腺病毒介导的炎性基因miRNAs干预小鼠暴发性肝炎和裸鼠人肝癌皮下移植瘤的研究
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摘要
[背景及目的]
我国是乙肝病毒(HBV)感染高发地区,2006年我国乙肝流行病学调查显示,目前我国1-59岁乙肝表面抗原(HbsAg)人群携带率为7.18%,儿童(5岁以下)HBsAg携带率为0.96%,据此估算,目前我国HBV慢性感染者约9300万人,其中约2000万患者为慢性乙肝。慢性乙肝患者中约1%发展为重型肝炎。由于多种原因所致肝细胞大量坏死,导致肝脏合成、代谢、生物转化、解毒、排泄等功能发生严重障碍,并出现肝性脑病、脑水肿、感染、出血等严重并发症的临床综合征称之为重型肝炎或肝功能衰竭,是目前临床常见的急危重症。在欧美国家,药物及酒精是引起重型肝炎的主要原因,在我国,重型肝炎则主要由肝炎病毒,尤其是HBV感染所致(约70%)。重型肝炎来势凶险、病情严重、发展迅速、病死率高,严重威胁患者生命。重型肝炎的发病机制尚未明了,目前临床上仍缺乏有效治疗手段,虽然部分患者可通过肝移植手术挽救生命,但移植后1年生存率也仅为50%-60%,因此绝大部分重型肝炎患者预后不良,死亡率可达到70%-80%。开发与重型肝炎发生、发展相关的基因治疗的方法正成为该领域的研究热点和发展方向。
原发性肝癌是恶性程度极高、预后极差的恶性肿瘤,居恶性肿瘤发生率的第五位、病死率的第三位。全世界每年约有65万人死于肝癌。全世界80%以上的肝癌患者集中在亚洲和非洲,其中中国占了约50%。在中国,肝癌多由乙肝所致肝硬化演变而来,而在西方国家,由于HCV感染和非酒精性脂肪肝患者的增加使得肝癌的发病率逐年增加。传统的肝癌治疗手段包括外科切除、肝移植、放疗和化疗。然而,无论是手术切除还是肝移植均存在着术后的高复发率问题,且肝移植供体贫乏,加之肝细胞癌对化疗和放疗均不敏感,使得肝癌的临床诊疗仍缺乏有效的治疗手段。因此,针对肝癌诊治新技术、新疗法如基因治疗和分子靶向治疗的研究则显得尤为重要和迫切。
在重型肝炎的发病过程中,涉及到多种炎性分子的异常表达,如Fas/FasL系统、TNFa/TNFR1系统介导的肝细胞凋亡和fgl2介导的肝细胞坏死。在重型肝炎时,体内残存的肝细胞过度表达Fas,同时大量激活的CTL异常高表达FasL,且血清可溶性FasL水平显著增高,从而导致肝细胞凋亡。TNFa由活化的巨噬细胞产生,它有两种受体,即TNFR1和TNFR2,有研究表明,TNFa及TNFR1的表达水平与重型肝炎的病变程度相一致,同时凋亡的肝细胞数目与TNFa的表达呈现非常显著的正相关。Fgl2凝血酶原酶被称为“免疫凝血”,它具有免疫调节功能和凝血功能。作为新型凝血酶原酶,fgl2可直接促进凝血酶的生成而不依赖于其它凝血因子,凝血酶可通过降解纤维蛋白原形成纤维蛋白导致血栓形成和纤维素的沉积。研究揭示在IFN-y等炎性因子的刺激下,fgl2在重型肝炎中高表达,是肝细胞坏死的重要分子机制；本研究所前期利用mfgl2反义质粒和shRNA干扰质粒可显著减轻暴发性肝炎小鼠肝组织病理损伤,减少炎性细胞的浸润、纤维素的沉积,从而提高暴肝小鼠生存率至33.3%。
Fgl2与肝癌的发生、发展也有密切关系。我们的前期研究发现常见实体肿瘤如肝癌、食管癌、肺癌、胃癌、结肠癌、宫颈癌、乳腺癌等多种恶性肿瘤的实质细胞、间质浸润细胞、血管内皮细胞及细胞外基质均有fgl2凝血酶原酶的广泛表达,并与纤维蛋白沉积有密切组织学关系。作为凝血酶的上游基因,我们推测fgl2在肿瘤生长、转移及血管新生中亦发挥了重要作用。我们应用裸鼠角膜血管新生模型和肿瘤皮下瘤移植模型显示fgl2基因沉寂后的人肝癌细胞诱导血管新生的能力显著降低；Fgl2敲除的高侵袭性人肝癌细胞HCCLM6细胞增殖能力、侵袭能力,耐受TNFa诱导的凋亡能力、表达促血管生成因子如IL-8、VEGF的能力较未干扰HCCLM6细胞及非相关对照HCCLM6细胞均显著下降,表明fgl2在肿瘤细胞中的高表达在肿瘤的生长和转移中发挥了重要作用。进一步研究表明：分泌型fgl2在肿瘤微环境中的表达上调通过激活p38-MAPK通路和抑制JNK通路实现。上述变化可增强单核巨噬细胞的致炎活性,抑制向树突状细胞分化,继而可能抑制肿瘤特异性T细胞免疫；而膜结合型fgl2在单核巨噬细胞的表达则可能通过活化p38-MAPK和ERK途径促进细胞的增殖。
RNA干扰(RNA interference, RNAi)是指内源性或外源性双链RNA (dsRNA)分子降解相应序列的mRNA,导致靶基因表达沉默的过程,是一种序列特异性的转录后基因沉默,具有高效、特异、快速等优点。作为一种自然防御机制,RNA干扰在许多物种如线虫、果蝇、植物以及哺乳动物中都存在,可防止生物体受到病毒或其它外源核酸的入侵,同时在生物生长发育中发挥着基因表达调控的作用。目前发现有两种非编码小干扰RNA：小的干涉RNA (siRNA)和微小RNA (miRNA). RNA干扰现象被发现以后,越来越多的研究者将其广泛应用于功能基因组学的研究。近年来,RNA干扰技术作为一种治疗工具,在预防和治疗人类疾病如感染性疾病、肿瘤、某些代谢性疾病等方面也取得了瞩目的成绩。
本研究旨在利用miRNA技术干预在重型肝炎、肝癌发生发展中起重要作用的致病基因,研究基因治疗的可行性,为重型肝炎、肝癌的防治开辟出新方法。具体研究内容如下：
1、针对与重型病毒性肝炎发生发展密切相关的分子mfgl2,构建腺病毒介导的miRNA即Ad-mfgl2-miRNA,探索其对目的基因的沉默效应以及联合应用(Ad-mfgl2-miRNA、Ad-mFas-mTNFR1-miRNA)对暴发性肝炎小鼠病程的干预作用；
2、利用Ad-hfgl2-miRNA沉默肝癌中高表达的fgl2,观察其对裸鼠人肝癌皮下移植瘤的靶向干预作用。
[研究方法]
1、通过Gateway技术构建产生小鼠fg12-miRNA的表达载体P-mfgl2-miRNA和非相关对照质粒,分别连入5型缺陷型腺病毒载体中,形成Ad-mfgl2-miRNA和Ad-neg-miRNA。细胞水平共转染Ad-mfgl2-miRNA和pcDNA3.1-mfg12表达质粒,通过实时荧光定量PCR及Western blot观察fgl2在基因转录水平及蛋白水平表达量的改变。
2、建立MHV-3诱导的Balb/cJ小鼠暴发性肝炎模型；通过尾静脉注射将Ad-mfg12-miRNA和／或Ad-mFas-mTNFR 1-miRNA(实验室保存)导入小鼠体内,取小鼠眼球血检测血清生化学指标的改变、肝组织HE染色检测小鼠肝组织病理变化,并观察暴发性肝炎小鼠生存率的变化；通过实时荧光定量PCR、Western blot及免疫组化检测各组小鼠肝组织中目的基因的表达情况；通过TUNNEL法检测各组小鼠肝组织中肝细胞凋亡情况。
3、建立裸鼠人肝癌皮下移植瘤模型,瘤内注射Ad-hfg12-miRNA(实验室保存),优化治疗剂量；观察裸鼠皮下瘤的动态生长变化,计算相对肿瘤增殖率；通过实时荧光定量PCR和Western blot检测瘤组织中fgl2的表达,免疫组化检测瘤组织中fgl2、纤维蛋白以及血管新生标志物CD31的表达,并计算瘤组织中微血管密度。
[结果]
1、成功构建了腺病毒介导的针对小鼠fgl2的miRNA和非相关对照miRNA,测序鉴定无误。在CHO细胞中,干预组fgl2在RNA水平及蛋白水平的表达均较非相关对照组和阳性对照组显著降低。
2、尾静脉注射Ad-mfg12-miRNA或Ad-mFas-mTNFR 1-miRNA后,可使暴发性肝炎小鼠生存率从0分别提高到38.9%和16.7%,而上述两种腺病毒联合干预后的暴肝小鼠生存率可提高到44.4%。干预组小鼠均可见肝组织病理学变化和血清学指标的明显改善；小鼠肝组织中相应靶基因的表达和肝细胞凋亡在干预组受到明显抑制。
3、经Ad-hfg12-miRNA处理后,干预组小鼠的整体状态好于非相关对照组和空白对照组小鼠,瘤块明显小于后两组小鼠的瘤块。与非相关对照及空白对照相比,其相对肿瘤增殖率分别达到29.9%和36.7%,同时,也检测到了注射腺病毒注射液后,受体组织肿瘤细胞中hfg12 mRNA水平和蛋白质水平均出现了抑制,证实了Ad-hfg12-miRNA对hfg12凝血酶原酶基因的沉默作用,此外,瘤组织中纤维蛋白沉积及新生血管也较对照组明显降低。
[结论]
1、本研究成功构建了Ad-mfg12-miRNA,体内外实验均观察到其高效特异的下调目的基因表达的作用。
2、本研究表明fgl2、Fas及TNFR1可作为重型肝炎基因治疗的靶点,基于重型肝炎相关基因的体内联合干预较单独干预显示出了更为显著的治疗效果,利用miRNA沉默fgl2、Fas及TNFR1基因为重型肝炎的治疗提供了新的策略。
3、本研究显示Ad-hfg12-miRNA可显著抑制裸鼠人肝癌皮下移植瘤生长,干预效果明显,为今后肝癌等恶性肿瘤的治疗提供了新思路。
[BACKGROUND&OBJECTIVE]
Hepatitis B virus (HBV) infection is highly prevalent in China. The seroepidemiological survey on HBV infection conducted in 2006 showed that HBsAg carrier rate was 7.18% in the group of age 1-59 years old and 0.96% in children of 1 to 4 years old. Accordingly there were about 93 million HBV carriers, and among them 20 million were patients with chronic hepatitis B. The characteristic of severe hepatitis or hepatic failure is the extreme rapidity of hepatocellular injury, resulting in widespread hepatocellular necrosis in weeks or even days and occurrence of hepatic encephalopathy. Severe hepatitis is still a common disease with acute and severe manifestation. In China, severe hepatitis is primarily due to hepatitis virus infection, especially HBV infection (about 70%), whereas in the West it is mainly due to drugs or alcohol. The pathogenesis of severe hepatitis is still not clearly known. Moreover, it is lack of specific and effective treatment. Even an liver transplantation can be applied, the 1 year survival rate is only 50-60% after liver transplantation. Therefore, the majority of patients suffered from fulminant viral hepatitis are with poor prognosis and the mortality of severe hepatitis is about 70-80%. Hence, new therapeutic approach such as gene therapy for severe hepatitis has become a research focus in this field.
Primary liver cancer, including hepatocellular carcinoma (HCC), is the fifth most common malignancy in the world and the third most common cause of cancer mortality, killing more than 650,000 individuals around the world every year. Over 80% of HCC occurs in Asia and Africa, among them China accounts for a total of 50% of HCC cases worldwide. In China, HCC is mainly due to HBV infection. In recent years, the incidence of HCC has also been rising in other regions because of increased hepatitis C virus (HCV) infection and non-alcoholic steatohepatitis (NASH). The conventional therapy includes surgical resection, liver transplantation, chemotherapy and radiotherapy. However, surgical resection is associated with a high incidence of post-operative recurrence. The prognosis after liver transplantation is also unsatisfactory because of the shortage of donor organs and recurrence. In addition, HCC is insensitive to chemotherapy and radiotherapy, and these therapies can lead to side-effects. Hence, new therapeutic strategies such as gene therapy and molecular targeted therapy for HCC must be explored.
The pathogenic process of severe hepatitis is associated with hepatocyte apoptosis induced by Fas/FasL, TNFα/TNFR1 and hepatocyte necrosis induced by fg12. Fas is overexpressed in hepatocyte and FasL is overexpressed in actived CTL from patients with severe hepatitis, which can induce hepatocyte apoptosis. TNFa, which is produced by actived macrophage, has two primary categories of receptors, TNFR1 and TNFR2. Studies have revealed the expression level of TNFa and TNFR1 was consistent with the pathological degree of severe hepatitis. What's more, the number of apoptotic hepatocytes was high correlated with expression of TNFa. As a member of the fibrinogen-related protein superfamily, fibrinogen-like protein 2 prothrombinase (also called fg12 prothrombinase) has serine protease activity and directly catalyzes prothrombin to activated thrombin in the absence of factor VII or factor X. Studies have shown that fg12, which is highly expressed at the cytomembrane of activated macrophages, lymphocytes, and endothelial cells, is associated with microthrombosis and intravascular fibrin deposition within the liver. Results from our research showed that interference of mfg12 with antisense and shRNA significantly decrease hepatocyte necrosis, improve fibrin deposition and inflammatory cell infiltration, and elevated the survival rate in Balb/cJ mice with MHV-3-induced fulminant hepatitis.
Fg12 is considered as an important link on tumorigenesis and tumor growth of human malignancies. Our previous research has also shown that fg12 is overexpressed in subcutaneous tumors and in colon carcinoma, breast cancer, lung cancer, gastric cancer, esophageal carcinoma, cervical cancer and many other malignant tumor tissues from patients. Fg12 is distributed in cancer cells, vascular endothelial cells and interstitial infiltrated cells. As the upstream target of thrombin, we proposed that fg12 influences tumor biology by participating in tumor angiogenesis, growth and metastasis. Our previous study demonstrated that fg12 knock down in HCCLM6 cells led to delayed tumorigenesis and an obvious suppression of angiogenesis both in HCCLM6 tumor-bearing nude mice and corneal micropocket transplanted model; fg12 knock down could inhibit proliferation of HCCLM6 cells and increase their sensitivity to TNFa induced apoptosis in vitro; high level of fg12 expression could induce VEGF and IL-8 expression at both mRNA and protein level; recombinant fg12 protein could lead to transient P38-MAPK activation and subsequently sustained ERK1/2 MAPK activation through thrombin generation.
RNA interference (RNAi) mediates sequence-specific inhibition of gene expression via a post-transcriptional gene silencing mechanism. It is a natural phenomenon occurring in a wide range of species, including plants, fungi and animals and can effectively and specifically degrade cognate mRNA. There are two primary categories of small non-coding RNAs, including small interfering RNAs (siRNA) that can be transfected directly into cells and microRNA (miRNA) that can be transfected into cells with the help of specific vectors. Since the discovery of RNAi, there has been substantial interest in using this technology for functional genomics. In recent years, attention has turned to assessing the potential therapeutic effect of RNAi, and researchers worldwide have utilized this technology as a therapeutic tool to prevent and treat human diseases such as tumors, infectious diseases and other disorders including some dominant genetic diseases.
The purpose of this study was to silence pathogenic gene which participates in the development and progression of severe hepatitis and HCC via RNA interference, as well as to explore the feasibility of gene therapy for the treatment of severe hepatitis and HCC. The concrete contents are as follows:
1. To construct an adenovirus vector expressing miRNA against hfg12 gene (Ad-mfgl2-miRNA) which can inhibit the expression of mfgl2 in vitro and to evaluate the therapeutic effects on Balb/cJ mice with MHV-3-induced fulminant hepatitis using combination therapy with Ad-mfg12-miRNA and Ad-mFas-mTNFR1-miRNA in vivo;
2. To evaluate the therapeutic effect of Ad-hfgl2-miRNA on the growth of HCCLM6 tumor xenograft in nude mice.
[METHODS]
1. P-mfg12-miRNA and P-neg-miRNA were constructed by Gateway technology, then they were transferred into recombinant adenoviral vector using Gateway technology, thus, Ad-hfgl2-miRNA and Ad-neg-miRNA were generated. Ad-mfgl2-miRNA and pcDNA3.1-mfg12 were cotransfected into CHO cells. The level of mfg12 mRNA transcript and protein were analysed by RT-PCR and Western blot.
2. Fulminant hepatitis model in Balb/cJ mice was established by intraperitoneally injection with MHV-3. Ad-mfgl2-miRNA and/or Ad-mFas-mTNFR1-miRNA were introduced into mice via tail intravenous injection. Then the serum biochemical disorder, hepatic pathological change and the survival rate of mice were examined; the mfg12 mRNA and protein levels in MHV-3-infected Balb/cJ mice were detected by qRT-PCR, Western blot analysis and immunohistochemistry respectively; the TUNEL method was utilized to estimate hepatocyte apoptosis in MHV-3 infected fulminant hepatitis in Balb/cJ mice.
3. The xenograft tumor model was established by subcutaneous inoculation with HCCLM6 cells in nude mice, and this was accompanied with intratumoral injection of Ad-hfgl2-miRNA when the tumor volume had reached 100 mm3. The volume of tumor xenograft was recorded twice a week and the relative tumor volume (RTV) was calculated; the hfg12 mRNA and protein levels, fibrin, as well as angiogenesis in the tumor xenograft were assessed by qRT-PCR, Western blot analysis and immunohistochemistry, respectively; the microvessel density (MVD) in tumor tissues was calculated.
[RESULTS]
1. Ad-mfg12-miRNA and Ad-neg-miRNA were successfully constructed as evidenced by sequence analysis; qRT-PCR and Western blot analyses showed that mfg12 expression in CHO cells treated with the Ad-mfg12-miRNA significantly decreased at the mRNA and protein levels compared with those of the irrelevant control and positive control groups.
2. After tail intravenous injection with Ad-mfgl2-miRNA or Ad-mFas-mTNFR1-miRNA, the survival rate reached to 38.9% and 16.7%, respectively; the survival rate elevated to 44.4% after combination therapy with the adenoviruses mentioned above; the administration of Ad-mfgl2-miRNA and/or Ad-mFas-mTNFR1-miRNA ameliorates liver pathology including fibrin deposition, inflammatory cell infiltration, hepatocyte apoptosis and necrosis. Additionally, the target genes expression were significantly reduced in vivo.
3. After intratumorally injection with Ad-hfgl2-miRNA, the Ad-hfgl2-miRNA-treated tumors exhibited a slower speed of growth than the Ad-neg-miRNA-and PBS-treated tumors, and all mice in the control groups became emaciated by day 28. Meanwhile, the tumor volumes were measured and showed a relative tumor proliferation rate T/C (%) of 36.7%(P<0.05 versus blank control) and 29.9%(P<0.01 versus the Ad-neg-miRNA group), respectively. In addition, fg12 gene expression, fibrin deposition and angiogenesis in the Ad-hfgl2-miRNA-treated tumors was significantly suppressed which confirmed by qRT-PCR, Western blot analysis and immunohistochemistry.
[CONCLUSION]
1. Ad-mfg12-miRNA was successfully constructed, which can downregulate mfg12 expression effectively and specifically in vitro and in vivo.
2. This study suggests that fg12, Fas and TNFR1 might be potential target for treatment of severe hepatitis. Furthermore, the combination of Ad-mfgl2-miRNA with Ad-mFas-mTNFR1-miRNA may enhance the therapeutic efficacy of this approach. Gene silencing using miRNA may be an alternative strategy for gene therapy.
3. Administration of Ad-hfgl2-miRNA exhibited significant antitumor effects for the treatment of HCC and that this therapeutic efficacy may be accomplished by inhibition of angiogenesis. Therefore, RNAi-based gene therapy targeting fg12 may be useful as an effective approach for management of HCC.
我国是乙肝病毒(HBV)感染高发地区,2006年我国乙肝流行病学调查显示,目前我国1-59岁乙肝表面抗原(HbsAg)人群携带率为7.18%,儿童(5岁以下)HBsAg携带率为0.96%,据此估算,目前我国HBV慢性感染者约9300万人,其中约2000万患者为慢性乙肝。慢性乙肝患者中约1%发展为重型肝炎。由于多种原因所致肝细胞大量坏死,导致肝脏合成、代谢、生物转化、解毒、排泄等功能发生严重障碍,并出现肝性脑病、脑水肿、感染、出血等严重并发症的临床综合征称之为重型肝炎或肝功能衰竭,是目前临床常见的急危重症。在欧美国家,药物及酒精是引起重型肝炎的主要原因,在我国,重型肝炎则主要由肝炎病毒,尤其是HBV感染所致(约70%)。重型肝炎来势凶险、病情严重、发展迅速、病死率高,严重威胁患者生命。重型肝炎的发病机制尚未明了,目前临床上仍缺乏有效治疗手段,虽然部分患者可通过肝移植手术挽救生命,但移植后1年生存率也仅为50%-60%,因此绝大部分重型肝炎患者预后不良,死亡率可达到70%-80%。开发与重型肝炎发生、发展相关的基因治疗的方法正成为该领域的研究热点和发展方向。
原发性肝癌是恶性程度极高、预后极差的恶性肿瘤,居恶性肿瘤发生率的第五位、病死率的第三位。全世界每年约有65万人死于肝癌。全世界80%以上的肝癌患者集中在亚洲和非洲,其中中国占了约50%。在中国,肝癌多由乙肝所致肝硬化演变而来,而在西方国家,由于HCV感染和非酒精性脂肪肝患者的增加使得肝癌的发病率逐年增加。传统的肝癌治疗手段包括外科切除、肝移植、放疗和化疗。然而,无论是手术切除还是肝移植均存在着术后的高复发率问题,且肝移植供体贫乏,加之肝细胞癌对化疗和放疗均不敏感,使得肝癌的临床诊疗仍缺乏有效的治疗手段。因此,针对肝癌诊治新技术、新疗法如基因治疗和分子靶向治疗的研究则显得尤为重要和迫切。
在重型肝炎的发病过程中,涉及到多种炎性分子的异常表达,如Fas/FasL系统、TNFa/TNFR1系统介导的肝细胞凋亡和fgl2介导的肝细胞坏死。在重型肝炎时,体内残存的肝细胞过度表达Fas,同时大量激活的CTL异常高表达FasL,且血清可溶性FasL水平显著增高,从而导致肝细胞凋亡。TNFa由活化的巨噬细胞产生,它有两种受体,即TNFR1和TNFR2,有研究表明,TNFa及TNFR1的表达水平与重型肝炎的病变程度相一致,同时凋亡的肝细胞数目与TNFa的表达呈现非常显著的正相关。Fgl2凝血酶原酶被称为“免疫凝血”,它具有免疫调节功能和凝血功能。作为新型凝血酶原酶,fgl2可直接促进凝血酶的生成而不依赖于其它凝血因子,凝血酶可通过降解纤维蛋白原形成纤维蛋白导致血栓形成和纤维素的沉积。研究揭示在IFN-y等炎性因子的刺激下,fgl2在重型肝炎中高表达,是肝细胞坏死的重要分子机制；本研究所前期利用mfgl2反义质粒和shRNA干扰质粒可显著减轻暴发性肝炎小鼠肝组织病理损伤,减少炎性细胞的浸润、纤维素的沉积,从而提高暴肝小鼠生存率至33.3%。
Fgl2与肝癌的发生、发展也有密切关系。我们的前期研究发现常见实体肿瘤如肝癌、食管癌、肺癌、胃癌、结肠癌、宫颈癌、乳腺癌等多种恶性肿瘤的实质细胞、间质浸润细胞、血管内皮细胞及细胞外基质均有fgl2凝血酶原酶的广泛表达,并与纤维蛋白沉积有密切组织学关系。作为凝血酶的上游基因,我们推测fgl2在肿瘤生长、转移及血管新生中亦发挥了重要作用。我们应用裸鼠角膜血管新生模型和肿瘤皮下瘤移植模型显示fgl2基因沉寂后的人肝癌细胞诱导血管新生的能力显著降低；Fgl2敲除的高侵袭性人肝癌细胞HCCLM6细胞增殖能力、侵袭能力,耐受TNFa诱导的凋亡能力、表达促血管生成因子如IL-8、VEGF的能力较未干扰HCCLM6细胞及非相关对照HCCLM6细胞均显著下降,表明fgl2在肿瘤细胞中的高表达在肿瘤的生长和转移中发挥了重要作用。进一步研究表明：分泌型fgl2在肿瘤微环境中的表达上调通过激活p38-MAPK通路和抑制JNK通路实现。上述变化可增强单核巨噬细胞的致炎活性,抑制向树突状细胞分化,继而可能抑制肿瘤特异性T细胞免疫；而膜结合型fgl2在单核巨噬细胞的表达则可能通过活化p38-MAPK和ERK途径促进细胞的增殖。
RNA干扰(RNA interference, RNAi)是指内源性或外源性双链RNA (dsRNA)分子降解相应序列的mRNA,导致靶基因表达沉默的过程,是一种序列特异性的转录后基因沉默,具有高效、特异、快速等优点。作为一种自然防御机制,RNA干扰在许多物种如线虫、果蝇、植物以及哺乳动物中都存在,可防止生物体受到病毒或其它外源核酸的入侵,同时在生物生长发育中发挥着基因表达调控的作用。目前发现有两种非编码小干扰RNA：小的干涉RNA (siRNA)和微小RNA (miRNA). RNA干扰现象被发现以后,越来越多的研究者将其广泛应用于功能基因组学的研究。近年来,RNA干扰技术作为一种治疗工具,在预防和治疗人类疾病如感染性疾病、肿瘤、某些代谢性疾病等方面也取得了瞩目的成绩。
本研究旨在利用miRNA技术干预在重型肝炎、肝癌发生发展中起重要作用的致病基因,研究基因治疗的可行性,为重型肝炎、肝癌的防治开辟出新方法。具体研究内容如下：
1、针对与重型病毒性肝炎发生发展密切相关的分子mfgl2,构建腺病毒介导的miRNA即Ad-mfgl2-miRNA,探索其对目的基因的沉默效应以及联合应用(Ad-mfgl2-miRNA、Ad-mFas-mTNFR1-miRNA)对暴发性肝炎小鼠病程的干预作用；
2、利用Ad-hfgl2-miRNA沉默肝癌中高表达的fgl2,观察其对裸鼠人肝癌皮下移植瘤的靶向干预作用。
[研究方法]
1、通过Gateway技术构建产生小鼠fg12-miRNA的表达载体P-mfgl2-miRNA和非相关对照质粒,分别连入5型缺陷型腺病毒载体中,形成Ad-mfgl2-miRNA和Ad-neg-miRNA。细胞水平共转染Ad-mfgl2-miRNA和pcDNA3.1-mfg12表达质粒,通过实时荧光定量PCR及Western blot观察fgl2在基因转录水平及蛋白水平表达量的改变。
2、建立MHV-3诱导的Balb/cJ小鼠暴发性肝炎模型；通过尾静脉注射将Ad-mfg12-miRNA和／或Ad-mFas-mTNFR 1-miRNA(实验室保存)导入小鼠体内,取小鼠眼球血检测血清生化学指标的改变、肝组织HE染色检测小鼠肝组织病理变化,并观察暴发性肝炎小鼠生存率的变化；通过实时荧光定量PCR、Western blot及免疫组化检测各组小鼠肝组织中目的基因的表达情况；通过TUNNEL法检测各组小鼠肝组织中肝细胞凋亡情况。
3、建立裸鼠人肝癌皮下移植瘤模型,瘤内注射Ad-hfg12-miRNA(实验室保存),优化治疗剂量；观察裸鼠皮下瘤的动态生长变化,计算相对肿瘤增殖率；通过实时荧光定量PCR和Western blot检测瘤组织中fgl2的表达,免疫组化检测瘤组织中fgl2、纤维蛋白以及血管新生标志物CD31的表达,并计算瘤组织中微血管密度。
[结果]
1、成功构建了腺病毒介导的针对小鼠fgl2的miRNA和非相关对照miRNA,测序鉴定无误。在CHO细胞中,干预组fgl2在RNA水平及蛋白水平的表达均较非相关对照组和阳性对照组显著降低。
2、尾静脉注射Ad-mfg12-miRNA或Ad-mFas-mTNFR 1-miRNA后,可使暴发性肝炎小鼠生存率从0分别提高到38.9%和16.7%,而上述两种腺病毒联合干预后的暴肝小鼠生存率可提高到44.4%。干预组小鼠均可见肝组织病理学变化和血清学指标的明显改善；小鼠肝组织中相应靶基因的表达和肝细胞凋亡在干预组受到明显抑制。
3、经Ad-hfg12-miRNA处理后,干预组小鼠的整体状态好于非相关对照组和空白对照组小鼠,瘤块明显小于后两组小鼠的瘤块。与非相关对照及空白对照相比,其相对肿瘤增殖率分别达到29.9%和36.7%,同时,也检测到了注射腺病毒注射液后,受体组织肿瘤细胞中hfg12 mRNA水平和蛋白质水平均出现了抑制,证实了Ad-hfg12-miRNA对hfg12凝血酶原酶基因的沉默作用,此外,瘤组织中纤维蛋白沉积及新生血管也较对照组明显降低。
[结论]
1、本研究成功构建了Ad-mfg12-miRNA,体内外实验均观察到其高效特异的下调目的基因表达的作用。
2、本研究表明fgl2、Fas及TNFR1可作为重型肝炎基因治疗的靶点,基于重型肝炎相关基因的体内联合干预较单独干预显示出了更为显著的治疗效果,利用miRNA沉默fgl2、Fas及TNFR1基因为重型肝炎的治疗提供了新的策略。
3、本研究显示Ad-hfg12-miRNA可显著抑制裸鼠人肝癌皮下移植瘤生长,干预效果明显,为今后肝癌等恶性肿瘤的治疗提供了新思路。
[BACKGROUND&OBJECTIVE]
Hepatitis B virus (HBV) infection is highly prevalent in China. The seroepidemiological survey on HBV infection conducted in 2006 showed that HBsAg carrier rate was 7.18% in the group of age 1-59 years old and 0.96% in children of 1 to 4 years old. Accordingly there were about 93 million HBV carriers, and among them 20 million were patients with chronic hepatitis B. The characteristic of severe hepatitis or hepatic failure is the extreme rapidity of hepatocellular injury, resulting in widespread hepatocellular necrosis in weeks or even days and occurrence of hepatic encephalopathy. Severe hepatitis is still a common disease with acute and severe manifestation. In China, severe hepatitis is primarily due to hepatitis virus infection, especially HBV infection (about 70%), whereas in the West it is mainly due to drugs or alcohol. The pathogenesis of severe hepatitis is still not clearly known. Moreover, it is lack of specific and effective treatment. Even an liver transplantation can be applied, the 1 year survival rate is only 50-60% after liver transplantation. Therefore, the majority of patients suffered from fulminant viral hepatitis are with poor prognosis and the mortality of severe hepatitis is about 70-80%. Hence, new therapeutic approach such as gene therapy for severe hepatitis has become a research focus in this field.
Primary liver cancer, including hepatocellular carcinoma (HCC), is the fifth most common malignancy in the world and the third most common cause of cancer mortality, killing more than 650,000 individuals around the world every year. Over 80% of HCC occurs in Asia and Africa, among them China accounts for a total of 50% of HCC cases worldwide. In China, HCC is mainly due to HBV infection. In recent years, the incidence of HCC has also been rising in other regions because of increased hepatitis C virus (HCV) infection and non-alcoholic steatohepatitis (NASH). The conventional therapy includes surgical resection, liver transplantation, chemotherapy and radiotherapy. However, surgical resection is associated with a high incidence of post-operative recurrence. The prognosis after liver transplantation is also unsatisfactory because of the shortage of donor organs and recurrence. In addition, HCC is insensitive to chemotherapy and radiotherapy, and these therapies can lead to side-effects. Hence, new therapeutic strategies such as gene therapy and molecular targeted therapy for HCC must be explored.
The pathogenic process of severe hepatitis is associated with hepatocyte apoptosis induced by Fas/FasL, TNFα/TNFR1 and hepatocyte necrosis induced by fg12. Fas is overexpressed in hepatocyte and FasL is overexpressed in actived CTL from patients with severe hepatitis, which can induce hepatocyte apoptosis. TNFa, which is produced by actived macrophage, has two primary categories of receptors, TNFR1 and TNFR2. Studies have revealed the expression level of TNFa and TNFR1 was consistent with the pathological degree of severe hepatitis. What's more, the number of apoptotic hepatocytes was high correlated with expression of TNFa. As a member of the fibrinogen-related protein superfamily, fibrinogen-like protein 2 prothrombinase (also called fg12 prothrombinase) has serine protease activity and directly catalyzes prothrombin to activated thrombin in the absence of factor VII or factor X. Studies have shown that fg12, which is highly expressed at the cytomembrane of activated macrophages, lymphocytes, and endothelial cells, is associated with microthrombosis and intravascular fibrin deposition within the liver. Results from our research showed that interference of mfg12 with antisense and shRNA significantly decrease hepatocyte necrosis, improve fibrin deposition and inflammatory cell infiltration, and elevated the survival rate in Balb/cJ mice with MHV-3-induced fulminant hepatitis.
Fg12 is considered as an important link on tumorigenesis and tumor growth of human malignancies. Our previous research has also shown that fg12 is overexpressed in subcutaneous tumors and in colon carcinoma, breast cancer, lung cancer, gastric cancer, esophageal carcinoma, cervical cancer and many other malignant tumor tissues from patients. Fg12 is distributed in cancer cells, vascular endothelial cells and interstitial infiltrated cells. As the upstream target of thrombin, we proposed that fg12 influences tumor biology by participating in tumor angiogenesis, growth and metastasis. Our previous study demonstrated that fg12 knock down in HCCLM6 cells led to delayed tumorigenesis and an obvious suppression of angiogenesis both in HCCLM6 tumor-bearing nude mice and corneal micropocket transplanted model; fg12 knock down could inhibit proliferation of HCCLM6 cells and increase their sensitivity to TNFa induced apoptosis in vitro; high level of fg12 expression could induce VEGF and IL-8 expression at both mRNA and protein level; recombinant fg12 protein could lead to transient P38-MAPK activation and subsequently sustained ERK1/2 MAPK activation through thrombin generation.
RNA interference (RNAi) mediates sequence-specific inhibition of gene expression via a post-transcriptional gene silencing mechanism. It is a natural phenomenon occurring in a wide range of species, including plants, fungi and animals and can effectively and specifically degrade cognate mRNA. There are two primary categories of small non-coding RNAs, including small interfering RNAs (siRNA) that can be transfected directly into cells and microRNA (miRNA) that can be transfected into cells with the help of specific vectors. Since the discovery of RNAi, there has been substantial interest in using this technology for functional genomics. In recent years, attention has turned to assessing the potential therapeutic effect of RNAi, and researchers worldwide have utilized this technology as a therapeutic tool to prevent and treat human diseases such as tumors, infectious diseases and other disorders including some dominant genetic diseases.
The purpose of this study was to silence pathogenic gene which participates in the development and progression of severe hepatitis and HCC via RNA interference, as well as to explore the feasibility of gene therapy for the treatment of severe hepatitis and HCC. The concrete contents are as follows:
1. To construct an adenovirus vector expressing miRNA against hfg12 gene (Ad-mfgl2-miRNA) which can inhibit the expression of mfgl2 in vitro and to evaluate the therapeutic effects on Balb/cJ mice with MHV-3-induced fulminant hepatitis using combination therapy with Ad-mfg12-miRNA and Ad-mFas-mTNFR1-miRNA in vivo;
2. To evaluate the therapeutic effect of Ad-hfgl2-miRNA on the growth of HCCLM6 tumor xenograft in nude mice.
[METHODS]
1. P-mfg12-miRNA and P-neg-miRNA were constructed by Gateway technology, then they were transferred into recombinant adenoviral vector using Gateway technology, thus, Ad-hfgl2-miRNA and Ad-neg-miRNA were generated. Ad-mfgl2-miRNA and pcDNA3.1-mfg12 were cotransfected into CHO cells. The level of mfg12 mRNA transcript and protein were analysed by RT-PCR and Western blot.
2. Fulminant hepatitis model in Balb/cJ mice was established by intraperitoneally injection with MHV-3. Ad-mfgl2-miRNA and/or Ad-mFas-mTNFR1-miRNA were introduced into mice via tail intravenous injection. Then the serum biochemical disorder, hepatic pathological change and the survival rate of mice were examined; the mfg12 mRNA and protein levels in MHV-3-infected Balb/cJ mice were detected by qRT-PCR, Western blot analysis and immunohistochemistry respectively; the TUNEL method was utilized to estimate hepatocyte apoptosis in MHV-3 infected fulminant hepatitis in Balb/cJ mice.
3. The xenograft tumor model was established by subcutaneous inoculation with HCCLM6 cells in nude mice, and this was accompanied with intratumoral injection of Ad-hfgl2-miRNA when the tumor volume had reached 100 mm3. The volume of tumor xenograft was recorded twice a week and the relative tumor volume (RTV) was calculated; the hfg12 mRNA and protein levels, fibrin, as well as angiogenesis in the tumor xenograft were assessed by qRT-PCR, Western blot analysis and immunohistochemistry, respectively; the microvessel density (MVD) in tumor tissues was calculated.
[RESULTS]
1. Ad-mfg12-miRNA and Ad-neg-miRNA were successfully constructed as evidenced by sequence analysis; qRT-PCR and Western blot analyses showed that mfg12 expression in CHO cells treated with the Ad-mfg12-miRNA significantly decreased at the mRNA and protein levels compared with those of the irrelevant control and positive control groups.
2. After tail intravenous injection with Ad-mfgl2-miRNA or Ad-mFas-mTNFR1-miRNA, the survival rate reached to 38.9% and 16.7%, respectively; the survival rate elevated to 44.4% after combination therapy with the adenoviruses mentioned above; the administration of Ad-mfgl2-miRNA and/or Ad-mFas-mTNFR1-miRNA ameliorates liver pathology including fibrin deposition, inflammatory cell infiltration, hepatocyte apoptosis and necrosis. Additionally, the target genes expression were significantly reduced in vivo.
3. After intratumorally injection with Ad-hfgl2-miRNA, the Ad-hfgl2-miRNA-treated tumors exhibited a slower speed of growth than the Ad-neg-miRNA-and PBS-treated tumors, and all mice in the control groups became emaciated by day 28. Meanwhile, the tumor volumes were measured and showed a relative tumor proliferation rate T/C (%) of 36.7%(P<0.05 versus blank control) and 29.9%(P<0.01 versus the Ad-neg-miRNA group), respectively. In addition, fg12 gene expression, fibrin deposition and angiogenesis in the Ad-hfgl2-miRNA-treated tumors was significantly suppressed which confirmed by qRT-PCR, Western blot analysis and immunohistochemistry.
[CONCLUSION]
1. Ad-mfg12-miRNA was successfully constructed, which can downregulate mfg12 expression effectively and specifically in vitro and in vivo.
2. This study suggests that fg12, Fas and TNFR1 might be potential target for treatment of severe hepatitis. Furthermore, the combination of Ad-mfgl2-miRNA with Ad-mFas-mTNFR1-miRNA may enhance the therapeutic efficacy of this approach. Gene silencing using miRNA may be an alternative strategy for gene therapy.
3. Administration of Ad-hfgl2-miRNA exhibited significant antitumor effects for the treatment of HCC and that this therapeutic efficacy may be accomplished by inhibition of angiogenesis. Therefore, RNAi-based gene therapy targeting fg12 may be useful as an effective approach for management of HCC.
引文
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[1]Ning Q, Brown D, Parodo J, et al. Ribavirin Inhibits Viral Induced Macrophage Production of TNF, IL-1, the Procoagulant fgl2 Prothrombinase and Preserves Th1 Cytokine Production but Inhibits Th2 Cytokine Response. The Journal of Immunology,1998,160:3487-3493.
[2]Ning Q., Liu M, Kongkham FP, et al. The Nucleocapsid Protein of Murine Hepatitis Virus Type 3 Induces Transcription of the Novel fg12 Prothrombinase Gene. The Journal of Biological Chemistry; 1999,274(15):9930-9936.
[3]Liu MF, Chan CW, McGilvray Y, et al. Fulminant viral hepatitis:Molecular and cellular basis, and clinical implications. Expert Reviews in Molecular Medicine,2001, March.
[4]宁琴,罗小平,雷延吕。鼠病毒性肝炎信号转录传导激活因子的研究。中华微生物与免疫学杂志,2002,22(6)：666-669。
[5]Ning, Q, Berger L, Luo XP, et al. STAT1 and STAT3 Splice-Form Activation Predicts Host Response in Mouse Hepatitis Virus Infection. The Journal of Medical Vioriology 2003 Mar, 69(3):306-12.
[6]Ning Q, Lakatoo S, Liu M, et al. Induction of prothrombinase fg12 by the nucleocapsid protein of virulent mouse hepatitis virus is dependent on host hepatic nuclear factor-4. The Journal of Biology Chemistry; 2003,278:15541-15549。
[7]陈悦,宁琴,王宝菊,等。重症乙型肝炎人纤维介素基因的表达及意义。中华医学杂志,2003,83(6)：446-450.
[8]宁琴,罗小平,汪之沫,等。Mfgl2凝血酶原酶/fibroleukin基因转录调控元件HNF4的研究。中华医学杂志,2003,83(8)：678-682.
[9]Marsden PA, Ning Q, Fung LS, et al. The Fg12/fibroleukin prothrombinase contributes to immunologically-mediated thrombosis in experimental and human viral hepatitis. Jounal of Clinical Investigation,2003,112:58-66. (* equal contribution).
[10]朱帆,宁琴,陈悦,等。重型乙型肝炎肝组织和外周血单个核细胞中人纤维介素基因的检测及其与临床转归的关系探讨。中华肝脏病杂志,2004,12(7)：385-388.
[11]Zhu CL, Sun Y, Luo XP, et al. Novel mfg12 antisense plasmid inhibits mfg12 expression and ameliorates MHV-3 induced fulminant hepatitis in Balb/cJ mice. Human Gene Therapy.2006
[12]Zhu CL, Yan WM, Zhu F, et al. fg12/fibroleukin expression and its correlation with disease progression in mice and human server viral hepatitis. World Journal of Gastroenterology.2005, 11(44):6936-6940.
[13]汪之沫,宁琴。重症肝炎相关基因mfg12 shRNA表达质料的构建及其体外RNA干扰研究。 2006中华肝脏病杂志。
[14]Brotin E, Meryet-Figuiere M, Simonin K, et al. Bcl-XL and MCL-1 constitute pertinent targets in ovarian carcinoma and their concomitant inhibition is sufficient to induce apoptosis. Int J Cancer. 2010,15;126(4):885-95.
[15]Gao S, Wang M, Ye HL, et al, Dual interference of novel gene mfg12 and mTNFRl ameliorates murine hepatitis virus type 3-induced fulminant hepatitis in BALB/cJ mice. Human Gene Ther, 2010,21(8):969-77.
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[1]Ning Q, Brown D, Parodo J, et al. Ribavirin Inhibits Viral Induced Macrophage Production of TNF, IL-1, the Procoagulant fgl2 Prothrombinase and Preserves Th1 Cytokine Production but Inhibits Th2 Cytokine Response. The Journal of Immunology,1998,160:3487-3493.
[2]Ning Q., Liu M, Kongkham FP, et al. The Nucleocapsid Protein of Murine Hepatitis Virus Type 3 Induces Transcription of the Novel fg12 Prothrombinase Gene. The Journal of Biological Chemistry; 1999,274(15):9930-9936.
[3]Liu MF, Chan CW, McGilvray Y, et al. Fulminant viral hepatitis:Molecular and cellular basis, and clinical implications. Expert Reviews in Molecular Medicine,2001, March.
[4]宁琴,罗小平,雷延吕。鼠病毒性肝炎信号转录传导激活因子的研究。中华微生物与免疫学杂志,2002,22(6)：666-669。
[5]Ning, Q, Berger L, Luo XP, et al. STAT1 and STAT3 Splice-Form Activation Predicts Host Response in Mouse Hepatitis Virus Infection. The Journal of Medical Vioriology 2003 Mar, 69(3):306-12.
[6]Ning Q, Lakatoo S, Liu M, et al. Induction of prothrombinase fg12 by the nucleocapsid protein of virulent mouse hepatitis virus is dependent on host hepatic nuclear factor-4. The Journal of Biology Chemistry; 2003,278:15541-15549。
[7]陈悦,宁琴,王宝菊,等。重症乙型肝炎人纤维介素基因的表达及意义。中华医学杂志,2003,83(6)：446-450.
[8]宁琴,罗小平,汪之沫,等。Mfgl2凝血酶原酶/fibroleukin基因转录调控元件HNF4的研究。中华医学杂志,2003,83(8)：678-682.
[9]Marsden PA, Ning Q, Fung LS, et al. The Fg12/fibroleukin prothrombinase contributes to immunologically-mediated thrombosis in experimental and human viral hepatitis. Jounal of Clinical Investigation,2003,112:58-66. (* equal contribution).
[10]朱帆,宁琴,陈悦,等。重型乙型肝炎肝组织和外周血单个核细胞中人纤维介素基因的检测及其与临床转归的关系探讨。中华肝脏病杂志,2004,12(7)：385-388.
[11]Zhu CL, Sun Y, Luo XP, et al. Novel mfg12 antisense plasmid inhibits mfg12 expression and ameliorates MHV-3 induced fulminant hepatitis in Balb/cJ mice. Human Gene Therapy.2006
[12]Zhu CL, Yan WM, Zhu F, et al. fg12/fibroleukin expression and its correlation with disease progression in mice and human server viral hepatitis. World Journal of Gastroenterology.2005, 11(44):6936-6940.
[13]汪之沫,宁琴。重症肝炎相关基因mfg12 shRNA表达质料的构建及其体外RNA干扰研究。 2006中华肝脏病杂志。
[14]Brotin E, Meryet-Figuiere M, Simonin K, et al. Bcl-XL and MCL-1 constitute pertinent targets in ovarian carcinoma and their concomitant inhibition is sufficient to induce apoptosis. Int J Cancer. 2010,15;126(4):885-95.
[15]Gao S, Wang M, Ye HL, et al, Dual interference of novel gene mfg12 and mTNFRl ameliorates murine hepatitis virus type 3-induced fulminant hepatitis in BALB/cJ mice. Human Gene Ther, 2010,21(8):969-77.
[1]Levy GA., Liu M., Ding J, et al. Molecular and functional analysis of the human prothrombinase gene (HFGL2) and its role in viral hepatitis. Am. J. Pathol.2000,156,1217-1225.
[2]Su K, Chen F, Yan WM, et al. Fibrinogen-like protein 2/fibroleukin prothrombinase contributes to tumor hypercoagulability via IL-2 and IFN-gamma. World J. Gastroenterol.2008,14,5980-5989.
[3]El-Serag, HB, Rudolph KL. Hepatocellular carcinoma:epidemiology and molecular carcinogenesis. Gastroenterology.2007,132,2557-2576.
[4]Mazzanti R., Gramantieri L, Bolondi L. Hepatocellular carcinoma:epidemiology and clinical aspects. Mol. Aspects Med.2008,29,130-143.
[5]Yang JD, Roberts LR. Hepatocellular carcinoma:a global view. Nat Rev Gastroenterol Hepatol. 2010,7,448-458
[6]Parkin DM, Bray F, Ferlay J, et al. Estimating the world cancer burden:globocan 2000. Int. J. Cancer.2001,94,153-156.
[7]El-Serag HB, Davila JA, Petersen NJ, et al. The continuing increase in the incidence of hepatocellular carcinoma in the United States:an update. Ann. Intern. Med.2003,139,817-823.
[8]Sherman M. Recurrence of hepatocellular carcinoma. N.Engl.J.Med.2008,359,2045-2047.
[9]Poon RT, Fan ST. Resection prior to liver transplantation for hepatocellular carcinoma:a strategy of optimizing the role of resection and transplantation in cirrhotic patients with preserved liver function. Liver Transpl.2004,10,813-815.
[10]Brechot C. Pathogenesis of hepatitis B virus-related hepatocellular carcinoma:Old and new paradigms. Gastroenterology.2004,127,56-61.
[11]Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet.2007,362,1907-1917.
[12]Ribatti D, Nico B, Crivellato E, et al. The history of the angiogenic switch concept. Leukemia.2007, 21,44-52
[13]Bruix J, Sherman M. Management of hepatocellular carcinoma. Hepatology.2005,42,1208-36.
[14]Liotta LA, Stracke ML. Tumor invasion and metastases:biochemical mechanisms. Cancer Treat. Rev.1998,40,223-238.
[15]Yao DF, Wu XH, Zhu Y, et al. Quantitative analysis of vascular endothelial growth factor, microvascular density and their clinicopathologic features in human hepatocellular carcinoma. Hepatobiliary Pancreat. Dis. Int.2005,4,220-226.
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