| BackgroundHepatocellular carcinoma (HCC) is a seriously threat the life and health of people around the world, about 90 million people worldwide are die from the disease every year, accounting for the third in the malignancy tumor. Currently surgical resection is still considered to be the preferred treatment method, but more than 80 percent of HCC patients was accompany with cirrhosis, which result in liver resection is subject to certain extent. And many tumors are adjacent to the first, second or third hepatic hilum in lots of HCC patients, which caused great difficulties to the surgery, so clinically surgery within HCC patients is less than 20%. Hepatic arterial infusion strategy is one of the main methods to treat HCC in clinical, while its long-term efficacy is still not poor, and recurrence and metastasis rate after surgery is still very high in clinical. Subsequently, researchers have found that the up-regulation of VEGF due to ischemia and hypoxia within liver cancer tissues after hepatic artery surgery is the critical reason for the low treatment effect.VEGF is one of the most powerful and most specific factor to stimulate endothelial cell proliferation. It is also known as the strongest vascular penetrant, which 50000 times greater than histamine. VEGF can increase the permeability of capillaries, especially the veins and small veins, and then promote blood vessel leakage. Therefore, expression of VEGF reflects the proliferation, migration and angiogenesis of tumor vascular endothelial cells, which directly suggests the growth rate and metastasis tendency of tumor. To reduce the recurrence and metastasis rate of hepatic arterial infusion, and to improve the survival rate, it is necessary to block the signal transduction pathway of VEGF and reduce the expression of VEGF, which can prevent the formation of tumor angiogenesis. It is urgent to combine hepatic arterial infusion with target VEGF to anti-angiogenesis. The discovery of RNA interference (RNAi) provides the scientific basis for the project.RNA interference (RNAi) is an evolutionarily conserved surveillance mechanism through which 21- and 22-nucleotide small interfering RNAs (siRNAs) induce sequence-specific post-transcriptional gene silencing in many organisms. Using the RNAi technology, previously studies showed that siRNAs targeting VEGF inhibited gastric cancer cells growth and gastric cancer growth in a nude mouse model of subcutaneous xenografts, inhibited the growth of malignant melanoma, and inhibited the tumor growth and tumor angiogenesis in orthotopic tumors in mice through intraperitoneal application of siRNA-VEGF. Researchers have found that siRNA can significantly block or down-regulate expression of target gene, which has high specificity, less investment, short period, simple operation and other advantages. It is likely to become the next generation of therapeutic drugs, and has broad prospects. Recent years, some studies have shown that tumor angiogenesis was efficiently inhibited in ovarian cancer, malignant melanoma, colon cancer, stomach cancer etc, when they targeted inhibit the expression of VEGF by RNAi. Unfortunately, the majority of such studies belong to in vitro experiments. Since the complex living environment of living tumor cells, the reports on using RNAi in vivo experiments is less, and a few experiments are still limited to surface organ or tumor local injection in vivo. Recently, Takeshita et al. reported that siRNA molecules targeted tumors animal by intravenous injection of siRNA, but accompany with drug toxicity, low local drug concentration in target organ.In order to overcome the disadvantages mentioned above, in our present study, we plan to use the transarterial embolization (TAE) technology to deliver the siRNAs for the first time and to evaluate the effect of VEGF knockdown on tumor growth of HCC in vivo via interventional treatment of HCC in clinical application with two aspects of advantages. One advantage is the fact that the unique affinity and avocation of HCC to lipiodol to improve the targeting property. Another advantage is that lipiodol may have the property to wrap up the siRNAs as an emulsifying agent to protect the siRNAs from degradation by the substances in blood.Materials and Methods1. Cell culture:VX2 cell line.2. Specific siRNA target for VEGF:designed the 19nt target sequence corresponding to siRNA design principles and according to the sequence of VEGF in Genebank.3. Cell Culture Conditions and Transfection of siRNAs using Lipofectamin 2000TM according to manufacture’s instructions.4. Cell proliferation assay with MTT assay.5. RT-qPCR Analysis for VEGF gene expression to in vitro.6. VEGF ELISA assay to detect the secretion of VEGF protein in vitro.7. Establishment of VX2 transplantation tumor within rabbit model.8. Spiral CT examination on the rabbit models to measure the sizes of transplantation tumor.9. Experimental grouping and TACE was performed.10. CT examination was performed to identify deposit of lipiodol and to measure tumor growth ratio.11. Immunohistochemical staining procedure for VEGF and CD34 to quantitate VEGF and Microvessel density.12. RT-qPCR and West blotting technologies were used to detect VEGF expression levels in vivo.13. Analyze the cell apoptotic rates by flow cytometry and double-stained with fluorescein APC-labeled Annexin-V and PI assay.14. The plasma AST, ALT, blood urea nitrogen and creatinine levels of the rabbits were measured for Assessment of Hepatic and Renal Toxicity.Results1. The viability inhibitory rate of VEGF siRNA no.1 and no.3 to VX2 cells were 38.5±7.3% and 30.0±5.8%, respectively, while the scr-siRNA have no marked effect.2. VEGF mRNA were significantly knocked out in the siRNA no.1 and no.3 group quantified using RT-qPCR technology and normalized to a non-transfected group.3. ELIS A results for VEGF revealed that the inhibitory rates of secreted VEGF protein by VEGF siRNA no.1 and no.3 were 58.1 ± 7.3% and 51.9 ± 7.9%, respectively, while the scr-siRNA showed no effect.4. It was obvious that lipiodol was accumulated at the margins of the liver tumor and that there was obvious necrosis in the (middle) center of tumor obtained from the results of the contrast-enhanced CT image at 28 days after TACE operation.5. The tumors growth ratios (GRs) at 28 days after TACE operation was 155.18 ± 19.42% in the control group, while the GRs in the low-dose group and high-dose group were 79.67± 19.63% and 36.09± 15.73%, respectively. There were significant differences among these three groups.6. Compare with the control group (57.88± 5.67 of MVDs,102.15± 18.33 of VEGF), the MVDs were 34.22± 4.01 and 22.63± 4.07 and the VEGF 67.40± 12.32 and 36.25±10.88 in the low-dose group and high-dose group, respectively.7. The expression levels of VEGF mRNA and VEGF protein of rabbits’livers at 28 day after TACE operation were significantly decreased in low-dose group and high-dose group, compared with corresponding control group.8. The apoptotic rates of VX2 cells were obviously increased at 28 day after TACE operation with VEGF-siRNA in low-dose group and high-dose group, compared with corresponding control group. These data implied that VEGF silencing could suppress the cell survival of VX2 cells by inducing cell apoptosis.9. VEGF siRNA delivered by TACE technology not only lacked of toxicity to liver and kidney but also had the ability to protect liver damage induced by tumor growth.Conclusion1. Targeting VEGF siRNAs could down-regulation the expression levels of VEGF mRNA and protein in vitro.2. Targeting VEGF siRNAs also could down-regulation the expression levels of VEGF mRNA and protein, inhibit tumor angiogenesis and tumor growth and promote the apoptosis of VX2 cells in vivo.3. Targeting VEGF via TACE-mediated siRNA-lipiodol emulsion delivery may become a novel and powerful option to effectively treatment of HCC in clinic. |
