| It has been reported that miR-145 showed wonderful results of inhibiting proliferation,migration and invasion by downregulating Oct4,ADAM17,CDK6 and Sp1 expression in many malignancies such as renal carcinoma,prostatic cancer and liver cancer.However,it was difficult to enter into cells through the membranal barrier due to the negative electricity of miR-145.Cuttently,the gene vectors used is including virus vectors and the nonviral vectors.The virus vectors had strong delivery efficiency but were restricted by the high cost and low safety to some extent.The nonviral vectors had been widely used because of their easy preparation,hypotoxicity as well as low immunogenicity,which involved liposome,micell and nanoparticle.Among them,the cationic liposome is one of the most widely used vectors currently.miR-145 pDNA was advantaged by its long co-expression of miR-145 and expression green fluorescein protein as reporter gene.In the present study,we used DOTAP and cholesterol to prepare CLs which used for miR-145 transfection.In order to optimize the tansfection system,different hydrous media,plasmid weight,DOTAP/Chol molar ratio and DOTAP/miR-145 pDNA weight ratio were extensively studied.And finally,5% glucose,8 ?g miR-145 pDNA,DOTAP/Chol=3(m/m)and DOTAP/miR-145 pDNA=3(w/w)were confirmed as the optimum conditions.The particle size and potential are 542±10.5 nm and 37.1±2.1 mV,respectively,with a transfection efficiency of 30.0±4.4% and harder DNase? degradation.To better understand the delivery mechanism of CLs/miR-145 pDNA,influence of temperature and endocytosis inhibitors on transfection system were also studied on 293 T cells.The results indicated that the present delivery system may act through the function of caveolin and pinocytosis.To solve the problem of low transfection of CLs/miR-145 pDNA delivery system,the cationic materials such as PEI,chitosan,PLL and spermidine were widely studied as the nucleic condenser in the following researches,to form a ternary delivery system.The particle size,potential,cytotoxicity and transfection efficiency are chosen as the final criteria.And finally,DOTAP/spermidine /miR-145 pDNA=3:1.1:1(w/w)was confirmed as the optimum condition,with a particle size of 283.7±13.6nm and potential of 23.9 ± 2.5 mV.And the final optimum condition exhibited a higher transfection of 47.3±1.4% and free of DNase?.However,through the particle size reduce to 283.7±13.6 nm,it still can not meet the requirements in vivo.So the miR-145 mimic(13300 Da)was chosen as the targeted nucleic acid to acquire a better proportion of DOTAP/Chol and DOTAP/miR-145 mimic on HepG2 cells.And finally,DOTAP/Chol=3(m/m)and DOTAP/miR-145 mimic=15:1(w/w)were confirmed as the optimum condition,with a particle size of 146.6 nm,potential of 27.3 mV and inhibitory rate of 26.7±3.0%.However,the present system was also lack of stability under the condition of serum.So mPEG2000-DSPE was added to prepare a long-circulating cationic liposomes.The results indicated that mPEG2000-DSPE has little influence on particle size but the potential was negative related with the molar percentage of mPEG2000-DSPE.And the molar content of mPEG2000-DSPE of 0.5% was finally confirmed as the optimum condition,with a similar inbititory rate in serum and serum-free medium,24.5±2.4% and 22.3±1.7%,respectively.The results of uptake kinetics indicated that 4 h after transfection,the condition reached an intake of saturated.Finally,the biological impact of miR-145 mimic on HepG2 cells were evaluated by the method of wound healing assay and transwell assay after miR-145 overexpression with our present system.The results indicated that the migrative ability and invasive ability of HepG2 were reduced by 70% after miR-145 overexpression.And furthermore,the results of western blotting indicated that E-cadherin and ZO-1 were upregulated nearly 200% after miR-145 overexpression,which indicatedthat E-cadherin and ZO-1 may be responsable for the anti-invasion function of miR-145 on HepG2 cells. |
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