Adenoviral Vector Mediated Co-expression Plasmid STAT3-siRNA-STAT1 In The Treatment Of Glioma In Vivo

Objective: To investigate the effect of recombinant adenovirus vector-mediated overexpression of STAT1 gene and RNAi interference of STAT3 gene on tumor growth of U87-MG xenograft model explore its possible mechanism,and provide new experimental basis for gene therapy of glioma.Methods:(1)Construction of recombinant adenovirus expressing STAT1 protein(Ad-stat1),si RNA against STAT3(Ad-stat3-si RNA),and the combination of both(Ad-stat1 / stat3-si RNA).the Corresponding empty backbone vector were used as negative controls,including Ad-stat1 backbone NCAd-stat1,Ad-stat3-si RNA backbone NCAd-stat3-si RNA,and Phosphate Base Saline control(PBS).(2)U-87 MG subcutaneous xenograft model were established using nude mice,and 36 animals were randomly divided into six groups:Ad-stat1,NCAd-stat1,Ad-stat3-si RNA,NCAd-stat3-si RNA,Ad-stat1 / stat3-si RNA,and PBS control.Viruses were administered with intratumoral injections once every other day for a total of 5 times.(3)Subcutaneous tumor tissue and major organs(Heart,Lung,Liver,Spleen,and Kidney)were harvested for histology.(4)Tumor expression of STAT1 and STAT3 m RNA was detected by Real Time-PCR(RT-PCR),and the protein level determined by immunohistochemistry and Western Blot.(5)Programmed cell death of the tumor cells were analyzed by Annexin-APC / PI dual color flow cytometry and TUNEL assay.Results: Recombinant adenovirus vector-mediated co-expression of STAT1 protein and STAT3-Si RNA subcutaneous gliomas in nude mice,but also has better therapeutic effect than single gene therapy.Recombinant adenoviruses are safe and reliable,and have no significant side effects.The results of Western blotting and RT-PCR showed that after co-expression plasmid treatment,STAT1 expression was significantly enhanced,while STAT3 gene expression was significantly down-reg?lated.Experiments with Annexin V and TUNEL showed that co-expression plasmid combination therapy can significantlypromote tumor cell apoptosis and necrosis.Conclusion:(1)Ad-stat1,Ad-stat3-si RNA and Ad-stat1/stat3-si RNA can significantly inhibit the growth of glioma transplanted tumors.(2)Recombinant adenoviruses are safe and reliable,with no significant side effects on the organs.(3)Ad-stat1/ stat3-si RNA dual gene tumor suppressive effect is significantly better than single gene,indicating that Ad-stat1/stat3-si RNA has the additive effect of cancer suppression and synergism.(4)The tumor suppressor mechanism of Ad-stat1 / stat3-si RNA may be related to the activation of apoptosis and necrosis,and the main mechanism to induce apoptosis and promote necrosis.