| Background:In recent years,the use of microbial vectors to deliver genes targeting inflammatory pathways has achieved good results in tumor treatment.Microbial vectors mainly include viruses and bacteria.Viral vectors can be divided into two categories: integrated and non-integrated viral vectors.Bacteria mainly include anaerobes and facultative anaerobes.Although a large number of studies have shown that the use of attenuated Salmonella to deliver genes in vivo can achieve good therapeutic effects,but the safety of biological vectors has limited its clinical transformation process.Recently,the delivery of anti-tumor genes by synthetic vectors has become a research hotspot.Synthetic carriers include liposomes and cationic polymers.Polyetherimide(PEI)is a classical cationic polymer and is widely used as the gold standard for cationic gene carrier research.However,PEI has high toxicity and low transfection efficiency in vivo,which makes the development of new cationic gene vectors become a research hotspot at the present stage.At present,a series of gene vectors have been developed for tumor therapy,but the related in vivo studies are limited to the tumor itself,and the comprehensive response of the body to the synthetic vector delivery gene anti-tumor is still unknown.Neuroblastoma(Neuroblastoma,NB)is the most common extracranial solid tumor in children,and the most common site is the adrenal gland.More than 50% of children have a survival rate of less than 40%.The search for effective therapeutic targets is a hot spot in neuroblastoma research.Clinically,neuroblastoma tissue is rich in blood supply,often early metastasis,accompanied by massive hemorrhage,necrosis and calcification.We speculate that inhibition of inflammation-causing signal pathways may play a significant role in the treatment of this tumor.Genes associated with retinoid-interferon-induced mortality 19(GRIM-19)can inhibit malignant tumors by inhibiting transcription factor signal transducer and activator of transcription-3(STAT3)pathway.Although recent studies have shown that the STAT3 signaling pathway is essential in children with neuroblastoma metastasis andchemotherapy resistance,there is still a lack of reports on the correlation between clinical pathological staging and GRIM-19.In vivo experiments on xenograft tumors suggest that inhibiting the STAT3 signaling pathway can inhibit the growth of neuroblastoma,but it is not sufficient to evaluate the changes in the tumor's local microenvironment after inhibiting this pathway.We speculate that in neuroblastoma allograft tumors,overexpression of GRIM-19 may inhibit tumor cell growth,promote apoptosis,and inhibit MMP-9 expression,reduce intratumoral hemorrhage,and improve treatment through the mechanism of inhibiting STAT3 signaling pathway.effect.It is well known that cisplatin,the first-line drug for most tumor chemotherapy,has obvious endothelial cell toxicity,and can significantly increase plasma von Willebrand factor(v WF)levels after treatment.Gene therapy for pathogenic mechanisms has the characteristics of high efficiency and low toxicity,and theoretically has less toxic and side effects on the body.Although many liposomes and cationic carriers have been developed for the treatment of neuroblastoma,how to achieve the maximum therapeutic effect in vivo still needs to be carefully evaluated.Methods:1.Synthesis and characterization of gene vectors: The amphiphilic block copolymer PCL-b-P(GMA-EA)(PCG)was synthesized by enzymatic polymerization.The chemical structure of PCG was detected by nuclear magnetic resonance.Gel electrophoresis was used to detect the binding ability of PCG to p GRIM-19.The particle size and zeta potential of PCG and p GRIM-19 complexes were evaluated by dynamic light scattering.The morphology of the complex was observed by electron microscopy.In human cervical cancer Hela cells,human liver cancer Hep G2 cells and murine neuroblastoma Neuro2 a cells,cell proliferation activity assay(CCK-8)was used to detect the cytotoxicity of PCG and p EGFP-N1 complexes,and the transfection efficiency of PCG was detected by fluorescence microscopy or flow cytometry.2.Analysis of clinical samples: The tumor tissues and paraneoplastic tissues of 28 children diagnosed with neuroblastoma and 8 adjacent tissues in the first hospital of Bethune Hospital of Jilin University were collected.The correlation between the expression of GRIM-19 and the age,gender,tumor size,INSS stage,INPC stage,lymph node and/or vascular metastasis of children with neuroblastoma was analyzed by immunohistochemistry.3.PCG mediated GRIM-19 gene therapy for neuroblastoma in vitro and in vivo:using PEI as a positive control,Western blot was used to detect the transfection efficiency of PCG-mediated GRIM-19 gene transfection in murine neuroblastoma Neuro2 a cells.The in vitro experiment was divided into 6 groups,namely the control group,PCG group,naked p GRIM-19 group,PCG/p CDNA group,PCG/p GRIM-19 group and PEI/p GRIM-19 group.The cell proliferation experiment(CCK-8),clone formation test,Brd U test,flow cytometry apoptosis test,immunofluorescence,scratch and Transwell assay were used to investigate the effect of PCG-mediated GRIM-19 overexpression on tumor proliferation,apoptosis and metastasis.In the in vivo experiment,it was divided into three groups: control group,PEI/p GRIM-19 group and PCG/p GRIM-19.Immunohistochemistry and Western blot were used to detect the transfection efficiency of GRIM-19,and the expression of PCNA was detected by immunohistochemistry.The apoptosis detection(TUNEL)and Western blot were used to analyze the changes in proliferation and apoptosis of transplanted tumors in nude mice after treatment.Further in vivo experiments were divided into three groups:control group,cisplatin group and PCG/p GRIM-19 group.The difference in the survival period of nude mice in each group was detected.Automatic hematology analyzer was used to detect hemoglobin in nude mice,enzyme-linked immunosorbent assay(ELISA)was used to detect v WF in plasma,HE staining was used to detect lung changes in tumor-bearing mice,immunohistochemistry was used to detect the expressions of p-STAT3 and MMP-9 in tumor tissues,and changes in tumor histology,intracranial hemorrhage,anemia,pulmonary embolism and other tumor-related complications were evaluated.Results:1.When N/P = 1(molar ratio of N for cationic polymer and P for DNA),PCG completely prevented the swimming of GRIM-19 plasmid DNA in gel electrophoresis.In the range of w/w of 5-50,PCG could compress the GRIM-19 plasmid into nanoparticles with a size of 200-400 nm and a surface potential of 25-30 m V.In Hela,Hep G2 and Neuro2 a cells,PCG(W/W =20)effectively delivered the EGFP-N1 plasmid in the cells,and showed little toxicity to the cells.2.The expression level of GRIM-19 protein in neuroblastoma tissue was down-regulated or absent compared with normal tissues adjacent to cancer.The expression level of GRIM-19 in tumors is negatively correlated with age(P=0.0012),INSS stage(P=0.0012),INPC stage(P=0.0299),lymph node and/or vascular metastasis(P = 0.0062),regardless of gender and the primary site of tumor.3.In vitro experiments,PCG vector could transfect GRIM-19 gene into Neuro2 a cells and improve the protein expression level of GRIM-19 gene,and its transfection efficiency was higher than PEI.Among the control group,PCG group,naked p GRIM-19 group,PCG/p CDNA group,PCG/p GRIM-19 group and PEI/p GRIM-19 group,the apoptosis increased and cell proliferation and migration were inhibited in the PCG/p GRIM-19 group and PEI/p GRIM-19 group.Overexpression of GRIM-19 protein mediated by PCG inhibited STAT3 signaling pathway and the expression of Cyclin D1,Bcl-2,MMP2 and MMP9.In vivo experiments,among the control group,PEI/p GRIM-19 group and PCG/p GRIM-19 group,PCG/p GRIM-19 group showed the most obvious inhibition on the growth of transplanted tumor.In this group,GRIM-19 protein expression increased,PCNA expression decreased,TUNEL staining and Cleaved caspase-3 upregulated.Among the control group,cisplatin group and PCG/p GRIM-19 group,the survival time of PCG/p GRIM-19 group was the longest.In this group,generally,the intratumoral hemorrhage was reduced,the hemoglobin of nude mice was increased,v WF was down-regulated,HE indicated that pulmonary embolism was reduced,p-STAT3 and MMP-9 in tumor tissue was down-regulated.Conclusion:1.The synthetic cationic polymer PCG has good DNA compression ability and can effectively transfect gene in vivo and in vitro,but its toxicity is much lower than that of PEI.2.The expression of GRIM-19 is related to the clinicopathological factors in children with neuroblastoma.The expression of GRIM-19 was down-regulated or deleted in tumor tissues,and its expression level was negatively correlated with age,INSS stage,INPC stage,lymph node and / or vascular metastasis.3.In the mouse model of transplanted tumor,the delivery of p GRIM-19,through cationic vector PCG in vivo can effectively inhibit the STAT3 signal pathway,not only directly inhibit the growth of tumor cells,but also reduce intratumoral bleeding by inhibiting the expression of MMP-9.Compared with the endothelial damagecaused by traditional cisplatin chemotherapy,v WF was down-regulated,pulmonary thrombosis was reduced and the survival time of mice was prolonged in gene delivery group.The therapeutic strategy of cationic PCG carrier delivery p GRIM-19 to inhibit neuroblastoma may provide a valuable reference for tumor therapy. |
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