Studies On Oncolytic Adenovirus Targeting Cancer Cells With MicroRNA For Cancer Therapy

In this thesis, we adhibited microRNAs which are missing or reduced expression specifically in cancers as a new targets of Oncolytic Adenovirus ,and we focused on the targeting and safety of this Oncolytic Adenovirus in cancer therapy .Back ground: microRNA, which is a research highlight in recent years, They negatively regulate gene expression in animals, by binding, with imperfect base pairing, to target sites in messenger RNAs (usually in 3' untranslated regions) thereby either reducing translational efficiency or determining transcript degradation.Studies have shown that reduced expression of some microRNAs in cancers had close relationship with the development and progression of tumors.The let-7miRNA was initially identified by genetic analysis of the developmental timing defects of mutants.The expression levels of the human let-7 gene have been shown to vary among various adult tissues, lung being one of the tissues with most abundant expression of let-7. However, studies in lung cancers shows that expression levels of let-7 are frequently reduced in lung cancers both in vitro and in vivo. Furthermore, lung cancer patients with reduced let-7 expression were found to have significantly worse prognosis after potentially curative resection[1].Yong Sun Lee and Anindya Dutta[50] found that HMGA2 was derepressed upon inhibition of let-7 in cells with high levels of the miRNA. Ectopic expression of let-7 reduced HMGA2 and cell proliferation in a lung cancer cell.Johnson, SM[51]and Christensen B. C.[52] let-7 family negatively regulates RAS. Yu F et al found that let-7 regulates multiple BT-IC stem cell-like properties by silencing more than one target.Therefore, a new Oncolytic Adenovirus carrying the let-7a target site was infected into the normal cell lines L02,WRL-68 with the constant expression of microRNA hsa-let-7a, and infected into hepatoma cell lines Hep3B,PLC with low expression of hsa-let-7a. We hope that the oncolytic adenovirus is able to basically do not replicate and proliferate in normal cells, while the specific replication and proliferation in tumor cells, so as to achieve the purpose that the oncolytic adenovirus can specifically target tumor cells and reduce the poisoning effect on normal cells.Methods And Results:Firstly, by using qRT-PCR, we screened the normal cell lines L02, WRL-68 with the constant expression of microRNA hsa-let-7a, and hepatoma cell lines Hep3B, PLC with low expression of hsa-let-7a.Let-7 target sequences were inserted into the E1A after the stop codon TAA to generate a new plasmid pXC1-let-7T. The plasmid pXC1-let-7T was co-transfected with pPE3-F11B into 293 cells to produce the new recombinant adenovirus by homologous recombination which was regulated by let-7 target sequences,we call it Ad5/11-Let-7T. The recombinant adenovirus was confirmed by PCR analysis using specific primers, amplified at a large scale and purified by cesium chloride gradient centrifugation. We got another recombinant adenovirus Ad5/11-Let-7MT ,which was regulated by let-7 mutational target sequences,by using the same method.WAd5,Ad5/11-Let-7T and Ad5/11-Let-7MT were infected into L02,WRL-68,Hep3B and PLC. qRT-PCR and Western blotting were used to evaluate the level of E1A protein expression, we found that only a small amount of protein expressed in normal cells which were infected with Ad5/11-Let-7T,while with high expression in tumor cells. Viral replication experiments were performed to evaluate the selective replication ability of Ad5/11-Let-7T. The replication of Ad5/11-Let-7T was remarkable in liver cancer cells, while hardly replicated in normal cells. When containing reporter gene green fluorescent protein gene, Ad5/11-Let-7T-EGFP was observed to replicate efficiently in liver cancer cells but poorly in normal cells under fluorescence microscope. MTT assay was performed to determine the killing ability of Ad5/11-Let-7T at various viral MOIs. Results showed that Ad5/11-Let-7T had a much better killing effect on hepatoma cells, than normal cells;but had a similar killing effect on hepatoma cells with the control viruses WAd5 and Ad5/11-Let-7MT.Hep3B tumor xenografts were established by a s.c. inoculation of 1×107 cells into the right flanks of BALB/c mice. When tumors reached 7~9 mm in diameter, mice were randomly divided into three treatment groups (n=5 in each group), namely as PBS, Ad5/11-Let-7T and Ad5/11-Let-7MT, respectively. The viruses (2×108 pfu) in 100ul adenovirus preservation medium were administrated intratumorally every other day for five times with a total dosage of 1×109 pfu. Compared with the control group, all treatment groups demonstrated significant tumor growth inhibition (P<0.05). Among three treatment groups, the treatment groups viruses had the similar antitumor effect(P<0.05).Conclusions:We successfully constructed an efficient replication-selective adenovirus Ad5/11-Let-7T, which selectively replicated in and lysed tumor cells,while hardly replicated in normal cells. Moreover, Ad5/11-Let-7T was superior to control grouop in the selective replication in tumor cells, and obvious tumor inhibition was observed in liver cancer xenografts in BALB/c mice. Meanwhile,we first adhibited microRNAs which are missing or reduced expression specifically in cancers as the new targets of Oncolytic Adenovirus , we provided a new strategy for the regulation of Oncolytic Adenovirus targeting. At the same time, the control method can combine with other targeted methods, and prepare bedding for the establishment of a higher targeting oncolytic adenovirus vector system.