The Effect Of Adenovirus Mediated MiRNAs Against Inflammatory Cytokines In Fulminant Hepatitis And HCCLM6 Tumor Xenograft

[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.