Construction Of Attenuated Salmonella Expressing RGD And MEL Gene Recombination And Its Targeted Therapeutic Effect On Melanoma

Melanoma was a skin malignant tumor with a high mortality rate,which was commonly seen in elderly animals in veterinary clinic,and had an increasing incidence worldwide.It was characterized by a high degree of malignancy,rapid progress,and easy to distant metastasis.It was a kind of malignant tumor that had strong resistance to conventional treatment drugs and which was quite troublesome in clinical treatment,and may caused great harm to food hygiene and public safety.In solid tumors,there were local immunosuppression,hypoxia,insufficient blood supply,tumor necrosis areas and other characteristics,resulting in immune escape in the tumor microenvironment,which leaded to the failure of chemotherapy drugs to reach and play a therapeutic role,but provided a favorable growth environment for a series of anaerobes and facultative anaerobes.This discovery indicated that some bacteria may be novel gene therapy vectors for tumor.Currently,the two key factors that affect the efficacy of tumor gene therapy were the treatment vector system and the selection of treatment genes.Therefore,finding suitable targeted vectors and therapeutic genes were the core and key point of tumor gene therapy,as well as the research purpose and scientific significance of this topic.Tumor targeting peptide(RGD)was a potent competitive antagonist of extracellular matrix binding to ?v?3 and was often used as a tumor targeting marker.Melittin(MEL)was the main component of melittin with pharmacological action and biological activity,and had significant effect in inhibiting tumor cell metastasis and invasion.In order to explore the antitumor effect of recombinant Salmonella LH430/p EGFP-RGD-MEL on mouse melanoma induced by fusion expression of RGD and MEL,this experiment constructed LH430/p EGFP-RGD-MEL,and infected B16 cells of mouse melanoma cultured in vitro to make the target gene expressed efficiently in B16 cells.CCK-8,Western Blotting,scanning electron microscopy and flow cytometry were used to detect the apoptosis and inhibitory effect of recombinant bacteria on B16 cells.CCK-8 results showed that LH430/p EGFP-RGD-MEL had a higher inhibition rate on B16 cells in vitro,which was dose-dependent and time-dependent.Its IC50 value(6.02×105 CFU/?L)was much lower than that of HEK293 cells(5.39×107CFU/?L).Western Blotting results showed that the expression of Caspase-3,Caspase-9,Bcl2 and Bax were significantly increased in B16 cells induced by recombinant bacteria(p<0.05).Scanning electron microscopy results showed that B16 cells had severe morphological changes with more protrusions and pseudopodia disappeared.Flow cytometry results showed that the recombinant bacteria could promote tumor cell apoptosis,and the survival rate of B16 cells was 24.7±5.81%.The effect of recombinant bacteria on migration,chemotaxis and invasion ability of B16 cells was detected by cell scratch assay and Transwells.The results showed that LH430/p EGFP-RGD-MEL could inhibit the migration of B16 cells and significantly inhibit the chemotaxis of FBS-mediated cells compared with the control group(p<0.05),and had an inhibitory effect on the invasion of B16 cells,although there was no significant difference between the two groups(p = 0.0507).In the experiment,an animal model of melanoma-bearing mice was successfully established.After intratumor injection of LH430/p EGFP-RGD-MEL(first injection,1×105 CFU per mouse: second injection,1×106 CFU per mouse),the body weight and tumor volume of tumor-bearing mice were measured in stages,and the expression and distribution of reporter genes in each organ were detected by biological imaging.The results showed that the fluorescence expression at the tumor site was higher than that in other tissues.When the tumor size exceeded 5% of body weight,mice were sacrificed,and the tumor,heart,liver,spleen,lung,kidney,brain and other tissues were taken for fluorescence protein expression detection,TUNEL staining,HE staining,immunohistochemical experiment and Q-PCR detection,respectively.The results showed that LH430/p EGFP-RGD-MEL treatment group could effectively inhibit the increase of tumor volume in mice,and the survival period of mice was significantly prolonged without significant weight loss(p<0.05).The tumor inhibition rate of LH430/p EGFP-RGD-MEL was up to 90.84±8.11%,which was significantly higher than that of RGD-MEL peptide and MEL peptide group.Although there was no significant difference between LH430/p EGFP-MEL,LH430/p EGFP and cisplatin groups in the tumor inhibition rate,the damage to internal organs was significantly lower than the three groups(p<0.01).At the same time,the results of biological imaging test and Q-PCR test showed that LH430/p EGFP-RGD-MEL had better targeting.The expression of exogenous fluorescent protein in tumor tissues of LH430/p EGFP-RGD-MEL treatment group was observed by frozen section.Tissue HE staining results showed that LH430/p EGFP-RGD-MEL treatment group showed no obvious pathological changes or tumor metastasis in liver,kidney,lung and other tissues and organs of mice,and the tumor tissue presented membrane-like structure,which was easy to be dissected.Immunohistochemical test results showed that the expressions of Caspase-3,P53 and GFP in tumor tissues of LH430/p EGFP-RGD-MEL treatment group were significantly increased.TUNEL staining showed that the apoptosis rate of tumor cells in the LH430/p EGFP-RGD-MEL treatment group was 48.31±1.26%,significantly higher than that in the control group(p<0.05).In order to explore the tumor suppressive mechanism of recombinant bacteria in the treatment of melanoma,RNA-seq was studied in tumor tissues.The results showed that the LH430/p EGFP-RGD-MEL treatment group,compared with the PBS group,mainly enriched the differentially expressed genes in the immune and tumor-related pathways in the body,and played a tumor suppressive role by destroying the immune escape in tumor tissues and activating the T-cell immune response in tumor tissues.In this study,the attenuated Salmonella LH430 with RGD-MEL double gene was successfully constructed,which can be used for stable passage and can effectively deliver and express target genes and proteins in vitro.In vitro experiments confirmed that LH430/p EGFP-RGD-MEL can promote apoptosis and had a strong inhibitory effect on proliferation,migration and chemotaxis of B16 cells.In vivo experiments on tumor-bearing mice showed that LH430/p EGFP-RGD-MEL mice injected with immune tumor-bearing mice could efficiently and stably express target genes and proteins in vivo,with good tumor targeting and less damage to other tissues and organs of the body.The results demonstrated the expression pattern of recombinant attenuated salmonella in tumor animal model and its targeted therapeutic effect on melanoma,providing a new research idea and method for the gene therapy of comparative medicine and human tumor.