.
Association of HIF-1α,VEGF and MVD in HCC with clinicopathological data
Significant association is found between HIF-1αand HCC differentiation degree,complication with portal venous tumor thrombus (PVTT)and tumor size.The strong positive rate of HIF-1αin poor differentiated,moderate differentiated and well differentiated HCC is 100.0%,62.5%and 16.7%,in HCC with PVTT and without PVTT is 100.0 %and 60.0%,and in tumor size more than 5cm and smaller than 5cm is 86.3%and 52.2%respectively.The expression of HIF-1αin HCC with extrahepatic metastasis showed increase tendency,but had no statistical significance.
The expression of VEGF in HCC with PVTT are significantly higher than those in HCC without PVTT,the positive rate is 90.0%and 42.9% respectively.The expression of VEGF in HCC with extrahepatic metastasis, poor differentiation and enhancing tumor size also showed increase tendency,but had no statistical significance.
The MVD value in poor differentiated HCC(61.0±17.2)was significant higher than that in well differentiated HCC(40.5±14.8).Compared with the MVD value in HCC without PVTT(46.8±16.0)and HCC without extrahepatic metastasis(49.7±18.5),those in HCC with PVTT(71.3±12.7) and in HCC with extrahepatic metastasis(64.1±12.2)were significant increased.The MVDvalue showed increase tendency with enhancing tumor size,but had no statistical significance.
Association of HIF-1α,VEGF and MVD in paraneoplastic tissue with clinicopathological data
In paraneoplastic tissue,significant difference of HIF-1α expression is found between HCC complicated with PVTT and without PVTT, the positive rate is 50.0%and 11.4%respectively.The expression of HIF-1αin paraneoplastic tissue complicated with extrahepatic metastasis,liver cirrhosis or decreased differentiation degree showed increase tendency,but had no statistical significance.
The expression of YEGFin paraneoplastic tissue complicated with PVTT, extrahepatic metastasis,liver cirrhosis or decreased differentiation degree also showed increase tendency,but had no statistical significance.
The MVDvalue in paraneoplastic tissue with poor differentiated HCC (28.5±10.6)was significant higher than those with well differentiated HCC(16.6±7.1)and moderate differentiated HCC(21.6±8.4).Compared with the MVD value in paraneoplastic tissue complicated without PVTT(20.2±8.6),without extrahepatic metastasis(21.4±8.7)and without liver cirrhosis(20.1±7.6),those with PVTT(33.7±5.6), extrahepatic metastasis(31.5±9.5)and liver cirrhosis(26.2±10.8) were significant increased.
Correlation of HIF-1αexpression with VEGF expression and
Spearman correlation analysis showed the expression of HIF-1αin HCC was correlated with the expression of VEGF(r=0.545),and the expression of HIF-1αand VEGF in HCC as correlated with the value of MVD(r=0.705 and 0.710,respectively).The MVD value in HCC with HIF-1αexpression determined as(+),(++)and(+++)were 36.5±7.9,53.8±14.3 and 75.3±9.1 respectively.MVD value in HCC with VEGF expression determined as(-)and(+)were 38.7±10.9 and 64.1±15.9 respectively. In paraneoplastic tissue,the expression of HIF-1αwas correlated with the expression of VEGF(r=0.784),and the expression of HIF-1αand VEGF in paraneoplastic tissue as correlated with the value of MVD(r=0.608 and 0.554,respectively).MVD value in paraneoplastic tissue with HIF-1αexpression determined as negative and positive was 20.0±7.6 and 36.0±6.2 respectively.MVD value in paraneoplastic tissue with VEGF expression determined as(-)and(+)was 20.9±7.9 and 38.5±6.0 respectively.
Conclusions
1.The expression of HIF-1αand VEGF and the value of MVD in HCC was significantly higher than those in paraneoplastic tissue and normal liver tissue,suggest HIF-1αand VEGF may play important roles in HCC progression and neovascularization.
2.Significant association was found between the expression of HIF-1αand HCC differentiation degree,complication with PVTT and tumor size; and was found between the expression of VEGF and HCC complication with PVTT.Significant association was also found between the value of MVD and HCC differentiation degree,complication with PVTT and extrahepatic metastasis.
3.In HCC and paraneoplastic tissue,the expression of HIF-1αand VEGF had closely correlation with the value of MVD.
Part 2 Inhibitory effect of HIF-1αAS-ODN on proliferation of HepG2 cells
Introduction
Hepatocellular carcinoma(HCC)is one of the most common malignancies worldwide,especially in Asia and Afria.Patients with HCC usually present very poor prognosis inspire of improvement of diagnostic modalities and treatment in the recent years.
The hypoxia-inducible factor-1(HIF-1),an oxygen-sensitive transcriptional activator,is a key regulator responsible for the induction of genes that facilitate adaptation and survival of cells and the whole organism from normoxia to hypoxia.And the role of HIF-1αin tumor angiogenesis and cellular adaptation to the hypoxic microenvironment has been well established.Antisense oligodeoxynucleotide(AS-ODN)is a single-strand DNA that is complementary to specific regions of mRNA and capable of inhibiting expression of that gene.AS-ODNs are widely used for the elucidation of gene and protein function and as therapeutic agents in clinical trials.
In the present study,we designed 6 AS-ODNs targeted against the HIF-1αgene to identify the most potent AS-ODN sequence that specifically inhibit HepG2 cells proliferation.And then the most potent AS-ODN sequence of HIF-1αwith various concentrations were employed respectively to inhibit the HIF-1αmRNA and protein in the HepG2 cells, and their inhibitory effect was examined.
Materials and Methods
Preparation of HIF-1αAS-ODN
AS-ODNs to the human sequence of HIF-1αmRNA were synthesized by using an automated DNA synthesizer.After deprotection,AS-ODNs were purified by HPLC.
Oligodeoxynucleotide Transfection
HepG2 cells at exponential phases of growth were inoculated into 96-well plates(4x10~3/100μl)in the presence of 10%fetal bovin serum(FBS). When cells grown to 40-60%confluence,the AS-ODNs were transfected into HepG2 cells with lipofectin reagent(Invitrogen,USA)according to the manufacturer's instructions.AS-ODNs with various concentrations, Liposomeand cell controls weredesigned,each group had fourwells.After transfectedfor 6h,the culture fluid were discarded,each well was added with 100μl DMEM supplement with 10%FBS,and incubated at hypoxic culture condition in humidified 5%CO_2 and air mixture at 37℃.
MTS analysis
The cells were harvested at 72h after transfection,each well was added with 20μl MTS(5mg/ml)and incubated in humidified 5%CO_2 and air mixture at 37℃for another 90min.The optical density(OD)values of the slides were read on enzyme-labeled reader at the wavelength of 492 nm. The proliferation inhibition rate were calculated. RT-PCR analysis
48h after transfection,total RNA was isolated from cells of each group by Trizol Reagent(GIBCO).Reverse transcription was carried out on 2μg of total RNA,20μl reaction system contained 0.5μl /mL oligodT 1μl,5×RT buffer 4μl,10mM dNTP 1μl,RNase 0.5μl,25mM MgCl_2 4μl,AMV 1.5μl,8μl ddH_2O,incubated at 42℃for 1h,heated to 72℃for 5 min to inactivate AMV reverse transcriptase.PCR reaction system contained 1μL RT products,15μl PCR mixture,0.5μl 20μmol/L forward primer and 0.5μl 20μmol/L reverse primer(forward primer:5'- CATTACCCA CCGCTGAAACGCC-3', reverse peimer:5'-CTGGGACTATTAGGCTCAGGTG-3'),0.5μl Taq DNA polymerase and 1.5μl ddH_2O.PCR amplification was conducted in following condition: pre-denaturation at 94℃for 4 min,denaturation at 94℃for 30 second, annealing at 56℃for 30 second,and extension at 72℃for 30s.After 30 amplification cycles the products were extended at 72℃for 5 min. The RT-PCR products were visualized byβ-actin and analyzed by 2%agarose gel electrophroesis.
Western Blot Analysis
48h after transfection,total protein was isolated and quantified by Lowry methods.Proteins(6Oμg/10μl)were boiled at 100℃in equal volume 2×SDS loading buffer for 5 min.Each sample was electrophoresed on a 8%of SDS-polyacriylamide gel,and the fractionated proteins were then transferred to PVDF membranes(Amersham Bioscience)by semi-dry transfer apparatus.The membranes were blocked with 1%skim milk in TBST for 1h at room temperature,and then incubated with primary antibodies against HIF-1αandβ-actin(1:400 dilution)for 1h at room temperature. After washing with TBST three times,the membranes were incubated for 1h with secondary antibody against HIF-1a andβ-actin(1:2000 dilution) and finally visualized using a chemiluminescence detection kit ECL.
Statistical analysis
Significance of statistical difference was analyzed by using ANOVA test,a value of P＜0.05 was considered as significant.All statistics are calculated through SPSS 13.0 software.
Results
Inhibition of cell proliferation by HIF-1αAS-ODN_(1-6)
HepG2 cell growth was inhibited by 6 HIF-1αAS-ODNs at various concentrations significantly.At the concentration of 0.8μmol/L,the proliferation inhibitory rate were 57.2±2.4%,61.8±1.9%,57.4±2.6%, 49.1±3.4%,53.3±3.1%and 53.7±2.7%respectively.Among them,HIF-1αAS-ODN_2 showed the most effective proliferation inhibition ability(P＜0.01),the proliferation inhibitory rate went up to 37.8±2.0%,49.2±1.7%,61.8±1.9%at the concentration of 0.2μmol/L,0.4μmol/L and 0.8μmol/L respectively.
Inhibition of cell proliferation by HIF-1αAS-ODN_2
Then the HIF-1αAS-ODN_2 with various concentrations(0.2umol/L, 0.4umol/L,0.8umol/L,1.0umol/L)were transfected into HepG2 cells.The inhibition effect of HIF-1αAS-ODN_2 in cell proliferation showed increase tendency with increased concentration.At concentration of 1.0μmol/L,the proliferation inhibitory rate was 89.7±3.8%.Compared with the liposome control group,there was no inhibitory effect of S-ODN in HepG2 cell proliferation(P＞0.01).
Inhibitory effect of AS-ODN_2 on HIF-1αmRNA expression
Compared with the cell control and the S-ODN(0.4umol/L)control, HIF-1αmRNA expression were down-regulated by AS-ODN_2 48h after transfection at both concentration of 0.4μmol/L and 0.8μmol/L.And there was no significant difference between 0.4μmol/L and 0.8μmol/L concentration on the inhibitory effect of HIP-1αAS-ODN_2.
Inhibitory effect of AS-ODN_2 on HIF-1αprotein expression
Compared with the cell control and the S-ODN(0.4umol/L)control, HIF-1αprotein synthesis were decreased by AS-ODN_2 48h after transfection at both concentration of 0.4μmol/L and 0.8μmol/L.And the inhibitory effect of AS-ODN_2 at concentration of 0.8μmol/L was higher than 0.4μmol/L.
Conclusions
1.The HepG2 cells proliferation were significantly inhibited by HIF-1αAS-ODN_(1-6)at different degree.At the concentration of 0.8μmol/L,the proliferation inhibitory rate were 57.2±2.4%,61.8±1.9%,57.4±2.6%, 49.1±3.4%,53.3±3.1%and 53.7±2.7%respectively.
2.Among them,HIF-1αAS-ODN_2 showed the most effective proliferation inhibition ability.At the concentration of 1.0μmol/L,the proliferation inhibitory rate was up to 89.7±3.8%.
3.The expression of HIF-1αmRNA and synthesis of protein were reduced by HIF-1αAS-ODN_2.suggested that the mechanism of inhibition action on HepG2 cells proliferation is through the inhibition of transcription and translation of HIF-1αmRNA.
缺氧诱导因子-1(hypoxia inducible factor-1,HIF-1)作为一种重要的转录激活因子,能诱导多种缺氧反应性表达,可促进包括血管内皮生长因子(Vascular Endothelial Growth Fatcor,VEGF)在内的多种血管生长相关基因转录,促进肿瘤血管生成,并在促进肿瘤细胞增殖和转移、调节糖代谢、增加肿瘤耐药性等方面发挥重要作用,与肿瘤的发展与转移密切相关。其中,HIF-1α是HIF-1的活性亚基。为进一步探讨HIF-1α在肝细胞癌(HepatocellularCarcinoma,HCC)发生、发展和转移中的作用,本研究将在第一部分采用免疫组化方法观察人HCC及癌旁组织中HIF-1α、VEGF及微血管密度(microvesseldensity,MVD)的表达及其临床意义。
材料和方法
1、一般资料收集2005年12月—2006年12月浙江大学医学院附属第一医院手术切除肝脏标本55例,并经病理检查证实。其中HCC组织45例,并取距离肿瘤边缘1cm处组织为癌旁组织。正常肝脏组织10例。
2、主要试剂HIF-1α鼠抗人多克隆抗体,购自Lab Vision公司:VEGF鼠抗人多克隆抗体,购自长岛生物技术公司;CD34鼠抗人多克隆抗体,购自长岛生物技术公司;PV9000试剂盒,购自北京中山生物技术公司。
3、实验方法HIF-1α、VEGF、CD34采用免疫组化PV9000二步法,染色步骤按试剂盒说明书进行。
4、结果判断HIF-1α染色综合染色强度和阳性细胞占总细胞数的百分比进行半定量评分。染色强度:1分,染色弱但明显强于阴性对照;2分,染色清晰,中等程度;3分,染色程度强。阳性细胞数:2分,阳性细胞占11-50%;3分,阳性细胞占51-80%;4分,阳性细胞＞81%。上述两项评分相加,不论染色强度,阳性细胞≤10%为(-),3分为(+),4-5分为(++),6-7分为(+++),其中以(++)和(+++)为强阳性。VEGF判别标准为:随机选择5个视野,计算100个细胞/视野,共计500个,阳性细胞≦10%为阴性,阳性细胞＞10%为阳性。微血管密度(MVD)计数方法:在200倍高倍镜下随机选择4个不同视野计数,计算每例标本的单位视野平均血管数。
5、统计学处理数据统计在SPSS 13.0统计软件上进行。样本率的比较采用X~2检验;计量资料两样本均数比较t检验:多个样本均数比较采用单因素方差分析;参数间相关性采用Spearman等级相关分析。以P＜0.05为差异有显著性。
结果
1、HIF-1α、VEGF与MVD在HCC、癌旁及正常组织中的表达HIF-1α在HCC组织中广泛表达,与正常及癌旁组织比较,HIF-1α表达显著增高,表达阳性率和强阳性率分别为91.1%和68.9%。在癌旁组织中,表达阳性率和强阳性率分别为20.0%和6.7%。10例正常组织中,仅1例阳性。癌旁组织与正常组织比较,差异无显著性。VEGF在HCC中表达阳性率为53.3%;在癌旁组织中,表达阳性率为13.3%。而正常组织中均为阴性表达。与正常及癌旁组织比较,HCC组织中VEGF表达显著增高。与正常组织比较,癌旁组织中VEGF表达差异无显著性。HCC组织MVD值较癌旁、正常组织明显增高,为52.2±18.3;癌旁组织与正常组织的MVD值分别为23.2±9.8和16.5±9.7,差异无显著性。
2、HCC中HIF-1α、VEGF表达及MVD与临床病理的关系在HCC中,HIF-1α表达强阳性率在高分化、中分化和低分化HCC分别为16.7%、62.5%和100.0%,差异具显著性;在伴门脉癌栓形成和无癌栓形成HCC组织中,HIF-1α强阳性率分别为100.0%和60.0%;在瘤体直径＞5cm和≤5cm HCC组织中,HIF-1α强阳性率分别为86.3%和52.2%,组间差异均具统计学意义。而随远处转移的发生,HIF-1α表达阳性程度有增高趋势,但无统计学意义。VEGF表达随HCC组织分型减低呈增高趋势,但无统计学意义。在伴门脉癌栓形成和无癌栓形成HCC组织中,VEGF表达阳性率分别为90.0%和42.9%,差异具显著性。VEGF表达随远处转移发生、肿瘤体积增大而增高,但无统计学意义。在高分化HCC和低分化HCC组织中,MVD均值分别为40.5±14.8和61.0±17.2,差异具显著性。在伴发门脉癌栓形成、远处转移发生HCC组织中,MVD均值分别为71.3±12.7和64.1±12.2,与无门脉癌栓形成(46.8±16.0)和远处转移发生(49.7±18.5)HCC组织相比,组间差异具有统计学意义。MVD均值随肿瘤体积增大而增高,但无统计学意义。
3、HCC癌旁HIF-1α、VEGF表达及MVD与临床病理的关系在伴门脉癌栓形成和无癌栓形成的癌旁组织中,HIF-1α表达阳性率分别为50.0%和11.4%,差异具显著性。随远处转移发生、伴发肝硬化、HCC组织分型减低,表达阳性程度有增高趋势,但无统计学意义。VEGF表达随门脉癌栓形成、远处转移发生、HCC组织分型减低、伴发肝硬化呈增高趋势,但无统计学意义。在低分化HCC癌旁组织,MVD均值为28.5±10.6,与高分化(16.6±7.1)和中分化(21.6±8.4)HCC癌旁组织相比,差异具有统计学意义。在伴发门脉癌栓形成、远处转移发生及肝硬化的癌旁组织中,MVD均值分别为33.7±5.6、31.5±9.5和26.2±10.8,与无门脉癌栓形成(20.2±8.6)、无远处转移发生(21.4±8.7)及不伴肝硬化(20.1±7.6)癌旁组织相比,组间差异具有统计学意义。
4、HIF-1α与VEGF、MVD表达的关系在HCC中,HIF-1α阳性表达程度与VEGF表达、MVD均值有显著的正相关性(r=0.545,r=0.705)。HIF-1α表达(+)、(++)和(+++)癌组织中MVl)分别为36.5±7.9、53.8±14.3、75.3±9.1。VEGF阳性表达的HCC组织其MVD(64.1±15.9)明显高于VEGF阴性HCC组织(38.7±10.9),VEGF阳性表达程度与MvD呈正相关(r=0.710)。在HCC癌旁组织中,HIF-1α表达与VEGF表达、MVD值同样具有相关性(r=0.784,r=0.608)。HIF-1α阴性与HIF-1α阳性表达癌旁组织的MVD值分别为20.0±7.6和36.0±6.2。VEGF阳性表达的癌旁组织MVD值为38.5±6.0,明显高于VEGF阴性癌旁组织(20.9±7.9),VEGF阳性表达程度与MVD呈正相关(r=0.554)。
本研究采用免疫组化PV9000二步法检测肝组织中HIF-1α,VEGF和MVD的表达,结果表明:
1、HIF-1α,VEGF和MVD在HCC肿瘤组织中表达上调,与癌旁组织以及正常肝组织有显著性差异。提示上述各种因子可能在HCC发生发展过程中起重要作用。
2、HIF-1α,VEGF和MVD表达上调与HCC临床病理特征有一定的相关性。其中,HIF-1α表达与肿瘤组织分型、肿瘤体积、门脉癌栓形成密切相关;VEGF表达与门脉癌栓形成密切相关;MVD与肿瘤组织分型、肿瘤远处转移、门脉癌栓形成密切相关。
3、HIF-1α,VEGF和MVD在HCC肿瘤组织及癌旁组织中的表达具有相关性。
第二部分HIF-1αAS-ODN对肝癌细胞HepG2增殖的抑制作用
由于HIF-1α在肝癌发生、发展与转移中的关键作用,HIF-1α可能成为肝癌治疗的新靶向,在抑制肝癌血管生成、抑制肝癌细胞增殖和转移、降低肝癌细胞对缺氧的耐受性等环节中发挥作用。反义寡核苷酸(antisenseoligodeoxynucleotides,AS-ODN)是人工合成的单链小片段核苷酸,能在转录水平或翻译水平特异性地抑制靶基因的表达,以达到治疗效果。
为进一步研究HIF-1αAS-ODN对肝癌细胞生长的抑制作用,本研究将在第二部分实验中设计和合成若干条HIF-1αAS-ODN,以脂质体介导转染HepG2细胞,观察AS-ODN对肝癌细胞增殖的抑制作用,选择出细胞生长抑制效应最强的AS-ODN,并进一步验证该AS-ODN对肝癌细胞靶基因HIF-1αmRNA转录和翻译的抑制作用。
材料与方法
1、实验材料HIF-1αAS-ODN_(1-6),由北京军事医学科学院放射研究所设计、合成、修饰和纯化。HIF-1αRT-PCR引物,北京军事医学科学院放射研究所合成。PCR反应试剂盒,购于美国Promega公司;Trizol RNA提取试剂盒、购于Gibco BRL公司;HIF-1α抗体(一抗和二抗),购自stressgen公司;β-actin抗体(一抗和二抗),购自Santa CruzBiotechnology公司。
2、实验方法
2.1脂质体寡核苷酸转染肝癌细胞HepG2培养至对数生长期后,将HepG2细胞接种到96孔板(4×10~3细胞/孔/100μL)。培养至细胞贴壁且丰度达到40%～60%时,吸弃各孔中的培养液。按脂质体说明书进行AS-ODN的转染。用无血清无抗生素DMEM细胞培养液分别配制各序列及浓度寡核苷酸和阳离子脂质体溶解液,室温静置40分钟。振荡并混和寡核苷酸液和阳离子脂质体液成转染液,使脂质体充分包裹寡核苷酸。转染液混匀后滴加至细胞上,置于培养箱转染6小时。每个浓度寡核苷酸设置4个平行孔。吸弃各孔转染液,加入100μl含血清的DMEM培养液。封闭缺氧的条件下置于37℃、5%CO_2孵箱继续培养。
2.2 MTS检测法脂质体寡核苷酸转染96孔板后继续培养72小时后,每孔加入20μl MTS(5mg/ml),在微量振荡器上混匀后,置于37℃、5%CO_2孵箱继续培养90分钟后,用多功能酶标仪(Wallac美国)在492nm测定吸光值,并计算细胞增殖抑制率(Ip)。
2.3 RT-PCR法脂质体寡核苷酸转染后继续培养48小时,提取HepG2细胞RNA,取2μg RNA加入20μl逆转录体系中,42℃孵育1小时反转录合成cDNA,72℃,5分钟,灭活逆转录酶,中止反应。PCR反应体系:cDNA1μl,Taq酶0.5μl,PCR mix 15μl,20μmol/L内参照β-actin和HIF-1α上、下游引物各0.5μl,去离子水补充至20μl。PCR扩增条件:94℃预循环4分钟;94℃变性30秒,56℃退火30秒,72℃延伸30秒,30个循环;最后72℃延伸5分钟。产物进行2%琼脂糖电泳。利用ImageQuant5.2软件读取各条电泳带的信号值,计算抑制率Im。
2.4 Western blot检测脂质体寡核苷酸转染后继续培养48小时,提取HepG2细胞蛋白,改良Lowry法进行蛋白定量后将各组蛋白提取物调至相同浓度。取60μg/10μl蛋白与等体积2×SDS上样缓冲液混合均匀,煮沸5分钟充分变性。用8%的SDS-聚丙烯酰胺凝胶进行电泳分离。凝胶行半干转移至PVDF膜。用含1%脱脂奶粉的TBST缓冲液室温封闭1小时。室温结合β-actin和HIF-1α一抗1小时,浓度比例(1:400),用TBST洗涤;室温结合β-actin和HIF-1α二抗1小时,浓度比例(1:2000),用TBST洗涤后加ECL显色剂反应2分钟,X光胶片成像。
3、统计学处理数据统计在SPSS 13.0统计软件上进行。计量资料多个样本均数比较采用单因素方差分析,以P＜0.05为差异有显著性。
结果
1、HIF-1αAS-ODN细胞水平活性筛选脂质体法AS-ODN转染HepG2细胞72小时后,采用MTS检测法测定各序列对HepG2细胞增殖的抑制率。结果显示,与脂质体对照组相比,AS-ODN_(1-6)各序列均可以显著抑制HepG2细胞的增殖(p＜0.01),随剂量增大,抑制作用呈增强趋势,当浓度为0.8μmol/L时,AS-ODN_(1-6)的细胞增殖抑制率分别达57.2±2.4%、61.8±1.9%、57.4±2.6%、49.1±3.4%、53.3±3.1%和53.7±2.7%。尤其是AS-ODN_2当浓度为0.2μmol/L、0.4μmol/L和0.8μmol/L时,细胞增殖抑制率分别为37.8±2.0%、49.2±1.7%、61.8±1.9%;与其它反义链相比,AS-ODN_2抑制HepG2细胞增殖作用更明显,具有显著性差异(p＜0.05)。因此我们筛选出AS-ODN_2作为反义药物继续以下研究。
2、HIF-1αAS-ODN_2对HepG2细胞增殖的抑制作用在AS-ODN_2对细胞增殖抑制作用的实验中,设置0.2、0.4、0.8、1.0μmol/L四个剂量,结果显示,与S-ODN相比,AS-ODN_2可以显著抑制HepG2细胞的增殖(p＜0.01);随剂量增大,抑制作用呈增强趋势,当浓度达到1.0μmol/L时,细胞增殖抑制率达89.7±3.8%。
3、HIF-1αAS-ODN_2对HepG2细胞HIF-1αmRNA表达的抑制作用脂质体法AS-ODN_2转染HepG2细胞48小时后,采用RT-PCR法,对AS-ODN_2抑制HepG2细胞HIF-1αmRNA表达进行检测。RT-PCR产物分析结果显示,与S-ODN相比,AS-ODN_2可以明显抑制HIF-1αmRNA的表达,浓度为0.4μmol/L和0.8μmol/L时,抑制率分别为79.5%和78.2%。
4、HIF-1αAS-ODN_2对HepG2细胞HIF-1α蛋白表达的抑制作用脂质体法AS-ODN_2转染HepG2细胞48小时后,采用Western blot印迹检测HIF-1αAS-ODN_2对HIF-1α蛋白表达的抑制。免疫印迹结果显示,与S-ODN和细胞对照组相比,AS-ODN_2可以显著抑制相应蛋白的表达。该抑制作用随浓度增加有增强趋势,当浓度为0.8μmol/L时,HIF-1α蛋白表达基本受到抑制。
结论
本研究合成6条HIF-1α硫代AS-ODN,阳离子脂质体法转染HepG2细胞,观察其对HepG2细胞增殖、HIF-1αmRNA转录及翻译的抑制作用,结果表明:
1、HIF-1αAS-ODN_(1-6)各序列均具有不同程度抑制HepG2细胞增殖的作用,当浓度为0.8μmol/L时,AS-ODN_(1-6)的细胞增殖抑制率分别达57.2±2.4%、61.8±1.9%、57.4±2.6%、49.1±3.4%、53.3±3.1%和53.7±2.7%。
2、与其它反义链相比,HIF-1αAS-ODN_2抑制HepG2细胞增殖作用最明显,当浓度达到1.0μmol/L时,细胞增殖抑制率达89.7±3.8%。
3、HIF-1αAS-ODN_2可以明显抑制HepG2细胞HIF-1αmRNA和HIF-1α蛋白的表达,提示HIF-1αAS-ODN_2对肝癌HepG2细胞增殖的抑制作用,由通过阻断HIF-1αmRNA的转录及翻译而实现。
Part 1 Expression and significance of HIF-1α, VEGF and MVD in hepatocellular carcinoma
Introduction
Hepatocellular carcinoma(HCC)is one of the most common malignancies in China.Owing to the improvement of surgical technique and early diagnostic methods,the resection rate of HCC has increased. However,the postoperative relapse and metastatic rate remains high. Neovascularization may play important roles in the relapse and metastasis of HCC.Hypoxia-inducible factor 1(HIF-1)is a heterodimeric transcriptional factor composed of theαandβsubunits.HIF-1αis the O_2-regulated subunit that determines HIF-1 activity.A series of genes and proteins that may increase the survival of tumor cells under hypoxia conditions,including vascular endothelial growth factor(VEGF),are regulated by HIF-1α.Thus,HIF-1αmay play important roles in tumor progression and neovascularization.The objective of this clinical study is to investigate the expression of HIF-1α,VEGF and microvessel density (MVD)in HCC and to explore their correlation with clinical pathological features.
Materials and Methods
Patients
Forty-five patients with HCC who underwent hepatic resection between Dec 2005 and Dec 2006 are included in this study.Paraneoplastic liver tissue is taken from non-cancerous tissue 1cm away from the tumor margin. Ten samples of normal liver tissue were also included.All the sections are re-evaluated and the diagnosis are confirmed.
Immunohistochemistry
Formalin-fixed,paraffin-embedded tissue specimens are obtained. Serial 4μmol/L sections are prepared,and one is stained with H&E. HIF-1α,VEGF and CD34 were detected by PV9000 immunohistochemistry according to the manufacturer's instruction.
Expression of HIF-1αwas determined by assessing semiquantitatively the percentage of stained tumor cells and the staining intensity.The percentage of positive cells was rated as follows:2 points,11-50% positive cells;3 points,51-80%positive cells;4 points,＞81% positive cells.The staining intensity was rated as follows:1 points, weak intensity;2 points,moderate intensity;3 points,strong intensity; points for expression and percentage of positive cells were added.(-), regardless of intensity,≤10%of cells stained positive;(+),3 points; (++),4-5 points;(+++),6-7 points.The results for VEGF are classified as follows:(-),≤10%positive cells;(+),＞10%positive cells. Microvessel density,assessed by immunostaining for CD34,is determined by counting all vessels at a total magnification of×200.
Statistical analysis
All statistics were calculated through SPSS 13.0 software. Chi-square test,t test,One-way ANOVA and Spearman coefficient of correlation are used as appropriate.P＜0.05 was considered statistically significant.
Results
Expression of HIF-1α,VEGF and MVD in liver tissue
The expression of HIF-1αin HCC was significantly higher than that in paraneoplastic tissue and normal liver tissue.The positive rate and strong positive rate of HIF-1αin HCC were 91.1%and 68.9%respectively, whereas the positive rate and strong positive rate of HIF-1αin paraneoplastic tissue are 20.0%and 6.7%respectively,and only 1 mild positive staining was observed in normal liver tissue.
Compared with paraneoplastic tissue and normal liver tissue,the expression of VEGF in HCC is significantly higher.The positive rate of VEGF in HCC and paraneoplastic tissue is 53.3%and 13.3%respectively, whereas no positive staining observed in normal liver tissue.
Compared with paraneoplastic tissue and normal liver tissue,the MVD value in HCC is significantly higher.The MVD value in HCC, paraneoplastic tissue and normal liver tissue is 52.2±18.3,23.2±9.8 and 16.5±9.7 respectively.
Association of HIF-1α,VEGF and MVD in HCC with clinicopathological data
Significant association is found between HIF-1αand HCC differentiation degree,complication with portal venous tumor thrombus (PVTT)and tumor size.The strong positive rate of HIF-1αin poor differentiated,moderate differentiated and well differentiated HCC is 100.0%,62.5%and 16.7%,in HCC with PVTT and without PVTT is 100.0 %and 60.0%,and in tumor size more than 5cm and smaller than 5cm is 86.3%and 52.2%respectively.The expression of HIF-1αin HCC with extrahepatic metastasis showed increase tendency,but had no statistical significance.
The expression of VEGF in HCC with PVTT are significantly higher than those in HCC without PVTT,the positive rate is 90.0%and 42.9% respectively.The expression of VEGF in HCC with extrahepatic metastasis, poor differentiation and enhancing tumor size also showed increase tendency,but had no statistical significance.
The MVD value in poor differentiated HCC(61.0±17.2)was significant higher than that in well differentiated HCC(40.5±14.8).Compared with the MVD value in HCC without PVTT(46.8±16.0)and HCC without extrahepatic metastasis(49.7±18.5),those in HCC with PVTT(71.3±12.7) and in HCC with extrahepatic metastasis(64.1±12.2)were significant increased.The MVDvalue showed increase tendency with enhancing tumor size,but had no statistical significance.
Association of HIF-1α,VEGF and MVD in paraneoplastic tissue with clinicopathological data
In paraneoplastic tissue,significant difference of HIF-1α expression is found between HCC complicated with PVTT and without PVTT, the positive rate is 50.0%and 11.4%respectively.The expression of HIF-1αin paraneoplastic tissue complicated with extrahepatic metastasis,liver cirrhosis or decreased differentiation degree showed increase tendency,but had no statistical significance.
The expression of YEGFin paraneoplastic tissue complicated with PVTT, extrahepatic metastasis,liver cirrhosis or decreased differentiation degree also showed increase tendency,but had no statistical significance.
The MVDvalue in paraneoplastic tissue with poor differentiated HCC (28.5±10.6)was significant higher than those with well differentiated HCC(16.6±7.1)and moderate differentiated HCC(21.6±8.4).Compared with the MVD value in paraneoplastic tissue complicated without PVTT(20.2±8.6),without extrahepatic metastasis(21.4±8.7)and without liver cirrhosis(20.1±7.6),those with PVTT(33.7±5.6), extrahepatic metastasis(31.5±9.5)and liver cirrhosis(26.2±10.8) were significant increased.
Correlation of HIF-1αexpression with VEGF expression and
Spearman correlation analysis showed the expression of HIF-1αin HCC was correlated with the expression of VEGF(r=0.545),and the expression of HIF-1αand VEGF in HCC as correlated with the value of MVD(r=0.705 and 0.710,respectively).The MVD value in HCC with HIF-1αexpression determined as(+),(++)and(+++)were 36.5±7.9,53.8±14.3 and 75.3±9.1 respectively.MVD value in HCC with VEGF expression determined as(-)and(+)were 38.7±10.9 and 64.1±15.9 respectively. In paraneoplastic tissue,the expression of HIF-1αwas correlated with the expression of VEGF(r=0.784),and the expression of HIF-1αand VEGF in paraneoplastic tissue as correlated with the value of MVD(r=0.608 and 0.554,respectively).MVD value in paraneoplastic tissue with HIF-1αexpression determined as negative and positive was 20.0±7.6 and 36.0±6.2 respectively.MVD value in paraneoplastic tissue with VEGF expression determined as(-)and(+)was 20.9±7.9 and 38.5±6.0 respectively.
Conclusions
1.The expression of HIF-1αand VEGF and the value of MVD in HCC was significantly higher than those in paraneoplastic tissue and normal liver tissue,suggest HIF-1αand VEGF may play important roles in HCC progression and neovascularization.
2.Significant association was found between the expression of HIF-1αand HCC differentiation degree,complication with PVTT and tumor size; and was found between the expression of VEGF and HCC complication with PVTT.Significant association was also found between the value of MVD and HCC differentiation degree,complication with PVTT and extrahepatic metastasis.
3.In HCC and paraneoplastic tissue,the expression of HIF-1αand VEGF had closely correlation with the value of MVD.
Part 2 Inhibitory effect of HIF-1αAS-ODN on proliferation of HepG2 cells
Introduction
Hepatocellular carcinoma(HCC)is one of the most common malignancies worldwide,especially in Asia and Afria.Patients with HCC usually present very poor prognosis inspire of improvement of diagnostic modalities and treatment in the recent years.
The hypoxia-inducible factor-1(HIF-1),an oxygen-sensitive transcriptional activator,is a key regulator responsible for the induction of genes that facilitate adaptation and survival of cells and the whole organism from normoxia to hypoxia.And the role of HIF-1αin tumor angiogenesis and cellular adaptation to the hypoxic microenvironment has been well established.Antisense oligodeoxynucleotide(AS-ODN)is a single-strand DNA that is complementary to specific regions of mRNA and capable of inhibiting expression of that gene.AS-ODNs are widely used for the elucidation of gene and protein function and as therapeutic agents in clinical trials.
In the present study,we designed 6 AS-ODNs targeted against the HIF-1αgene to identify the most potent AS-ODN sequence that specifically inhibit HepG2 cells proliferation.And then the most potent AS-ODN sequence of HIF-1αwith various concentrations were employed respectively to inhibit the HIF-1αmRNA and protein in the HepG2 cells, and their inhibitory effect was examined.
Materials and Methods
Preparation of HIF-1αAS-ODN
AS-ODNs to the human sequence of HIF-1αmRNA were synthesized by using an automated DNA synthesizer.After deprotection,AS-ODNs were purified by HPLC.
Oligodeoxynucleotide Transfection
HepG2 cells at exponential phases of growth were inoculated into 96-well plates(4x10~3/100μl)in the presence of 10%fetal bovin serum(FBS). When cells grown to 40-60%confluence,the AS-ODNs were transfected into HepG2 cells with lipofectin reagent(Invitrogen,USA)according to the manufacturer's instructions.AS-ODNs with various concentrations, Liposomeand cell controls weredesigned,each group had fourwells.After transfectedfor 6h,the culture fluid were discarded,each well was added with 100μl DMEM supplement with 10%FBS,and incubated at hypoxic culture condition in humidified 5%CO_2 and air mixture at 37℃.
MTS analysis
The cells were harvested at 72h after transfection,each well was added with 20μl MTS(5mg/ml)and incubated in humidified 5%CO_2 and air mixture at 37℃for another 90min.The optical density(OD)values of the slides were read on enzyme-labeled reader at the wavelength of 492 nm. The proliferation inhibition rate were calculated. RT-PCR analysis
48h after transfection,total RNA was isolated from cells of each group by Trizol Reagent(GIBCO).Reverse transcription was carried out on 2μg of total RNA,20μl reaction system contained 0.5μl /mL oligodT 1μl,5×RT buffer 4μl,10mM dNTP 1μl,RNase 0.5μl,25mM MgCl_2 4μl,AMV 1.5μl,8μl ddH_2O,incubated at 42℃for 1h,heated to 72℃for 5 min to inactivate AMV reverse transcriptase.PCR reaction system contained 1μL RT products,15μl PCR mixture,0.5μl 20μmol/L forward primer and 0.5μl 20μmol/L reverse primer(forward primer:5'- CATTACCCA CCGCTGAAACGCC-3', reverse peimer:5'-CTGGGACTATTAGGCTCAGGTG-3'),0.5μl Taq DNA polymerase and 1.5μl ddH_2O.PCR amplification was conducted in following condition: pre-denaturation at 94℃for 4 min,denaturation at 94℃for 30 second, annealing at 56℃for 30 second,and extension at 72℃for 30s.After 30 amplification cycles the products were extended at 72℃for 5 min. The RT-PCR products were visualized byβ-actin and analyzed by 2%agarose gel electrophroesis.
Western Blot Analysis
48h after transfection,total protein was isolated and quantified by Lowry methods.Proteins(6Oμg/10μl)were boiled at 100℃in equal volume 2×SDS loading buffer for 5 min.Each sample was electrophoresed on a 8%of SDS-polyacriylamide gel,and the fractionated proteins were then transferred to PVDF membranes(Amersham Bioscience)by semi-dry transfer apparatus.The membranes were blocked with 1%skim milk in TBST for 1h at room temperature,and then incubated with primary antibodies against HIF-1αandβ-actin(1:400 dilution)for 1h at room temperature. After washing with TBST three times,the membranes were incubated for 1h with secondary antibody against HIF-1a andβ-actin(1:2000 dilution) and finally visualized using a chemiluminescence detection kit ECL.
Statistical analysis
Significance of statistical difference was analyzed by using ANOVA test,a value of P＜0.05 was considered as significant.All statistics are calculated through SPSS 13.0 software.
Results
Inhibition of cell proliferation by HIF-1αAS-ODN_(1-6)
HepG2 cell growth was inhibited by 6 HIF-1αAS-ODNs at various concentrations significantly.At the concentration of 0.8μmol/L,the proliferation inhibitory rate were 57.2±2.4%,61.8±1.9%,57.4±2.6%, 49.1±3.4%,53.3±3.1%and 53.7±2.7%respectively.Among them,HIF-1αAS-ODN_2 showed the most effective proliferation inhibition ability(P＜0.01),the proliferation inhibitory rate went up to 37.8±2.0%,49.2±1.7%,61.8±1.9%at the concentration of 0.2μmol/L,0.4μmol/L and 0.8μmol/L respectively.
Inhibition of cell proliferation by HIF-1αAS-ODN_2
Then the HIF-1αAS-ODN_2 with various concentrations(0.2umol/L, 0.4umol/L,0.8umol/L,1.0umol/L)were transfected into HepG2 cells.The inhibition effect of HIF-1αAS-ODN_2 in cell proliferation showed increase tendency with increased concentration.At concentration of 1.0μmol/L,the proliferation inhibitory rate was 89.7±3.8%.Compared with the liposome control group,there was no inhibitory effect of S-ODN in HepG2 cell proliferation(P＞0.01).
Inhibitory effect of AS-ODN_2 on HIF-1αmRNA expression
Compared with the cell control and the S-ODN(0.4umol/L)control, HIF-1αmRNA expression were down-regulated by AS-ODN_2 48h after transfection at both concentration of 0.4μmol/L and 0.8μmol/L.And there was no significant difference between 0.4μmol/L and 0.8μmol/L concentration on the inhibitory effect of HIP-1αAS-ODN_2.
Inhibitory effect of AS-ODN_2 on HIF-1αprotein expression
Compared with the cell control and the S-ODN(0.4umol/L)control, HIF-1αprotein synthesis were decreased by AS-ODN_2 48h after transfection at both concentration of 0.4μmol/L and 0.8μmol/L.And the inhibitory effect of AS-ODN_2 at concentration of 0.8μmol/L was higher than 0.4μmol/L.
Conclusions
1.The HepG2 cells proliferation were significantly inhibited by HIF-1αAS-ODN_(1-6)at different degree.At the concentration of 0.8μmol/L,the proliferation inhibitory rate were 57.2±2.4%,61.8±1.9%,57.4±2.6%, 49.1±3.4%,53.3±3.1%and 53.7±2.7%respectively.
2.Among them,HIF-1αAS-ODN_2 showed the most effective proliferation inhibition ability.At the concentration of 1.0μmol/L,the proliferation inhibitory rate was up to 89.7±3.8%.
3.The expression of HIF-1αmRNA and synthesis of protein were reduced by HIF-1αAS-ODN_2.suggested that the mechanism of inhibition action on HepG2 cells proliferation is through the inhibition of transcription and translation of HIF-1αmRNA.
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