| Purpose:Melanoma,also known as malignant melanoma,is a fatal malignant tumor caused by the pathogenesis of melanocytes.Melanoma is common in the skin,but also in the mucosa,choroid and other parts of the eye.Although melanoma has a lower incidence than other malignancies,it is one of the fastest growing tumors of all malignancies.At present,the traditional treatment method has brought great pain to the patients,but the effect on the development of the tumor has little effect.Colon cancer is a common malignant tumor of the digestive tract that occurs in the colon,and is common in the junction of the rectum and the sigmoid colon.The incidence of colon cancer ranks third in gastrointestinal tumors,and has become one of the most common cancers in lung cancer,stomach cancer and liver cancer in China.Traditional surgical resection and radiotherapy have little effect on the prolongation of survival of colon cancer patients,and it is easy to cause poor prognosis.Gene therapy is currently considered to be a new and effective strategy in tumor immunotherapy.DNA-based tumor immunotherapy has been widely used in preclinical and clinical research.However,due to the size of the plasmid vector and the heterologous type,the DNA of the target DNA is low in efficiency,low in effect,and easily triggers the immune response of the body,thereby causing cytotoxic effects to the patient's body,hindering the wide range of promotion and application of DNA-based tumor immunotherapy.Based on this,the development of other effective forms of treatment is the key to genetic immunotherapy.Existing studies have shown that signaling transduction and activators of transcription(STATs)3 plays an important role in the immune escape of tumor cells.The proliferation of cancer cells is initiated by the STAT gene and transduced by JAK/STAT signaling(JAK/STAT signaling)and CDK regulation can inhibit the natural apoptosis of cancer cells and promote the division and reproduction of cancer cells.In this paper,we used the cationic liposome Liposome(hereinafter referred to as LP)to carry out the STAT3 siRNA(LP-P-siRNA-complex)complexed with protamine(P),and explore its silencing inhibition effect through a series of in vitro experiments.Mice with melanoma were subsequently treated by intravenous injection to verify their in vivo efficacy.Methods:We first characterized LP and LP-P-siRNA-complex,and examined the particle size and potential of LP-P-siRNA-complex at different complex ratios and their ability to load siRNA,and finally determined the LP-P-siRNA-complex composite ratio used.Subsequently,in vitro,we transfected B16 and CT26 cells with LP-P-siRNA-complex and measured the transfection efficiency of the complex and detected the toxicity of LP,P and LPP composites by CCK-8 method.In addition,we also performed apoptosis,plate cloning,STAT3 mRNA transcription level detection and STAT3 protein expression level changes in B16 cells and CT26 cells after complex transfection.In the in vivo experiment,we first constructed a B16 mouse tumor model and treated the mice by intravenous delivery using LP-P-siRNA-complex.During the treatment,the experimental mice including the treated mice group were monitored for body weight and the tumor size of the mice was photographed after the end of the treatment period,and then the experimental mice were sacrificed and the heart,liver,spleen,lung and kidney were performed.H&E staining was used to investigate the safety of the composite material.Results:The particle size potential test showed that when the concentration of LP was 1mg/ml,the particle size was 36.12 ± 2.98 nm and the potential was 42.8 ± 1.2 mv.Gel retardation(EMSA)results showed that LPP completely blocked siRNA when the mass ratio of LP:P:siRNA was 1:2:1.The 1 mg/ml LP and P and Scramble siRNA were quantified in a mass ratio of 1:2:1 to obtain a complex having a particle diameter and a potential of 43.82 ± 0.82 nm and 28.7 ± 1.5 mv,respectively.The results of transfection efficiency test showed that the transfection efficiency of B16 cells was 68.76% after transfected with Cy3 for 24 hours,which was much higher than that of the control group(P < 0.0001).When transfected with CT26 cells,the transfection efficiency was about 81%,and it also had a high transfection efficiency,which was also significantly statistically significant compared with the control group(P < 0.0001).LPP showed no obvious cytotoxicity in B16 and CT26 cells,which proved that the apoptosis of tumor cells was indeed due to the important role of STAT3 siRNA.After transfection of B16 and CT26 cells by LPP-delivered STAT3 siRNA,qPCR results showed that the transcription level of STAT3 mRNA was significantly down-regulated(P < 0.05),and the expression of STAT3 protein was decreased by Western blot.After the transfection of both cells with LPP-delivered STAT3 siRNA,the cell clone formation rate of the experimental group was significantly reduced,and there was a statistically significant difference(P < 0.001).In addition,the results of apoptosis assay showed that LPP can significantly inhibit the proliferation of B16 and CT26 cells(P < 0.01).The results of in vivo experiments showed that the mice with melanoma were significantly less than the control group after treatment with LPP/siSTAT3 complex,and the tumor was significantly reduced,and the H&E staining of the heart,liver,spleen,lung andkidney did not show the complex body toxicity.Conclusion:The experimental data indicate that the LP-P-STAT3 siRNA-complex exhibits good targeted therapeutic properties and excellent delivery efficiency,and the LPP-STAT3 siRNA-complex has good stability and low toxicity;LPP-STAT3siRNA-complex can effectively promote tumor cell apoptosis after tumor;the LPP-STAT3 siRNA-complex also shows good anti-tumor effect and can effectively inhibit tumor growth by intravenous injection. |
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