Design Of Bi-functional ShRNA Vector Targeting Pim-3and The Therapy In Hepatoma

ObjectiveIn recent years, many scholars began to study the development and metastasis of tumor cells at the molecular level, due to the development of molecular biology and immunology. The abnormal expression of some proto-oncogenes has received extensive attention in the development of tumor. Pepole began to seek a new method to combine the gene therapy and biological treatment into cancer elimination. RNAi (RNA interference) has been a feasible fashion in tumor treatment. RNA interference decreases the expression of targeting gene as part of post-transcriptional gene silencing (PTGS), in virtue of shRNA (short-hairpin RNA) embraced in the expressing plasmid, or siRNA (small interfering RNA) which is a class of double-stranded RNA molecules,20-25nucleotides in length. Similar to other tumors, in the tumorigenesis process of hepatocellular carcinoma, expression of some pro-oncogenes is also out of the way, as consequently, to induce the cell biological characteristics to be changed toward to be malignance. In mouse hepatoma cell lines, we focused on proto-oncogene Pim-3which is a member of Pim family. In previous reports, it has been proved that the silence of Pim-3can restain the tumor growth located in some organs such as colon and pancreas but the contribution of Pim-3to hepato-carcinogenesis remained unknown.Besides abrrent expression of proto-oncogene, the hypofunction of host immune system also should be responsible for the development and metastasis of malignant tumors. It has been proved that the mechanisms of immune suppression mediated by tumor cells include the up-regulation of immuno-suppressive factors such as IL-10and TGF-β, the activation of Treg cells, and even the modify of immune environment. We have previously noticed that TLR7stimulation via ssRNA (single-strain RNA) can directly inspire innate immune response followed by the activation of NF-κB signal pathway, proceeding further activation of adaptive immune system. So in this report, we constructed a bi-functional vector which can impede Pim-3expression and stimulate TLR7simultaneously to reverse tumor progression from two aspects including tumor self and immune circumstances. We hope that dual function plasmid can be regarded as a good attempt in tumor therapy, because biological characteristics of tumor cells can be changed and the immune response can be triggered.Methods1. RT-PCR and Q-PCR analysis were carried out to confirm the gene expression of Pim-3in Hepal-6, H22and BNL.CL2cells respectively. The expression of apoptotic-related molecular such as Bim, BCL-2and type I interferons in Hepal-6cells was also identified via RT-PCR.2. Western blot analysis was performed to test the endogenous expression of Pim-3,some PRRs and p-Bad in Hepal-6cells after transfection with bi-functional vector for was also determined via western-blot.3. Induction of apoptosis in Hepal-6cells was measured via flow cytometry with AnnexinV-FITC/PI staining and immuno-histochemistry with TUNEL staining.4. ELISA was performed to detect the concentration of type I interferons and inflammatory factors.5. MTT assay was applied to confirm the cytotoxity of NK cells from splenic lymphocytes targeting Hepal-6cells in vitro.6. C57BL/6mice were administrated with1×106Hepal-6tumor cells subcutaneous injection to construct the hepatoma model.7. CD4+T and NK cells were depleted via injection intraperitoneal with PK136and mT1mAb.8. Flow Cytometer analysis was used to judge the abundance of immune molecules such as CD3, CD69, NK1.1, CD4, NKG2D, PD-1and NKG2A.9. Lymphocyte infiltration in tumor tissues was assessed via H&E staining.Results 1. The oncogene Pim-3was highly expressed in mouse hepatoma cell lines but nearly no expression in the normal liver cell lines.2. Bi-functional vector bearing ssRNA and Pim-3-specific shRNA was successfully constructed.3. Transfection with shRNA and dual functional vector both promoted tumor cell apoptosis in vitro.4. Pim-3-specific gene silencing could re-adjust the expression of anti-apoptotic and pro-apoptotic proteins synergistically to promote tumor cell apoptosis.5. Treatment with bifunctional vector could restrain subcutaneous tumor growth and stimulate the immune response of NK cells and CD4+T cells in vivo.6. Efficient anti-tumor effect mediated by bifunctional vector was depended upon the activation of splenic NK in vivo.7. Splenic NK cells were involved in the anti-tumor effect mediated by bi-functional vector via killing tumor cells directly.8. The existence of splenic CD4+T cells was important for maintaining NK activation after treatment with bi-functional vector.9. TLR7expression was indispensable for the stimulation with dual functional vector to exert anti-tumor effect.Conclusions1. The bi-functional vector targeting Pim-3and expressing immuno-stimulatory RNA (ssRNA) is constructed successfully.2. The endogenous expression of Pim-3can be down-regulated and the secretion of IFN-α and IFN-β in Hepal-6cells can be induced after transfection with bi-functional vector.3. Apoptosis of Hepal-6cells can be induced in vitro after transfection with bi-functional vector4. Treatment with bi-functional vector delays tumor growth in vivo.5. Treatment with bi-functional vector stimulates activation of splenic NK and CD4+T cells. 6. The existence splenic NK is indispensable for the tumor treatment mediated by bifunctional vector in vivo and CD4+T cells provide helpful role for NK cell activation..7. Treatment mediated by bi-functional vector is dependent on TLR7stimulation.