CSB1-Lipo-DOX-miR101-targeted Therapy For Breast Cancer And Its Anti-drug Effect

Background and PurposeBreast cancer(BC)is the most common cancer in women.Together with lung cancer and colon cancer,it is considered to be the three most common cancers in the world,with high incidence and mortality.The molecular classification of breast cancer is complex and is closely related to clinical treatment and prognosis.According to Human epidermal growth factor receptor 2(HER2),Estrogen receptor(ER),and Progestrogen receptor(PR)tests,breast cancer is divided into several types.Among them,the triple-negative and HER-2 negative types are difficult to use the most effective hormone therapy for breast cancer.These types have hidden onset,rapid progress,easy to miss and cause early metastasis,so these types of patients often lose the opportunity for surgery or cannot surgery,so chemotherapy becomes the only means of treatment.However,the current clinical chemotherapy for breast cancer leads to huge drug toxicity,unobvious efficacy,and poor prognosis due to the following reasons:(1)the drug lacks or insufficient targeting;(2)more than 50%of cases will quickly produce Multidrug resistance(MDR)of the drug.Therefore,improving the targeting and anti-MDR properties of chemotherapeutic drugs is an effective focus on the development of a new generation of chemotherapeutic drugs.Doxorubicin(DOX)is the first-line broad-spectrum anti-tumor drugs applied clinically.It is widely used in the treatment of solid tumors and hematological malignancies and has obvious killing effects on tumor cells.However,DOX's huge cardiotoxicity,bone marrow suppression,and digestive system and dermal reactions have severely affected its application and efficacy.To improve the efficacy of DOX,Lipo-DOX,a liposome formulation of DOX has already been launched internationally,and it has also been listed in China.However,Lipo-DOX only solves the problem of increasing the intake of drugs due to easy fusion with cell membranes.It still does not have obvious cancer cell targeting and anti-MDR properties.Therefore,Lipo-DOX is highly toxic and its efficacy is still not ideal.Based on the two basic requirements of cancer cell targeting and anti-MDR,we designed the following breast cancer polypeptide-oriented DOX liposome drug delivery system with MDR inhibitory effect.The drug-carrying system's in vitro therapeutic effect on breast cancer was studied,and its mechanism of enhancing cancer cell killing and MDR inhibition was analyzed.The preliminary screening experiment of our laboratory obtained the BC-targeting 12 amino acid peptide CSB1.A series of tests proved that CSB1 has high binding specificity and sensitivity to breast cancer cells/tissues.Therefore,this study uses CSB1 as the Lipo-DOX guide elements of breast cancer.Previous research in this laboratory,literature reports,and database analysis have suggested that miRNA101 can inhibit the expression of several genes related to the formation of MDR.Therefore,this study uses its precursor(Pre-miRNA101)expression plasmid as the MDR inhibitory element.Based on the above analysis and considerations,this study designed and constructed a liposome drug delivery system-CSB1-Lipo-DOX-miR101,which can target BC cells and reverse the MDR effect.We hope that the system can achieve high efficiency,low toxicity,and weak MDR therapeutic effect.This design meets the needs of the bottleneck problem urgently needed to be solved in the current clinical breast cancer treatment!Methods1.Use the network database to analyze the physical and chemical properties of BC cell targeting peptide CSB1,and to predict and analyze its target.2.The CSB1 modified and linked liposomes were synthesized by thin-film ultrasonic dispersion technology,and DOX drugs were encapsulated with ammonium sulfate active drug loading technology,and the negatively charged Pre-miRNA 101 expression plasmid was linked to the cationic liposomes through electrostatic adsorption.Get the DOX liposome delivery system CSB1-Lipo-DOX-miR101,which is targeted by BC cells and has MDR inhibitory properties.3.Use laser particle size analyzer,transmission and scanning electron microscope,fluorescence spectrophotometer,and other methods to detect the particle size,morphological uniformity,encapsulation efficiency,and other indicators of CSB1-Lipo-DOX-miR101.4.Use a plasmid expressing GFP instead of the Pre-miRNA101 expression plasmid(pcDNA6.2-GW/miRNA)to evaluate the transfection efficiency and targeting of CSB1-Lipo-DOX-miR101.5.Transfection of MCF-7 and DOX-resistant MCF-7(MCF-7/ADR)cells:divided into wild cell group,CSB1-Lipo group,CSB1-Lipo-DOX group,CSB1-Lipo-DOX-miR101 group,a total of 8 groups.6.Use the MTT method,damage repair method,Transwell,nuclear staining,scanning electron microscope,and other techniques to detect the lethality of CSBl-Lipo-DOX-miR101 on MCF-7 and MCF-7/ADR cells,and the inhibition of their proliferation and motility,and its influence on the apoptosis and morphology of these cells.7.Network database analysis:analysis of miRNA101 potential MDR-related target genes and BC malignant characterization related genes.8.Combined with the results of database analysis,Western blot was used to analyze the expression levels of possible target genes that CSB1-Lipo-DOX-miR101 could act on,and the possible BC treatment mechanism of CSB1-Lipo-DOX-miR101.Results1.Combined with the previous results of this laboratory,further prediction and analysis of this study indicate that CSB1 has a specific affinity for BC and its target protein FLAP is highly expressed on the surface of BC cell line MCF-7.2.The CSB1-Lipo-DOX-miR101 drug delivery system was successfully prepared.The liposomes are spherical,uniform in size,with a particle size of 122-141 nm,high DOX drug encapsulation efficiency(89%),good stability,and performance.It is released quickly in a simulated tumor microenvironment,which meets actual needs.3.The CSB1-Lipo-DOX-miR101 system transfects MCF-7 cells with a transfection efficiency of 80%,and CSB1 has a good orientation.4.MTT,in vitro damage repair,Transwell,DAPI staining,MitoScene 633 staining,Coomassie brilliant blue staining,scanning electron microscopy,cell plate cloning,etc.The results show that CSB1-Lipo-DOX-miR101 is resistant to BC cells,especially DOX The BC cells(MCF-7/ADR)have significant lethality,which strengthens the ability of DOX to inhibit cell proliferation,movement,and migration promotes the apoptosis process of DOX-induced BC cells and inhibits the formation of MDR.5.Through network predictive analysis,miRNA101 can be combined with the seed regions of multiple MDR-related genes.Combined with the results of Western blot,the CSB1-Lipo-DOX-miR101 liposomes significantly inhibited the following genes related to MDR and EMT:ABCC5,EZH2,APEX1,ITGB1,PCNA,VIM,Snaill,MMP2,etc.Conclusions1.Preliminary experimental results prove that CSB1-Lipo-DOX-miR101 is a new type of drug delivery system that specifically targets BC cells,and has obvious lethality to MCF-7,especially MCF-7/ADR.2.CSB1-Lipo-DOX-miR101 has a significant inhibitory effect on the formation of DOX-induced MDR so that the drug can continue to play a killing effect.This is of great significance in improving the clinical therapeutic effect of DOX on BC,reducing toxic and side effects,and reducing the recurrence rate.3.The clinical therapeutic effect of CSB1-Lipo-DOX-miR101 on BC and the mechanism of MDR inhibition may be achieved by inhibiting the expression of the following genes:ABCC5,EZH2,APEX1,ITGB1,PCNA,VIM,Snail1,MMP2,etc.