GRIM-19及其靶基因产物p-STAT3与人肝细胞肝癌的相关性研究
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摘要
研究背景与目的
肝细胞肝癌(hepatocellular carcinoma, HCC)是源自肝细胞的恶性肿瘤,是原发性肝癌中最常见的一种病理类型,世界上平均每年死于HCC的人数约有1,250,000,HCC的发生、发展是一个多基因参与的复杂过程。目前针对HCC的治疗,主要是外科手术切除病灶或者是通过介入手段进行局部化疗栓塞治疗,对HCC患者进行放疗和全身化疗的疗效均不理想。随着分子生物学的发展,免疫治疗和基因治疗等生物治疗方法作为继手术、化疗、放疗之后的第四种治疗模式,日益受到重视。
GRIMs(genes associated with retinoid-IFN-induced mortality)是新近由Kalvakolanu在研究干扰素(interferon, IFN)和维甲酸(retinoic acid, RA)合并诱导细胞凋亡时发现的一组参与细胞凋亡的基因,共24种,GRIM-19是其中最具代表性的一种。GRIM-19编码含144个氨基酸残基的蛋白质,分子量约16KD,定位于19p13.1,是一种保守基因,可以在多种正常组织中表达。目前GRIM-19蛋白的细胞定位存在争议,有研究认为GRIM-19主要定位于细胞核,也有研究认为GRIM-19主要表达于胞浆中的线粒体。Lu fei等则认为GRIM-19的定位因细胞种类不同而存在差异,目前国内外对GRIM-19在肝癌组织中的表达及功能都尚未见报道。研究表明,GRIM-19参与细胞增殖、凋亡的调控过程,该基因表达的降低或者位点的突变可以导致细胞的异常增殖和恶性转化,GRIM-19在肝细胞癌变的过程中发挥了怎样的作用,值得我们深入探讨。
GRIM-19可以特异性的与组成性活化的STAT3偶联,从而抑制其转录活性,并抑制肿瘤生长,对肿瘤治疗有巨大的潜在价值。这一机制在肾细胞癌、部分前列腺癌、HPV感染的宫颈癌等部分肿瘤组织中都有所报道。但在肝癌方面,只在体外培养的HepG2细胞中有过报道。STAT3可以被多种病毒致癌蛋白所激活,STAT3的组成性活化能促进细胞的恶性转化并阻断其凋亡,促进肿瘤细胞的增殖,血管形成,因此,阻断STAT3勺表达对肿瘤的治疗具有巨大的潜在价值。深入研究GRIM-19与P-STAT3的关系,探讨其对细胞转化、增殖、凋亡的调控机制,将有利于阐明肝癌的发生机制。
GRIM-19是由RA和IFNs合并诱导细胞时发现的参与细胞凋亡的基因。维甲酸是维生素A在体内产生的衍生物,属于分化诱导剂类,具有逆转癌前病变,诱导肿瘤细胞分化,提高肿瘤细胞对化疗的敏感性等作用；干扰素则通过与细胞表面受体结合,激活包括JAK-STAT途径、干扰素调节因子在内的多种信号转导途径,直接抑制肿瘤细胞的增殖。在临床上,单用维甲酸和干扰素治疗肿瘤的研究较多,联合治疗肿瘤的研究比较少,两药联合对肝癌细胞的影响及其诱导凋亡的机制尚不清楚。目前针对GRIM-19基因的生物学功能及作用机制的研究尚处于起步阶段,进一步认识其蛋白的功能、调节方式及其作用机制,将为深入了解肿瘤细胞的发生、发展提供新的线索,为肿瘤的诊断、治疗提供新的靶点和研究方向。
第一部分GRIM-19及P-STAT3在人肝细胞肝癌中的表达及临床意义
研究目的
1.检测GRIM-19在原发性肝细胞肝癌和癌旁组织中的表达及定位。
2.检测P-STAT3在原发性肝细胞肝癌和癌旁组织中的表达及定位。
3.分析GRIM-19及其靶基因STAT3、p-STAT3、VEGF在肝癌和癌旁组织中的相关性。
4.分析GRIM-19与原发性肝癌的病因、病理类型、分化程度等临床特征的相关性。
研究方法
1.收集55例手术之前未经任何治疗的原发性肝癌患者的临床资料及经手术切除的肝细胞癌组织及邻近的非癌肝组织(邻近非癌肝组织：肉眼观正常,且距离肿瘤组织边缘5cm以上),组织标本于手术切除后尽快采集,经脱水、石蜡包埋后切片,采用免疫组织化学染色法,检测GRIM-19和P-STAT3在肝癌组织和非癌肝组织中的表达和定位。
2.分别提取肝癌及癌旁肝组织的总蛋白和核蛋白,通过Western Blot的方法检测GRIM-19蛋白的表达情况。
3.分别提取肝癌及癌旁肝组织的RNA,通过RT-PCR检测GRIM-19在mRNA水平的表达情况。
4.分别提取肝癌及癌旁肝组织的总蛋白,通过Western Blot的方法检测GRIM-19及其靶基因STAT3、p-STAT3、VEGF的表达情况。
5.分析GRIM-19与临床病理特征的相关性。
结果
1.免疫组织化学染色结果判读
免疫组织化学染色结果由与本研究无关的两名病理医生评定,每张切片观察5-6个400×视野,计数不少于100个细胞,依据染色强度和阳性细胞数进行分级,染色强度记分为四个等级：阴性：阳性细胞数少于5%,以“-”表示；弱阳性：阳性细胞数在5%-25%之间,以“+”表示；中等阳性：阳性细胞数在25%-50%之间,以“十+”表示；强阳性：阳性细胞数在50%-100%之间,以“+++”表示。
2.GRIM-19在肝细胞癌、非癌肝组织中的表达和定位。
①免疫组织化学染色结果显示：在非癌肝组织中,GRIM-19蛋白阳性产物主要分布在细胞质,亦有少量阳性产物分布在细胞核,表现为不同染色程度的黄色或棕黄色颗粒；而存肝细胞癌组织中,GRIM-19阳性产物只存在于细胞质。
②分别提取肝细胞癌及癌旁组织的核蛋白,Western Blot检测结果显示GRIM-19蛋白在肝癌细胞核蛋白中的表达明显低于癌旁肝细胞的核蛋白(P<0.001).
3.GRIM-19在肝细胞癌和癌旁肝组织中的表达水平。
①免疫组织化学染色法结果显示：GRIM-19蛋白在肝细胞癌组织中的表达明显低于癌旁肝组织。对照组4例正常肝组织中GRIM-19表达阳性的有3例(3／4,75%)；55例癌旁肝组织中GRIM-19表达阳性的有40例(40／55,72.7%)；而55例肝细胞癌组织中GRIM-19表达阳性的只有25例(25／55,45.5%)。16例不伴随肝纤维化的癌旁组织中有12例GRIM-19表达阳性(12／16,75%)；39例伴随肝纤维化的癌旁组织中有28例GRIM-19表达阳性(28／39,71.8%),不伴随肝纤维化的癌旁组织与伴随肝纤维化的癌旁组织比较,GRIM-19蛋白的表达无明显差异(p>0.05)。GRIM-19在肝癌组织中的表达明显低于癌旁组织(p<0.05),甚至表达缺失。另外,65例GRIM-19表达阳性的肝组织中有23例为++～＋＋+(按结果判读标准分为中等阳性),在这23例中等阳性标本中有20例是癌旁组织(20／23,87%)。
②Western Blot和RT-PCR检测,结果进行灰度分析显示：GRIM-19蛋白在肝细胞癌组织中的表达水平明显低于邻近的非癌肝组织(p<0.001)。普通RT-PCR检测结果显示,GRIM-19mRNA在肝细胞癌组织中的表达水平明显低于邻近的非癌肝组织(p<0.001)。QRT-PCR检测结果显示,GRIM-19mRNA在肝细胞癌组织中的表达水平约为癌旁组织的1／6～1／2不等。
4. p-STAT3在肝细胞癌、非癌肝组织中的表达及定位
免疫组织化学染色结果显示：在肝癌组织中,P-STAT3蛋白阳性产物主要定位在细胞核,多呈散在或灶状分布；P-STAT3蛋白在肝细胞癌组织中的表达明显高于癌旁肝组织。55例肝癌组织中p-STAT3表达阳性的有47例(47／55,85.5%),55例癌旁肝组织中p-STAT3表达阳性的只有14例(14／55,25.5%)。12例组织病理分级为Ⅰ级的肝癌组织中p-STAT3表达阳性的有8例(8／12,66.7%),43例组织病理分级为Ⅱ～Ⅲ级的肝细胞癌组织中p-STAT3表达阳性的有39例(39／43,90.7%),39例非癌肝纤维化组织中p-STAT3表达阳性的有14例(14／39,35.9%),而在16例非癌、非纤维化的肝组织中无p-STAT3的表达阳性者。在61例p-STAT3表达阳性的病例中有40例染色为++～+++(按结果判读标准为中等阳性,40／61,65.6%),在这40例中等阳性的病例中有35例为肝癌组织(35／L40,87.5%)。
5. GRIM-19与p-STAT3在原发性肝细胞肝癌及癌旁组织中表达的相关性。
提取55例患者肝癌及癌旁组织的总蛋白,通过Western Blot检测了GRIM-19和p-STAT3蛋白的表达情况,条带经灰度值分析,检测结果经配对t检验分析显示：肝细胞癌组织中GRIM-19/β-actin灰度比值(0.29±0.18)明显低于癌旁组织(0.61±0.11)(p<0.05),p-STAT3/β-actin的灰度比值,肝细胞癌中(0.94±0.36)明显高于癌旁组织(0.23±0.12)(p<0.05),两者经Pearson相关检验,相关系数r=一0.59,表明肝细胞肝癌组织中GRIM-19蛋白的表达与p-STAT3蛋白的表达呈负相关,差异有统计学意义(p<0.05)。
6. GRIM-19.p-STAT3.STAT3及VEGF在原发性肝细胞肝癌及癌旁组织中的表达。
随机抽取了20例患者肝癌及癌旁组织,提取总蛋白,通过Western Blot检测GRIM-19.p-STAT3.STAT3以及p-STAT3的下游蛋白VEGF的表达情况,条带经灰度值分析,检测结果经配对t检验分析显示：肝癌组织中GRIM-19/β-actin灰度比值(0.28±0.12)明显低于癌旁组织(0.64±0.21)(p<0.05)；肝细胞肝癌中p-STAT3/β-actin的灰度比值(0.92±0.27)明显高于癌旁组织(0.31±0.14)(p<0.05),与前期得出的实验结果一致。肝癌组织中STAT3/β-actin灰度比值(0.85±0.13)明显高于癌旁组织(0.32±0.15)(p=0.008)；肝细胞肝癌中VEGF/β-actin灰度比值(0.79±0.21)也明显高于相应的癌旁肝组织(0.36±0.20)(p=0.012)。
7.肝细胞癌组织中GRIM-19mRNA的表达水平与临床病理特征的相关性。
肝细胞癌组织中GRIM-19勺表达水平与肝癌组织病理分级呈负相关(p=0.001)、血管侵袭呈负相关(p=0.032)、与临床分期呈负相关(p=0.024)；而与年龄、性别、HBV感染、甲胎蛋白(Serum α-fetoprotein).纤维化水平、肿瘤大小、淋巴结转移等其他临床病理特征无相关性。
结论
1.在正常肝组织中,GRIM-19主要分布在细胞质,少量分布在细胞核；在肝癌组织中GRIM-19表达明显低于癌旁组织,并且出现核表达缺失。
2.在肝癌及癌旁组织中,p-STAT3主要分布在细胞核,p-STAT3在肝癌组织中的表达水平明显高于癌旁组织。
3.肝细胞肝癌组织中GRIM一19蛋白的表达与p-STAT3蛋白的表达呈明显的负相关。
4.肝细胞癌组织中GRIM-19的表达水平与肝癌组织病理分级、血管侵袭、与临床分期呈负相关；而与年龄、性别、HBV感染、甲胎蛋白、纤维化水平、肿瘤大小、淋巴结转移等其他临床病理特征无相关性。
第二部分全反式维甲酸联合干扰素β对体外培养的人肝癌细胞增殖、凋亡及GRIM-19分子表达的影响
研究目的
1.检测GRIM-19在不同的肝癌细胞株中的表达情况。
2.探讨维甲酸和干扰素β对肝癌细胞HepG2增殖、凋亡的影响。
3.探讨维甲酸和干扰素β对肝癌细胞HepG2GRIM-19表达的影响。
4.检测维甲酸和干扰素β对肝癌细胞HepG2GRIM-19的靶基因STAT3、p-STAT3及VEGF表达水平的影响。
研究方法
1.取传代培养的肝癌细胞株HepG2、MHCC-97H,同时以正常肝细胞株L02作对照,检测三种细胞株中内源性的GRIM-19的表达情况。
2.取传代培养的HepG2细胞,实验组加入不同浓度的干扰素β(interferon-β,IFN-β)和维甲酸(retinoic acid, RA),同时设空白对照组,培养不同的时间(24h、48h和72h),通过MTT法检测细胞增殖变化,找到干扰素β和维甲酸抑制肝癌细胞增殖的最适合的浓度和时间。
3.取传代培养的HepG2细胞,根据MTT的结果选择适宜的作用时间,加入不同浓度的维甲酸和干扰素β,流式细胞仪检测维甲酸和干扰素β对细胞凋亡的影响。
4.取传代培养的HepG2细胞,根据MTT的结果选择适宜的作用时间,加入不同浓度的维甲酸和干扰素β,通过免疫荧光染色比较维甲酸和干扰素β对HepG2细胞中GRIM-19表达的影响。通过Western Bot分别检测实验组和对照组细胞总蛋白和核蛋白中GRIM-19表达水平的变化。
5.Western Bot检测实验组和对照组GRIM-19勺靶基因STAT3、p-STAT3及VFGF的表达变化。
结果
1GRIM-19在不同的肝癌细胞株中的表达
1)免疫荧光检测结果显示：正常人肝细胞株L02及肝癌细胞株HepG2和MHCC-97H中均有内源性GRIM-19的表达,其阳性产物主要定位在细胞质,此外,正常人肝细胞L02亦有少量阳性产物分布于细胞核内,而肝癌细胞株HepG2和MHCC-97H则无细胞核表达,这一结果与前期肝癌及癌旁组织中GRIM-19蛋白的表达定位一致。另外,肝细胞株L02中GRIM-19的荧光强度明显高于肝癌细胞株HepG2和MHCC-97H.
2)通过Western Blot检测内源性GRIM-19蛋白在正常人肝细胞株L02及肝癌细胞株HepG2和MHCC-97H中的表达水平,结果显示：内源性的GRIM-19蛋白在正常人肝细胞株L02中表达水平最高,在肝癌细胞株MHCC-97H中表达水平最低,在肝癌细胞株HepG2勺表达水平介于L02细胞和MHCC-9711细胞之间,三组比较,差异具有统计学意义(p<0.001)。而STAT3、p-STAT3和VEGF蛋白在三种细胞株中的表达,则以MHCC-97H细胞最高,L02细胞最低,HepG2细胞介于L02细胞和MHCC-97H细胞之间,三组比较,差异具有统计学意义(p<0.001)。
3)通过RT-PCR检测内源性GRIM-19mRNA在正常人肝细胞株L02及肝癌细胞株HepG2和MHCC-97H中的表达水平,结果与Western Blot检测结果一致GRIM-19mRNA在正常人肝细胞L02中表达水平最高,在肝癌细胞MHCC-97H中表达水平最低,而在肝癌细胞HepG2中的表达水平介于L02细胞和MHCC-97H细胞之间,三组比较,差异具有统计学意义(p<0.001)。
2不同浓度的I FN-β及RA单独或共同作用对HepG2细胞的增殖抑制
MTT结果显示：不同浓度的IFN-β、RA单独或联合加入肝癌细胞株HepG2(?)培养液中,对HepG2细胞的增殖均有一定程度的抑制作用,并且呈明显的浓度依赖性和作用时间相关性。此外,联合用药比单独用药对细胞增殖的抑制作用更强,联合用药组对HepG2细胞的增殖抑制率分别与相同浓度相同作用时间的单独用药组比较,差异均具有统计学意义(p<0.05)。我们同时发现IFN-β/RA联合用药浓度过大或作用时间过长均对HepG2细胞有毒性反应,在IFN-β4500u/ml+RA9μmol/L联合作用组和所有72h组均观察到较多的死亡细胞,而在IFN-β1500u/ml+RA3μmol/L联合作用48h组未看到明显的死亡细胞。因此,在以下实验中,我们选择采用IFN-β1500u/ml+RA3μmol/L作为最佳干预浓度,48h作为最佳干预时间进行实验。
3IFN-β/RA联合用药可促进HepG2细胞凋亡
流式细胞仪检测结果显示：不同浓度的IFN-β+RA联合加入肝癌细胞株HepG2的培养液中作用48h时,HepG2细胞凋亡指数随药物浓度的增加而增加,对照组llepG2细胞的凋亡指数平均为4.27±1.02, IFN-β500u/ml+RA1μmol/L共同作用48h组,HepG2细胞凋亡指数平均为9.32±1.16:IFN-β1500u/ml+RA3μmol/L共同作用48h组,HepG2细胞凋亡指数平均为16.34±2.71；IFN-β4500u/ml+RA9μmol/L共同作用48h组,HepG2细胞凋亡指数平均为39.61±6.55。四组凋亡指数比较,差异具有统计学意义(p<0.05)；三个处理组分别与对照组比较,差异均具有统计学意义(p<0.05)。
4IFN-β/RA可诱导HepG2细胞内源性GRIM-19表达增加,并出现GRIM-19的核表达阳性
1)细胞免疫荧光检测HepG2细胞在IFN-β／RA作用前后GRIM-19蛋白的表达变化,结果显示：对照组HepG2细胞中GRIM-19蛋白主要定位于细胞质,无细胞核表达;而IFN-β／RA处理组除了细胞体积增大外,出现GRIM-19的核表达阳性GRIM-19荧光强度比对照组明显增强,并且随IFN-β／RA浓度增加,GRIM一19蛋白的荧光强度逐渐增强。
2)收集经IFN-β1500u/ml+RA3μmol/L作用48h后的HepG2细胞分别提取总蛋白和核蛋白,通过Western Blot检测了GRIM-19蛋白的表达情况,条带经灰度值分析,检测结果经t检验分析显示：实验组总蛋白中GRIM-19/β-actin灰度比值(1.36±0.37)明显高于对照组(0.78±0.21)(p<0.01)；实验组核蛋白中GRIM-19/β-actin灰度比值(0.56+0.19)亦明显高于对照组(0.14+0.05)(p<0.01)。
3)收集经IFN-β/RA处理后的HepG2细胞抽提mRNA行RT-PCR检测,结果显示：IFN-β500u/ml+RA1umol/L作用48h时,GRIM-19mRNA表达较对照组增加,约为对照组的1.8倍；当IFN-β1500u/ml+RA3μmol/L作用48h时,GRIM-19mRNA表达约为对照组的3.9倍；当IFN-β4500u/ml+RA9μmol/L作用48h时,GRIM-19mRNA表达程度最高,约为对照组的5.7倍。
5实验组GRIM-19的表达增加伴随p-STAT3、STAT3、VEGF的表达下降
收集经不同浓度的IFN-β/RA作用48h的HepG2细胞提取总蛋白行Western Blot检测,条带经灰度值分析,检测结果显示：实验组GRIM-19的表达水平均高于对照组(p<0.05),并且GRIM-19的表达量随药物浓度的增加而增加；而p-STAT3、STAT3、 VEGF蛋白在实验组中的表达均明显低于对照组(p<0.05),并且表达水平随药物浓度的增加而降低。
结论
1.内源性GRIM-19在正常肝细胞株及肝癌细胞株中表达和定位不同。
2. IFN-β和RA联合作用可抑制肝癌细胞HepG2增殖,促进其凋亡；同时,还可诱导肝癌细胞内源性GRIM-19的表达,下调p-STAT3介导的VEGF的表达,抑制肝癌的发展。
3. GRIM-19有可能成为肝细胞肝癌基因治疗的一个新靶点。
Background and Objective
Hepatocellular carcinoma (HCC) is the fifth most common malignancy in the world, and it is estimated to cause approximately half a million deaths annually. Although the underlying mechanisms of hepatocellular carcinogenesis remain to be investigated, accumulating evidence has indicated that signal transducer and activator of transcription (STAT) proteins, in particular STAT3, may be directly or indirectly involved in HCC development. Constitutively activated STAT3, through its phosphorylation at tyrosine residue705. has been reported in many human cancers, including carcinomas of the breast, lung, prostate, ovary, and melanoma. In general, activated STAT3participates in carcinogenesis by promoting angiogenesis or stimulating cell proliferation. Recently, activated STAT3has been shown in the progression of viral hepatitis and oncogenesis associated with hepatitis B virus (HBV) and hepatitis C virus (HCV) in the liver. In addition, STAT3activity is also increased in chemically-induced HCC. Taken together, these findings suggest that constitutively activated STAT3may play a crucial role in HCC development. However, the consequences of constitutive phosphorylation of STAT3and the mechanisms that trigger this constitutive activation require further investigation.
In recent years, several studies have evaluated the gene associated with retinoid-interferon-induced mortality-19(GRIM-19). GRIM-19is a novel gene product involved in interferon-β/retinoic acid (IFN-β/RA)-induced apoptosis, and it suppresses STAT3-induced gene expression. In addition, GRIM-19plays a major role in the control of cell growth through regulation of STAT3. Lufei and others have reported that GRIM-19binds to STAT3and inhibits its transcriptional activation function. Inhibition of STAT3activation by GRIM-19appears to be an important step in oncogenesic suppression. Furthermore, constitutive activation of STATS promotes anti-apoptotic gene expression to support tumor survival. Low expression levels of GRIM-19have been shown in renal, prostate, esophageal, and colonic tumors. However. GRIM-19expression levels and the associated pathologies have not been reported for liver tumors. In the current study, we demonstrate a significant downregulation of GRIM-19protein levels in HCC that was associated with increased expression of p-STAT3and downstream target genes. In addition, we present novel findings that downregulation of GRIM-19and hyperactivation of p-STAT3expression in HCC lesions were closely correlated with an increased histological grading in HCC,
Part I Expression and Clinical value of GRIM-19and P-STAT3in Hepatocellular Carcinoma
Objective
l.To detect the location and the expression levels of GRIM-19and p-STAT3in hepatocellular carcinoma and adjacent nontumorous liver tissues.
2. To detect the expression level of GRIM-19. STAT3, p-STAT3and VEGF in hepatocellular carcinoma and adjacent nontumorous liver tissues.
3. To evaluated the correlation between expression level of the GRIM-19mRNA in HCC tissues between different patient subgroups according to various clinicopathological parameters.
Methods
1. Detect the location of GRIM-19and p-STAT3in hepatocellular carcinoma and adjacent nontumorous liver tissues by immunohistochemistry.
2. Detect the expression level of GRIM-19and p-STAT3in hepatocellular carcinoma and adjacent nontumorous liver tissues by immunohistochemistry. western blot analysis, and RT-PCR.
3. Detect the expression level of GRIM-19, STAT3, p-STAT3and VEGF in hepatocellular carcinoma and adjacent nontumorous liver tissues by western blot analvsis.
Results
1. The percentage of cells positively stained were categorized
The percentage of cells positively stained in each section were categorized as follows: negative (samples with≤5%positive cells), low (5-25%positive cells), moderate (25-50%positive cells), and strong (50-100%positive) were indicated as-,+,++, and+++, respectively.
2. Cellular Localization of GRIM-19in Liver Tissue
We first analyzed the localization of GRIM-19in liver tissues using a purified GRIM-19antibody and an immunohistochemistry assay. GRIM-19protein expression was predominantly located in the cytoplasm with weak staining in the nucleus in ANLT, but only located in the cytoplasm in HCC tissues. To confirm this observation, pure nuclear protein was extracted separately from HCC tissues and ANLT, and GRIM-19protein expression was determined by western blot analysis. Consistent with the immunohistochemistry results, GRIM-19expression levels in the nucleus was significantly higher in ANLT compared to HCC tissues,(p<0.001).
3. Lower Expression Levels of GRIM-19in HCC Compared to Adjacent Nontumorous Liver Tissue
GRIM-19-positive staining was found in3/4(75%) of the normal liver tissues,40/55(72.7%) of the adjacent nontumorous liver tissues, and25/55(45.5%) of the HCC tissues. GRIM-19expression in the ANLT tissues was not significantly different between the nontumorous, noncirrhotic specimens (12/16,75%) and the nontumorous but cirrhotic specimens (28/39,71.8%). In HCC tissues, however, GRIM-19expression was extremely reduced, and in some cases, even absent. In the cases with positive GRIM-19expression,23/65(35.4%) were determined to be++to+++(by the ranking system described in the methods section); and among them,20/23(87%) were ANLT. Thus, GRIM-19protein expression was extremely lower in HCC tissues than in the ANLT. To verify this finding, we examined the expression of GRIM-19mRNA using RT-PCR. Notably, the GRIM-19mRNA expression levels were significantly lower in HCC tissues compared to the ANLT (p<0.001).
3. Downregulation of GRIM-19was Associated with Increased p-STAT3in HCC Tissue
GRIM-19is known to play an important role in the control of cell growth exerted through STAT3, which is a transcription factor known to be inhibited by GRIM-19binding. To explore the relationship between GRIM-19and p-STAT3expression in HCC, we evaluated the expression status of p-STAT3in HCC samples. In HCC tissues, immunopositive p-STAT3was confined to the nuclei of cancerous cells. Expression of p-STAT3in nonneoplastic. noncirrhotic liver was absent, while positive staining was found in14/39(35.9%) of the cases with liver cirrhosis and in47/55(85.5%) of the HCC samples. In the positively stained cases,40/61(65.6%) were determined to be++to+++, and among them,35/40(87.5%) cases were HCC tissues. These findings indicate that p-STAT3protein expression is highly elevated in HCC tissues compared to the ANLT. These results suggest that an inverse correlation existed between the expression of GRIM-19and p-STAT3in HCC tissues and the ANLT.
To investigate this relationship further, we examined the expression of GRIM-19and p-STAT proteins in HCC tissues (n=55) and the ANLT (n=55) by western blot analysis. The expression of GRIM-19protein was significantly decreased in all grades of HCC tissues (p<0.05), and the expression of p-STAT3protein was significantly increased in all grades of HCC tissues (p<0.05) compared to the β-actin control. Next, we examined the expression of total STAT3and the p-STAT3target protein VEGF in HCC tissues (n=20) and the ANLT (n=20). Consistent with the reduced GRIM-19expression and the increased p-STAT3expression that were found, total STAT3expression (p=0.008) and VEGF expression (p=0.012) were significantly upregulated in the HCC tissues compared to the ANLT. Together, these results suggest that downregulation of GRIM-19may be associated with increased p-STAT3activity in HCC tissues.
4. Correlation of GRIM-19Expression Levels with Clinicopathological Parameters We evaluated possible correlations of GRIM-19mRNA expression in HCC tissues between different patient subgroups according to various clinicopathological parameters (Table1). Two significant correlations of GRIM-19expression were observed for vascular invasion (p=0.032) and histological grade (p=0.001). No significant correlation of GRIM-19mRNA expression was found for age, gender. HBV infection, presence of cirrhosis, tumor size, serum a-fetoprotein levels, or local lymph node metastasis.
Conclusion
1、GRIM-19protein expression was predominantly located in the cytoplasm with weak staining in the nucleus in ANLT, but only located in the cytoplasm in HCC tissues.
2、HCC samples exhibited low levels of GRIM-19and moderate to high levels of p-STAT3expression. In contrast,ANLT was characterized by high levels of GRIM-19and low levels of p-STAT3expression.
3、Downregulation of GRIM-19was closely correlated with increased histological grade in HCC.
Part Ⅱ The Affection of Retinoic Acid combined with Beta-Interferon Synergize in Inducing Growth Inhibition, Apoptosis and GRIM-19Expression in Human Hepatoma Carcinoma Cell in Vitro
Objective
1. To detect the location and expression level of the GRIM-19in different hepatocellular carcinoma cell lines.
2. To research growth inhibition and apoptosis of hepatocellular carcinoma cell (HepG2) intervened by RA combined with IFN-β.
3. To investigate the changes in the expression level of GRIM-19and proto-oncogene STAT3、p-STAT3、VEGF intervened by RA combined with IFN-β.
Methods
1. Immunofluorescence、western blot and RT-PCR were used to detect the location and the expression of GRIM-19in LO2cell line and HepG2、MHCC-97H cell lines, respectively.
2. MTT and flow cytometry were used to research growth inhibition and apoptosis of hepatocellular carcinoma cell (HepG2) intervened by RA combined with IFN-β
3. Immunofluorescence、western blot and RT-PCR were used to detect the expression level of the GR1M-19in HepG2intervened by RA combined with IFN-β.
4. Western blot was used to detect the expression level of the GRIM-19、STAT3、p-STAT3and VEGF in HepG2intervened by RA combined with IFN-β.
Results
1. Cellular Localization of GRIM-19in hepatocellular carcinoma cell lines
In our previous studies, we first analyzed the localization of GRIM-19in liver tissues using a purified GRIM-19antibody and an immunohistochemistry assay. GRIM-19protein expression was predominantly located in the cytoplasm with weak staining in the nucleus in ANLT, but only located in the cytoplasm in HCC tissues. To confirm this observation, we analyzed the cellular localization of endogenous GRIM-19in LO2cells (a human hepatic cell line), HepG2cells and MHCC-97H cells (a human hepatocellular carcinoma cell line) using a purified GRIM-19antibody and an immunofluorescence assay. Positive staining for GRIM-19was observed in the cytoplasm of all cells, which was intensified near the peri-nuclear region. In addition, weak staining was also noticeable in the nucleus of the LO2cells but not in the nucleus of the HepG2cells and MHCC-97H.
2. Expression levels of GRIM-19in different hepatocellular carcinoma cell lines
Since GRIM-19was identified as a potential tumor suppressor in recent studies, we evaluated the expression status of GRIM-19in primary liver cancers using immunohistochemistry. Briefly, GRIM-19protein expression was extremely lower in HCC tissues than in the ANLT. To explore the relationship between human hepatocellular carcinoma cell lines and hepatic cell lines, we evaluated the expression status of endogenous GRIM-19expression levels in LO2cells, HepG2cells and MHCC-97H cells by immunofluorescence experiments at equal pace. Endogenous GRIM-19expression in HepG2cells was significantly lower than in LO2cells, but it was higher than in the MHCC-97H cells. These findings were confirmed by determination of the endogenous GRIM-19protein expression levels by western blot analysis and endogenous GRIM-19mRNA levels were determined by QRT-PCR. Consistent with the immunofluorescence data, endogenous GRIM-19expression in HepG2cells was higher than in MHCC-97H cells but lower than in LO2cells. In addition, endogenous STATS、p-STAT3and VEGF expression in HepG2cells was significantly higher than in LO2cells, but it was lower than in the MHCC-97H cells.
3. The growth inhibition of HepG2intervened by RA combined with IFN-β
The combination of RA and IFN-βhas been shown to inhibit cell growth in other cell lines. To determine if IFN/RA exerts similar growth-suppressive effects in the hepatocellular carcinoma cells, human hepatocellular carcinoma HepG2cells grown in culture dishes were treated either with either IFN or RA or a combination of IFN/RA. MTT assays were performed. As shown in Table2, exposure to RA or IFN-β alone can inhibit the growth of HepG2cells, but RA combined with IFN-β can greatly increase it, and this is associated with concentration and time. We found that too much RA/IFN-β or too long time can induce cell death in HepG2cell lines. Exposure to RA or IFN-β alone did not cause cell death. Similarly, combined exposure to IFN/RA for24h and48h did not cause significant cell death, where as combined exposure to IFN/RA for72h caused significant cell death. So, we chose48h as best exposure time to study the biology change of HepG2cells combined exposure to different concentration of IFN-β and RA in following.
4. The apoptosis of HepG2intervened by RA combined with IFN-β
Human hepatocellular carcinoma HepG2cells at a low density and then exposing to different concentration of IFN/RA for48h. To examine the effect of RA combined with IFN-P on the apoptosis of HepG2cells, flow cytometry were used. The result showed that IFN/RA exhibits a dual effect on cell growth:inhibition of cell growth and an induction of cell apoptosis. RA combined with IFN-P can promote the apoptosis of HepG2cells, and this is associated with concentration.
5. GRIM-19expression increased and appear nuclear expression in HepG2after exposed to RA/IFN-p
To determine if the cell apoptosis could be correlated with the expression of GRIM-19, we analyzed the cellular localization of GRIM-19in HepG2cells after intervened by different concentration of IFN/RA for48h using the purified GRIM-19antibody by immunofluorescence. Positive staining of GRIM-19was observed in the cytoplasm of untreated HepG2cells which was intensified at the peri-nuclear region. In addition, a positive staining was also noticeable in the nucleus after the IFN-β/RA treatment, which was significantly increased following the increased of the concentration. To verify these results further, pure nuclear protein was extracted in HepG2cells which absence or presence of IFN-β/RA treatment, and GRIM-19expression was determined. Consistent with the immunofluorescence results, the expression of GRIM-19in the nucleus was increased slightly after IFN-β/RA treatment.
6. STAT3, p-STAT3and VEGF expression decreased after exposed to RA/IFN-β We detected the expression of GRIM-19in HepG2cells after exposed to different concentration of IFN/RA for48h using the purified GRIM-19antibody by Western blot. The result indicated that GRIM-19protein expression increased than control group(p <0.05). More importantly, the lower expression of STAT3、p-STAT3、VEGF were found in treatment groups than control cells.
Conclusion
1. The location of GRIM-19in human hepatic cell lines was different with the hepatocellular carcinoma cell lines. And the expression of GRIM-19in human hepatic cell lines was higher than hepatocellular carcinoma cell lines.
2. RA combination with IFN-β can inhibit the growth and promote the apoptosis of HepG2cells.
3. RA combination with IFN-β can up-regulate of GRIM-19protein expression and down-regulate of STAT3、p-STAT3、VEGF expression.
4. GRIM-19may be a new target spot in liver cancer gene therapy.
肝细胞肝癌(hepatocellular carcinoma, HCC)是源自肝细胞的恶性肿瘤,是原发性肝癌中最常见的一种病理类型,世界上平均每年死于HCC的人数约有1,250,000,HCC的发生、发展是一个多基因参与的复杂过程。目前针对HCC的治疗,主要是外科手术切除病灶或者是通过介入手段进行局部化疗栓塞治疗,对HCC患者进行放疗和全身化疗的疗效均不理想。随着分子生物学的发展,免疫治疗和基因治疗等生物治疗方法作为继手术、化疗、放疗之后的第四种治疗模式,日益受到重视。
GRIMs(genes associated with retinoid-IFN-induced mortality)是新近由Kalvakolanu在研究干扰素(interferon, IFN)和维甲酸(retinoic acid, RA)合并诱导细胞凋亡时发现的一组参与细胞凋亡的基因,共24种,GRIM-19是其中最具代表性的一种。GRIM-19编码含144个氨基酸残基的蛋白质,分子量约16KD,定位于19p13.1,是一种保守基因,可以在多种正常组织中表达。目前GRIM-19蛋白的细胞定位存在争议,有研究认为GRIM-19主要定位于细胞核,也有研究认为GRIM-19主要表达于胞浆中的线粒体。Lu fei等则认为GRIM-19的定位因细胞种类不同而存在差异,目前国内外对GRIM-19在肝癌组织中的表达及功能都尚未见报道。研究表明,GRIM-19参与细胞增殖、凋亡的调控过程,该基因表达的降低或者位点的突变可以导致细胞的异常增殖和恶性转化,GRIM-19在肝细胞癌变的过程中发挥了怎样的作用,值得我们深入探讨。
GRIM-19可以特异性的与组成性活化的STAT3偶联,从而抑制其转录活性,并抑制肿瘤生长,对肿瘤治疗有巨大的潜在价值。这一机制在肾细胞癌、部分前列腺癌、HPV感染的宫颈癌等部分肿瘤组织中都有所报道。但在肝癌方面,只在体外培养的HepG2细胞中有过报道。STAT3可以被多种病毒致癌蛋白所激活,STAT3的组成性活化能促进细胞的恶性转化并阻断其凋亡,促进肿瘤细胞的增殖,血管形成,因此,阻断STAT3勺表达对肿瘤的治疗具有巨大的潜在价值。深入研究GRIM-19与P-STAT3的关系,探讨其对细胞转化、增殖、凋亡的调控机制,将有利于阐明肝癌的发生机制。
GRIM-19是由RA和IFNs合并诱导细胞时发现的参与细胞凋亡的基因。维甲酸是维生素A在体内产生的衍生物,属于分化诱导剂类,具有逆转癌前病变,诱导肿瘤细胞分化,提高肿瘤细胞对化疗的敏感性等作用；干扰素则通过与细胞表面受体结合,激活包括JAK-STAT途径、干扰素调节因子在内的多种信号转导途径,直接抑制肿瘤细胞的增殖。在临床上,单用维甲酸和干扰素治疗肿瘤的研究较多,联合治疗肿瘤的研究比较少,两药联合对肝癌细胞的影响及其诱导凋亡的机制尚不清楚。目前针对GRIM-19基因的生物学功能及作用机制的研究尚处于起步阶段,进一步认识其蛋白的功能、调节方式及其作用机制,将为深入了解肿瘤细胞的发生、发展提供新的线索,为肿瘤的诊断、治疗提供新的靶点和研究方向。
第一部分GRIM-19及P-STAT3在人肝细胞肝癌中的表达及临床意义
研究目的
1.检测GRIM-19在原发性肝细胞肝癌和癌旁组织中的表达及定位。
2.检测P-STAT3在原发性肝细胞肝癌和癌旁组织中的表达及定位。
3.分析GRIM-19及其靶基因STAT3、p-STAT3、VEGF在肝癌和癌旁组织中的相关性。
4.分析GRIM-19与原发性肝癌的病因、病理类型、分化程度等临床特征的相关性。
研究方法
1.收集55例手术之前未经任何治疗的原发性肝癌患者的临床资料及经手术切除的肝细胞癌组织及邻近的非癌肝组织(邻近非癌肝组织：肉眼观正常,且距离肿瘤组织边缘5cm以上),组织标本于手术切除后尽快采集,经脱水、石蜡包埋后切片,采用免疫组织化学染色法,检测GRIM-19和P-STAT3在肝癌组织和非癌肝组织中的表达和定位。
2.分别提取肝癌及癌旁肝组织的总蛋白和核蛋白,通过Western Blot的方法检测GRIM-19蛋白的表达情况。
3.分别提取肝癌及癌旁肝组织的RNA,通过RT-PCR检测GRIM-19在mRNA水平的表达情况。
4.分别提取肝癌及癌旁肝组织的总蛋白,通过Western Blot的方法检测GRIM-19及其靶基因STAT3、p-STAT3、VEGF的表达情况。
5.分析GRIM-19与临床病理特征的相关性。
结果
1.免疫组织化学染色结果判读
免疫组织化学染色结果由与本研究无关的两名病理医生评定,每张切片观察5-6个400×视野,计数不少于100个细胞,依据染色强度和阳性细胞数进行分级,染色强度记分为四个等级：阴性：阳性细胞数少于5%,以“-”表示；弱阳性：阳性细胞数在5%-25%之间,以“+”表示；中等阳性：阳性细胞数在25%-50%之间,以“十+”表示；强阳性：阳性细胞数在50%-100%之间,以“+++”表示。
2.GRIM-19在肝细胞癌、非癌肝组织中的表达和定位。
①免疫组织化学染色结果显示：在非癌肝组织中,GRIM-19蛋白阳性产物主要分布在细胞质,亦有少量阳性产物分布在细胞核,表现为不同染色程度的黄色或棕黄色颗粒；而存肝细胞癌组织中,GRIM-19阳性产物只存在于细胞质。
②分别提取肝细胞癌及癌旁组织的核蛋白,Western Blot检测结果显示GRIM-19蛋白在肝癌细胞核蛋白中的表达明显低于癌旁肝细胞的核蛋白(P<0.001).
3.GRIM-19在肝细胞癌和癌旁肝组织中的表达水平。
①免疫组织化学染色法结果显示：GRIM-19蛋白在肝细胞癌组织中的表达明显低于癌旁肝组织。对照组4例正常肝组织中GRIM-19表达阳性的有3例(3／4,75%)；55例癌旁肝组织中GRIM-19表达阳性的有40例(40／55,72.7%)；而55例肝细胞癌组织中GRIM-19表达阳性的只有25例(25／55,45.5%)。16例不伴随肝纤维化的癌旁组织中有12例GRIM-19表达阳性(12／16,75%)；39例伴随肝纤维化的癌旁组织中有28例GRIM-19表达阳性(28／39,71.8%),不伴随肝纤维化的癌旁组织与伴随肝纤维化的癌旁组织比较,GRIM-19蛋白的表达无明显差异(p>0.05)。GRIM-19在肝癌组织中的表达明显低于癌旁组织(p<0.05),甚至表达缺失。另外,65例GRIM-19表达阳性的肝组织中有23例为++～＋＋+(按结果判读标准分为中等阳性),在这23例中等阳性标本中有20例是癌旁组织(20／23,87%)。
②Western Blot和RT-PCR检测,结果进行灰度分析显示：GRIM-19蛋白在肝细胞癌组织中的表达水平明显低于邻近的非癌肝组织(p<0.001)。普通RT-PCR检测结果显示,GRIM-19mRNA在肝细胞癌组织中的表达水平明显低于邻近的非癌肝组织(p<0.001)。QRT-PCR检测结果显示,GRIM-19mRNA在肝细胞癌组织中的表达水平约为癌旁组织的1／6～1／2不等。
4. p-STAT3在肝细胞癌、非癌肝组织中的表达及定位
免疫组织化学染色结果显示：在肝癌组织中,P-STAT3蛋白阳性产物主要定位在细胞核,多呈散在或灶状分布；P-STAT3蛋白在肝细胞癌组织中的表达明显高于癌旁肝组织。55例肝癌组织中p-STAT3表达阳性的有47例(47／55,85.5%),55例癌旁肝组织中p-STAT3表达阳性的只有14例(14／55,25.5%)。12例组织病理分级为Ⅰ级的肝癌组织中p-STAT3表达阳性的有8例(8／12,66.7%),43例组织病理分级为Ⅱ～Ⅲ级的肝细胞癌组织中p-STAT3表达阳性的有39例(39／43,90.7%),39例非癌肝纤维化组织中p-STAT3表达阳性的有14例(14／39,35.9%),而在16例非癌、非纤维化的肝组织中无p-STAT3的表达阳性者。在61例p-STAT3表达阳性的病例中有40例染色为++～+++(按结果判读标准为中等阳性,40／61,65.6%),在这40例中等阳性的病例中有35例为肝癌组织(35／L40,87.5%)。
5. GRIM-19与p-STAT3在原发性肝细胞肝癌及癌旁组织中表达的相关性。
提取55例患者肝癌及癌旁组织的总蛋白,通过Western Blot检测了GRIM-19和p-STAT3蛋白的表达情况,条带经灰度值分析,检测结果经配对t检验分析显示：肝细胞癌组织中GRIM-19/β-actin灰度比值(0.29±0.18)明显低于癌旁组织(0.61±0.11)(p<0.05),p-STAT3/β-actin的灰度比值,肝细胞癌中(0.94±0.36)明显高于癌旁组织(0.23±0.12)(p<0.05),两者经Pearson相关检验,相关系数r=一0.59,表明肝细胞肝癌组织中GRIM-19蛋白的表达与p-STAT3蛋白的表达呈负相关,差异有统计学意义(p<0.05)。
6. GRIM-19.p-STAT3.STAT3及VEGF在原发性肝细胞肝癌及癌旁组织中的表达。
随机抽取了20例患者肝癌及癌旁组织,提取总蛋白,通过Western Blot检测GRIM-19.p-STAT3.STAT3以及p-STAT3的下游蛋白VEGF的表达情况,条带经灰度值分析,检测结果经配对t检验分析显示：肝癌组织中GRIM-19/β-actin灰度比值(0.28±0.12)明显低于癌旁组织(0.64±0.21)(p<0.05)；肝细胞肝癌中p-STAT3/β-actin的灰度比值(0.92±0.27)明显高于癌旁组织(0.31±0.14)(p<0.05),与前期得出的实验结果一致。肝癌组织中STAT3/β-actin灰度比值(0.85±0.13)明显高于癌旁组织(0.32±0.15)(p=0.008)；肝细胞肝癌中VEGF/β-actin灰度比值(0.79±0.21)也明显高于相应的癌旁肝组织(0.36±0.20)(p=0.012)。
7.肝细胞癌组织中GRIM-19mRNA的表达水平与临床病理特征的相关性。
肝细胞癌组织中GRIM-19勺表达水平与肝癌组织病理分级呈负相关(p=0.001)、血管侵袭呈负相关(p=0.032)、与临床分期呈负相关(p=0.024)；而与年龄、性别、HBV感染、甲胎蛋白(Serum α-fetoprotein).纤维化水平、肿瘤大小、淋巴结转移等其他临床病理特征无相关性。
结论
1.在正常肝组织中,GRIM-19主要分布在细胞质,少量分布在细胞核；在肝癌组织中GRIM-19表达明显低于癌旁组织,并且出现核表达缺失。
2.在肝癌及癌旁组织中,p-STAT3主要分布在细胞核,p-STAT3在肝癌组织中的表达水平明显高于癌旁组织。
3.肝细胞肝癌组织中GRIM一19蛋白的表达与p-STAT3蛋白的表达呈明显的负相关。
4.肝细胞癌组织中GRIM-19的表达水平与肝癌组织病理分级、血管侵袭、与临床分期呈负相关；而与年龄、性别、HBV感染、甲胎蛋白、纤维化水平、肿瘤大小、淋巴结转移等其他临床病理特征无相关性。
第二部分全反式维甲酸联合干扰素β对体外培养的人肝癌细胞增殖、凋亡及GRIM-19分子表达的影响
研究目的
1.检测GRIM-19在不同的肝癌细胞株中的表达情况。
2.探讨维甲酸和干扰素β对肝癌细胞HepG2增殖、凋亡的影响。
3.探讨维甲酸和干扰素β对肝癌细胞HepG2GRIM-19表达的影响。
4.检测维甲酸和干扰素β对肝癌细胞HepG2GRIM-19的靶基因STAT3、p-STAT3及VEGF表达水平的影响。
研究方法
1.取传代培养的肝癌细胞株HepG2、MHCC-97H,同时以正常肝细胞株L02作对照,检测三种细胞株中内源性的GRIM-19的表达情况。
2.取传代培养的HepG2细胞,实验组加入不同浓度的干扰素β(interferon-β,IFN-β)和维甲酸(retinoic acid, RA),同时设空白对照组,培养不同的时间(24h、48h和72h),通过MTT法检测细胞增殖变化,找到干扰素β和维甲酸抑制肝癌细胞增殖的最适合的浓度和时间。
3.取传代培养的HepG2细胞,根据MTT的结果选择适宜的作用时间,加入不同浓度的维甲酸和干扰素β,流式细胞仪检测维甲酸和干扰素β对细胞凋亡的影响。
4.取传代培养的HepG2细胞,根据MTT的结果选择适宜的作用时间,加入不同浓度的维甲酸和干扰素β,通过免疫荧光染色比较维甲酸和干扰素β对HepG2细胞中GRIM-19表达的影响。通过Western Bot分别检测实验组和对照组细胞总蛋白和核蛋白中GRIM-19表达水平的变化。
5.Western Bot检测实验组和对照组GRIM-19勺靶基因STAT3、p-STAT3及VFGF的表达变化。
结果
1GRIM-19在不同的肝癌细胞株中的表达
1)免疫荧光检测结果显示：正常人肝细胞株L02及肝癌细胞株HepG2和MHCC-97H中均有内源性GRIM-19的表达,其阳性产物主要定位在细胞质,此外,正常人肝细胞L02亦有少量阳性产物分布于细胞核内,而肝癌细胞株HepG2和MHCC-97H则无细胞核表达,这一结果与前期肝癌及癌旁组织中GRIM-19蛋白的表达定位一致。另外,肝细胞株L02中GRIM-19的荧光强度明显高于肝癌细胞株HepG2和MHCC-97H.
2)通过Western Blot检测内源性GRIM-19蛋白在正常人肝细胞株L02及肝癌细胞株HepG2和MHCC-97H中的表达水平,结果显示：内源性的GRIM-19蛋白在正常人肝细胞株L02中表达水平最高,在肝癌细胞株MHCC-97H中表达水平最低,在肝癌细胞株HepG2勺表达水平介于L02细胞和MHCC-9711细胞之间,三组比较,差异具有统计学意义(p<0.001)。而STAT3、p-STAT3和VEGF蛋白在三种细胞株中的表达,则以MHCC-97H细胞最高,L02细胞最低,HepG2细胞介于L02细胞和MHCC-97H细胞之间,三组比较,差异具有统计学意义(p<0.001)。
3)通过RT-PCR检测内源性GRIM-19mRNA在正常人肝细胞株L02及肝癌细胞株HepG2和MHCC-97H中的表达水平,结果与Western Blot检测结果一致GRIM-19mRNA在正常人肝细胞L02中表达水平最高,在肝癌细胞MHCC-97H中表达水平最低,而在肝癌细胞HepG2中的表达水平介于L02细胞和MHCC-97H细胞之间,三组比较,差异具有统计学意义(p<0.001)。
2不同浓度的I FN-β及RA单独或共同作用对HepG2细胞的增殖抑制
MTT结果显示：不同浓度的IFN-β、RA单独或联合加入肝癌细胞株HepG2(?)培养液中,对HepG2细胞的增殖均有一定程度的抑制作用,并且呈明显的浓度依赖性和作用时间相关性。此外,联合用药比单独用药对细胞增殖的抑制作用更强,联合用药组对HepG2细胞的增殖抑制率分别与相同浓度相同作用时间的单独用药组比较,差异均具有统计学意义(p<0.05)。我们同时发现IFN-β/RA联合用药浓度过大或作用时间过长均对HepG2细胞有毒性反应,在IFN-β4500u/ml+RA9μmol/L联合作用组和所有72h组均观察到较多的死亡细胞,而在IFN-β1500u/ml+RA3μmol/L联合作用48h组未看到明显的死亡细胞。因此,在以下实验中,我们选择采用IFN-β1500u/ml+RA3μmol/L作为最佳干预浓度,48h作为最佳干预时间进行实验。
3IFN-β/RA联合用药可促进HepG2细胞凋亡
流式细胞仪检测结果显示：不同浓度的IFN-β+RA联合加入肝癌细胞株HepG2的培养液中作用48h时,HepG2细胞凋亡指数随药物浓度的增加而增加,对照组llepG2细胞的凋亡指数平均为4.27±1.02, IFN-β500u/ml+RA1μmol/L共同作用48h组,HepG2细胞凋亡指数平均为9.32±1.16:IFN-β1500u/ml+RA3μmol/L共同作用48h组,HepG2细胞凋亡指数平均为16.34±2.71；IFN-β4500u/ml+RA9μmol/L共同作用48h组,HepG2细胞凋亡指数平均为39.61±6.55。四组凋亡指数比较,差异具有统计学意义(p<0.05)；三个处理组分别与对照组比较,差异均具有统计学意义(p<0.05)。
4IFN-β/RA可诱导HepG2细胞内源性GRIM-19表达增加,并出现GRIM-19的核表达阳性
1)细胞免疫荧光检测HepG2细胞在IFN-β／RA作用前后GRIM-19蛋白的表达变化,结果显示：对照组HepG2细胞中GRIM-19蛋白主要定位于细胞质,无细胞核表达;而IFN-β／RA处理组除了细胞体积增大外,出现GRIM-19的核表达阳性GRIM-19荧光强度比对照组明显增强,并且随IFN-β／RA浓度增加,GRIM一19蛋白的荧光强度逐渐增强。
2)收集经IFN-β1500u/ml+RA3μmol/L作用48h后的HepG2细胞分别提取总蛋白和核蛋白,通过Western Blot检测了GRIM-19蛋白的表达情况,条带经灰度值分析,检测结果经t检验分析显示：实验组总蛋白中GRIM-19/β-actin灰度比值(1.36±0.37)明显高于对照组(0.78±0.21)(p<0.01)；实验组核蛋白中GRIM-19/β-actin灰度比值(0.56+0.19)亦明显高于对照组(0.14+0.05)(p<0.01)。
3)收集经IFN-β/RA处理后的HepG2细胞抽提mRNA行RT-PCR检测,结果显示：IFN-β500u/ml+RA1umol/L作用48h时,GRIM-19mRNA表达较对照组增加,约为对照组的1.8倍；当IFN-β1500u/ml+RA3μmol/L作用48h时,GRIM-19mRNA表达约为对照组的3.9倍；当IFN-β4500u/ml+RA9μmol/L作用48h时,GRIM-19mRNA表达程度最高,约为对照组的5.7倍。
5实验组GRIM-19的表达增加伴随p-STAT3、STAT3、VEGF的表达下降
收集经不同浓度的IFN-β/RA作用48h的HepG2细胞提取总蛋白行Western Blot检测,条带经灰度值分析,检测结果显示：实验组GRIM-19的表达水平均高于对照组(p<0.05),并且GRIM-19的表达量随药物浓度的增加而增加；而p-STAT3、STAT3、 VEGF蛋白在实验组中的表达均明显低于对照组(p<0.05),并且表达水平随药物浓度的增加而降低。
结论
1.内源性GRIM-19在正常肝细胞株及肝癌细胞株中表达和定位不同。
2. IFN-β和RA联合作用可抑制肝癌细胞HepG2增殖,促进其凋亡；同时,还可诱导肝癌细胞内源性GRIM-19的表达,下调p-STAT3介导的VEGF的表达,抑制肝癌的发展。
3. GRIM-19有可能成为肝细胞肝癌基因治疗的一个新靶点。
Background and Objective
Hepatocellular carcinoma (HCC) is the fifth most common malignancy in the world, and it is estimated to cause approximately half a million deaths annually. Although the underlying mechanisms of hepatocellular carcinogenesis remain to be investigated, accumulating evidence has indicated that signal transducer and activator of transcription (STAT) proteins, in particular STAT3, may be directly or indirectly involved in HCC development. Constitutively activated STAT3, through its phosphorylation at tyrosine residue705. has been reported in many human cancers, including carcinomas of the breast, lung, prostate, ovary, and melanoma. In general, activated STAT3participates in carcinogenesis by promoting angiogenesis or stimulating cell proliferation. Recently, activated STAT3has been shown in the progression of viral hepatitis and oncogenesis associated with hepatitis B virus (HBV) and hepatitis C virus (HCV) in the liver. In addition, STAT3activity is also increased in chemically-induced HCC. Taken together, these findings suggest that constitutively activated STAT3may play a crucial role in HCC development. However, the consequences of constitutive phosphorylation of STAT3and the mechanisms that trigger this constitutive activation require further investigation.
In recent years, several studies have evaluated the gene associated with retinoid-interferon-induced mortality-19(GRIM-19). GRIM-19is a novel gene product involved in interferon-β/retinoic acid (IFN-β/RA)-induced apoptosis, and it suppresses STAT3-induced gene expression. In addition, GRIM-19plays a major role in the control of cell growth through regulation of STAT3. Lufei and others have reported that GRIM-19binds to STAT3and inhibits its transcriptional activation function. Inhibition of STAT3activation by GRIM-19appears to be an important step in oncogenesic suppression. Furthermore, constitutive activation of STATS promotes anti-apoptotic gene expression to support tumor survival. Low expression levels of GRIM-19have been shown in renal, prostate, esophageal, and colonic tumors. However. GRIM-19expression levels and the associated pathologies have not been reported for liver tumors. In the current study, we demonstrate a significant downregulation of GRIM-19protein levels in HCC that was associated with increased expression of p-STAT3and downstream target genes. In addition, we present novel findings that downregulation of GRIM-19and hyperactivation of p-STAT3expression in HCC lesions were closely correlated with an increased histological grading in HCC,
Part I Expression and Clinical value of GRIM-19and P-STAT3in Hepatocellular Carcinoma
Objective
l.To detect the location and the expression levels of GRIM-19and p-STAT3in hepatocellular carcinoma and adjacent nontumorous liver tissues.
2. To detect the expression level of GRIM-19. STAT3, p-STAT3and VEGF in hepatocellular carcinoma and adjacent nontumorous liver tissues.
3. To evaluated the correlation between expression level of the GRIM-19mRNA in HCC tissues between different patient subgroups according to various clinicopathological parameters.
Methods
1. Detect the location of GRIM-19and p-STAT3in hepatocellular carcinoma and adjacent nontumorous liver tissues by immunohistochemistry.
2. Detect the expression level of GRIM-19and p-STAT3in hepatocellular carcinoma and adjacent nontumorous liver tissues by immunohistochemistry. western blot analysis, and RT-PCR.
3. Detect the expression level of GRIM-19, STAT3, p-STAT3and VEGF in hepatocellular carcinoma and adjacent nontumorous liver tissues by western blot analvsis.
Results
1. The percentage of cells positively stained were categorized
The percentage of cells positively stained in each section were categorized as follows: negative (samples with≤5%positive cells), low (5-25%positive cells), moderate (25-50%positive cells), and strong (50-100%positive) were indicated as-,+,++, and+++, respectively.
2. Cellular Localization of GRIM-19in Liver Tissue
We first analyzed the localization of GRIM-19in liver tissues using a purified GRIM-19antibody and an immunohistochemistry assay. GRIM-19protein expression was predominantly located in the cytoplasm with weak staining in the nucleus in ANLT, but only located in the cytoplasm in HCC tissues. To confirm this observation, pure nuclear protein was extracted separately from HCC tissues and ANLT, and GRIM-19protein expression was determined by western blot analysis. Consistent with the immunohistochemistry results, GRIM-19expression levels in the nucleus was significantly higher in ANLT compared to HCC tissues,(p<0.001).
3. Lower Expression Levels of GRIM-19in HCC Compared to Adjacent Nontumorous Liver Tissue
GRIM-19-positive staining was found in3/4(75%) of the normal liver tissues,40/55(72.7%) of the adjacent nontumorous liver tissues, and25/55(45.5%) of the HCC tissues. GRIM-19expression in the ANLT tissues was not significantly different between the nontumorous, noncirrhotic specimens (12/16,75%) and the nontumorous but cirrhotic specimens (28/39,71.8%). In HCC tissues, however, GRIM-19expression was extremely reduced, and in some cases, even absent. In the cases with positive GRIM-19expression,23/65(35.4%) were determined to be++to+++(by the ranking system described in the methods section); and among them,20/23(87%) were ANLT. Thus, GRIM-19protein expression was extremely lower in HCC tissues than in the ANLT. To verify this finding, we examined the expression of GRIM-19mRNA using RT-PCR. Notably, the GRIM-19mRNA expression levels were significantly lower in HCC tissues compared to the ANLT (p<0.001).
3. Downregulation of GRIM-19was Associated with Increased p-STAT3in HCC Tissue
GRIM-19is known to play an important role in the control of cell growth exerted through STAT3, which is a transcription factor known to be inhibited by GRIM-19binding. To explore the relationship between GRIM-19and p-STAT3expression in HCC, we evaluated the expression status of p-STAT3in HCC samples. In HCC tissues, immunopositive p-STAT3was confined to the nuclei of cancerous cells. Expression of p-STAT3in nonneoplastic. noncirrhotic liver was absent, while positive staining was found in14/39(35.9%) of the cases with liver cirrhosis and in47/55(85.5%) of the HCC samples. In the positively stained cases,40/61(65.6%) were determined to be++to+++, and among them,35/40(87.5%) cases were HCC tissues. These findings indicate that p-STAT3protein expression is highly elevated in HCC tissues compared to the ANLT. These results suggest that an inverse correlation existed between the expression of GRIM-19and p-STAT3in HCC tissues and the ANLT.
To investigate this relationship further, we examined the expression of GRIM-19and p-STAT proteins in HCC tissues (n=55) and the ANLT (n=55) by western blot analysis. The expression of GRIM-19protein was significantly decreased in all grades of HCC tissues (p<0.05), and the expression of p-STAT3protein was significantly increased in all grades of HCC tissues (p<0.05) compared to the β-actin control. Next, we examined the expression of total STAT3and the p-STAT3target protein VEGF in HCC tissues (n=20) and the ANLT (n=20). Consistent with the reduced GRIM-19expression and the increased p-STAT3expression that were found, total STAT3expression (p=0.008) and VEGF expression (p=0.012) were significantly upregulated in the HCC tissues compared to the ANLT. Together, these results suggest that downregulation of GRIM-19may be associated with increased p-STAT3activity in HCC tissues.
4. Correlation of GRIM-19Expression Levels with Clinicopathological Parameters We evaluated possible correlations of GRIM-19mRNA expression in HCC tissues between different patient subgroups according to various clinicopathological parameters (Table1). Two significant correlations of GRIM-19expression were observed for vascular invasion (p=0.032) and histological grade (p=0.001). No significant correlation of GRIM-19mRNA expression was found for age, gender. HBV infection, presence of cirrhosis, tumor size, serum a-fetoprotein levels, or local lymph node metastasis.
Conclusion
1、GRIM-19protein expression was predominantly located in the cytoplasm with weak staining in the nucleus in ANLT, but only located in the cytoplasm in HCC tissues.
2、HCC samples exhibited low levels of GRIM-19and moderate to high levels of p-STAT3expression. In contrast,ANLT was characterized by high levels of GRIM-19and low levels of p-STAT3expression.
3、Downregulation of GRIM-19was closely correlated with increased histological grade in HCC.
Part Ⅱ The Affection of Retinoic Acid combined with Beta-Interferon Synergize in Inducing Growth Inhibition, Apoptosis and GRIM-19Expression in Human Hepatoma Carcinoma Cell in Vitro
Objective
1. To detect the location and expression level of the GRIM-19in different hepatocellular carcinoma cell lines.
2. To research growth inhibition and apoptosis of hepatocellular carcinoma cell (HepG2) intervened by RA combined with IFN-β.
3. To investigate the changes in the expression level of GRIM-19and proto-oncogene STAT3、p-STAT3、VEGF intervened by RA combined with IFN-β.
Methods
1. Immunofluorescence、western blot and RT-PCR were used to detect the location and the expression of GRIM-19in LO2cell line and HepG2、MHCC-97H cell lines, respectively.
2. MTT and flow cytometry were used to research growth inhibition and apoptosis of hepatocellular carcinoma cell (HepG2) intervened by RA combined with IFN-β
3. Immunofluorescence、western blot and RT-PCR were used to detect the expression level of the GR1M-19in HepG2intervened by RA combined with IFN-β.
4. Western blot was used to detect the expression level of the GRIM-19、STAT3、p-STAT3and VEGF in HepG2intervened by RA combined with IFN-β.
Results
1. Cellular Localization of GRIM-19in hepatocellular carcinoma cell lines
In our previous studies, we first analyzed the localization of GRIM-19in liver tissues using a purified GRIM-19antibody and an immunohistochemistry assay. GRIM-19protein expression was predominantly located in the cytoplasm with weak staining in the nucleus in ANLT, but only located in the cytoplasm in HCC tissues. To confirm this observation, we analyzed the cellular localization of endogenous GRIM-19in LO2cells (a human hepatic cell line), HepG2cells and MHCC-97H cells (a human hepatocellular carcinoma cell line) using a purified GRIM-19antibody and an immunofluorescence assay. Positive staining for GRIM-19was observed in the cytoplasm of all cells, which was intensified near the peri-nuclear region. In addition, weak staining was also noticeable in the nucleus of the LO2cells but not in the nucleus of the HepG2cells and MHCC-97H.
2. Expression levels of GRIM-19in different hepatocellular carcinoma cell lines
Since GRIM-19was identified as a potential tumor suppressor in recent studies, we evaluated the expression status of GRIM-19in primary liver cancers using immunohistochemistry. Briefly, GRIM-19protein expression was extremely lower in HCC tissues than in the ANLT. To explore the relationship between human hepatocellular carcinoma cell lines and hepatic cell lines, we evaluated the expression status of endogenous GRIM-19expression levels in LO2cells, HepG2cells and MHCC-97H cells by immunofluorescence experiments at equal pace. Endogenous GRIM-19expression in HepG2cells was significantly lower than in LO2cells, but it was higher than in the MHCC-97H cells. These findings were confirmed by determination of the endogenous GRIM-19protein expression levels by western blot analysis and endogenous GRIM-19mRNA levels were determined by QRT-PCR. Consistent with the immunofluorescence data, endogenous GRIM-19expression in HepG2cells was higher than in MHCC-97H cells but lower than in LO2cells. In addition, endogenous STATS、p-STAT3and VEGF expression in HepG2cells was significantly higher than in LO2cells, but it was lower than in the MHCC-97H cells.
3. The growth inhibition of HepG2intervened by RA combined with IFN-β
The combination of RA and IFN-βhas been shown to inhibit cell growth in other cell lines. To determine if IFN/RA exerts similar growth-suppressive effects in the hepatocellular carcinoma cells, human hepatocellular carcinoma HepG2cells grown in culture dishes were treated either with either IFN or RA or a combination of IFN/RA. MTT assays were performed. As shown in Table2, exposure to RA or IFN-β alone can inhibit the growth of HepG2cells, but RA combined with IFN-β can greatly increase it, and this is associated with concentration and time. We found that too much RA/IFN-β or too long time can induce cell death in HepG2cell lines. Exposure to RA or IFN-β alone did not cause cell death. Similarly, combined exposure to IFN/RA for24h and48h did not cause significant cell death, where as combined exposure to IFN/RA for72h caused significant cell death. So, we chose48h as best exposure time to study the biology change of HepG2cells combined exposure to different concentration of IFN-β and RA in following.
4. The apoptosis of HepG2intervened by RA combined with IFN-β
Human hepatocellular carcinoma HepG2cells at a low density and then exposing to different concentration of IFN/RA for48h. To examine the effect of RA combined with IFN-P on the apoptosis of HepG2cells, flow cytometry were used. The result showed that IFN/RA exhibits a dual effect on cell growth:inhibition of cell growth and an induction of cell apoptosis. RA combined with IFN-P can promote the apoptosis of HepG2cells, and this is associated with concentration.
5. GRIM-19expression increased and appear nuclear expression in HepG2after exposed to RA/IFN-p
To determine if the cell apoptosis could be correlated with the expression of GRIM-19, we analyzed the cellular localization of GRIM-19in HepG2cells after intervened by different concentration of IFN/RA for48h using the purified GRIM-19antibody by immunofluorescence. Positive staining of GRIM-19was observed in the cytoplasm of untreated HepG2cells which was intensified at the peri-nuclear region. In addition, a positive staining was also noticeable in the nucleus after the IFN-β/RA treatment, which was significantly increased following the increased of the concentration. To verify these results further, pure nuclear protein was extracted in HepG2cells which absence or presence of IFN-β/RA treatment, and GRIM-19expression was determined. Consistent with the immunofluorescence results, the expression of GRIM-19in the nucleus was increased slightly after IFN-β/RA treatment.
6. STAT3, p-STAT3and VEGF expression decreased after exposed to RA/IFN-β We detected the expression of GRIM-19in HepG2cells after exposed to different concentration of IFN/RA for48h using the purified GRIM-19antibody by Western blot. The result indicated that GRIM-19protein expression increased than control group(p <0.05). More importantly, the lower expression of STAT3、p-STAT3、VEGF were found in treatment groups than control cells.
Conclusion
1. The location of GRIM-19in human hepatic cell lines was different with the hepatocellular carcinoma cell lines. And the expression of GRIM-19in human hepatic cell lines was higher than hepatocellular carcinoma cell lines.
2. RA combination with IFN-β can inhibit the growth and promote the apoptosis of HepG2cells.
3. RA combination with IFN-β can up-regulate of GRIM-19protein expression and down-regulate of STAT3、p-STAT3、VEGF expression.
4. GRIM-19may be a new target spot in liver cancer gene therapy.
引文
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20. Dutta A. Sen T, Banerji A. et al. Studies on Multifunctional Effect of All-Trans Retinoic Acid (ATRA) on Matrix Metalloproteinase-2 (MMP-2) and Its Regulatory Molecules in Human Breast Cancer Cells (MCF-7). J Oncol 2009; 2009:627840.
21. Meng X. Wang L. Liu Z. Cloning of differentiation-related genes induced by all-transretinoic acid(ATRA) from human lung cancer GLC-82 cell line. Zhonghua Zhongliu Zazhi 1999:21(2):85-88.
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2. SCHAFER DR SORRELL MF. Hepatocellular carcinoma. Lancet 1999: 353:1253-1257.
3. Ozturk M. Genetic aspects of hepatocellular carcinogenesis. Semin Liver Dis 1999; 19(3):235-242.
4. Kalvakolanu DV. The GRIMs:a new interface between cell death regulation and interferon/retinoid induced growth suppression. Cytokine Growth Factor Rev 2004; 15(2-3):169-194.
5. Hofmann ER. Boyanapalli M, Lindner DJ, et al. Mol Cell Biol 1998; 18:6493-6504.
6. ANGELL J E, HNDNER D J, SHAPIRO P S, et al. Identification of GRIM·19. a novel cell death-regulatory gene induced by the interfemn-beta and retinoic acid combination using a genetic appreach. JBiol Chem 2000; 275(43):33416-33426.
7. Hu J, ANGELL JE, ZHANG J. et at. Characterization of monoclonal antibodies against GRIM-19, a novel IFN-beta and retinoic acid activated regulator of cell death. J Interferon Cytokine Res 2002; 22(10):1017-1026.
8. Huang G, Lu H, Hao A, et al. GRIM-19, a cell death regulatory protein, is essential for assembly and function of mitochondrial complex. Mol Cell Biol 2004;24(19):8447-8456.
9. 周爱民,赵继京,叶静,等GRIM-19蛋白在非小细胞肺癌组织中的表达及临床意义.癌症2009；28(4)：431-435.
10. Lufei C. Ma J, Huang G, et al. GRIM-19, a death-regulatory gene product, suppresses stat3 activity via functional interaction. EMBO J 2003; 22(6):1325-1335.
11. Lekili M, Ergen A. Celebi I. Zinc plasma levels in prostatic carcinoma and BPH. International Urology & Nephrology 1991; 23(2):151-154.
12. Maximo V. Botelho T, Capela J. et al. Somatic and germline mutation in GRIM-19, a dual function gene involved in mitochondrial metabolism and cell death, is linked to mitochondrion-rich (Hurthle cell) tumoursof the thyroid. Br J Cancer 2005:92:1892-1898.
13. Zhou Y. Li M. Wei Y. et al. Down-regulation of GRIM-19 expression is associated with hyperactivation of STAT3-induced gene expression and tumor growth in human cervical cancers. J Inierferon Cytokine Res 2009: 29(10):695-703.
14. Ferrantini M. Capone I. Belardelli F. Interferon-alpha and cancer:mechanisms of action and new perspectives of clinical use. Biochimie 2007.89(6-7):884-93.
15. Zhang D, Holmes WF. Wu S, et al. Retinoids and ovarian cancer. Journal of cellularphysiology 2000; 185(1):1-20.
16. Tangrea JA. Edwards BK, Taylor PR, et al. Long-term therapy with low-doseisotretinoin for prevention of basal cell carcinoma:a multicenter clinical trial. Journal of the National Cancer Institute 1992; 84(5):328-332.
17. Dutta A. Sen T, Banerji A. et al. Studies on Multifunctional Effect of All-Trans Retinoic Acid (ATRA) on Matrix Metalloproteinase-2 (MMP-2) and Its Regulatory Molecules in Human Breast Cancer Cells (MCF-7). J Oncol 2009; 2009:627840.
18.李冰,田波.等.全反式维甲酸对肝癌细胞生物学行为的影响.中华实验外科杂志2003：4：31-32.
19. Meng X. Wang L, Liu Z. Cloning of differentiation-related genes induced by all-transretinoic acid(ATRA) from human lung cancer GLC-82 cell line. Zhonghua Zhongliu Zazhi 1999; 21(2):85-88.
20. Arce F. Gatjens-Boniche O. Vargas E, et al. Apoptotic events induced by naturally occurring retinoids ATRA and 13-cis retinoic acid on human hepatoma cell lines Hep3Band HepG2. Cancer Lett 2005; 229(2):271-281.
21. Zou C, Zhou J, Qian L. et al. Comparing the effect of ATRA,4-HPR, and CD437 in bladder cancer cells. Front Biosci 2006; 11:2007-2016.
22. Dragnev KH. Rigas JR. Dmitrovsky E. The retinoids and cancer prevention mechanisms. Oncologist 2000; 5(5):361-368.
23. Chen W. YanC. Hou J. et al. ATRA enhances bystander effect of suicide gene therapy in the treatment of prostate cancer. Urol Oncol 2008; 26(4):397-405.
24. Nagler A, Berger R, Ackerstein A, et al. A randomized controlled multicenter study comparing recombinant interleukin 2 (IL-2) in conjunction with recombinant interferonalpha (IFN-alpha) versus no immunotherapy for patients with malignant lymphomapostautologous stem cell transplantation. J Immunother 2010,33(3):326-33.
25. Grigg AR Stone J, Milner AD, et al. Phase Ⅱ study of autologous stem cell transplant using busulfan-melphalan chemotherapy-only conditioning followed by interferon for relapsed poor prognosis follicular non-Hodgkin lymphoma. Leuk Lymphoma 2010,51(4):641-649.
26. Arce F, Gatjens-Boniche O, Vargas E, et al. Apoptotic events induced by naturally occurring retinoids ATRA and 13-cis retinoic acid on human hepatoma cell lines Hep3B and HepG2. Cancer Lett 2005,229(2):271-81.
27. Hu W, Verschraegen CF, Wu WQ, et al. Activity of ALRT 1550, a new retinoid, withinterferon-gamma on ovarian cancer cell lines. Int J Gynecol Cancer 2002, 12(2):202-207.
28. SEO T, LEE D, SHIM YS, et al. Viral interferon regulatory factorl of Kaposi's sarcoma-associated herpesvirus interacts with a cell feath regulator, GRIM-19, and inhibits interferon/retinoic acid-induced cell death. J Virol 2002; 76(17):8797-8807.
29. REEVES M B, DAVIES A A, MCSHARRY B P, et al. Complex Ⅰ binding by a virally encoded RNA regulates mitochondria-induced cell death. Science 2007; 316(5829):1345-1348.
30. Hua Y, Richard J. The STATs of cancer-new molecular targets come of ag. Nature(Review Cancer) 2004; 2(4):97-105.
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