MiR-129-5p Affects The Sensitivity Of Breast Cancer MCF-7 Cells To Taxol By Regulating ABCB1

Taxol is one of the most commonly used drugs for breast cancer,ovarian cancer and non-small cell lung cancer.Since the clinical application in the 1990s,Taxol has greatly prolonged the life cycle and increased survival rate of cancer patients.However,tumor cells gradually lose their sensitivity to Taxol during treatment,which has become the main reason for the failure of chemotherapy in most patients.Similarly,in the treatment of breast cancer,the resistance of breast cancer cells to Taxol is becoming increasingly prominent.How to reverse the drug resistance of cancer cells to Taxol and other tumor drugs has become an important problem for clinically choosing medicine.ally choosing medicineThe multidrug resistance-associated gene ABCB1?ATP Binding Cassette Subfamily B Member 1,ABCB1?belongs to the most important transporter member of the ABC transporter family and glycosylated ABCB1 encode P-glycoprotein?P-gp?with a molecular weight of 170KDa.The six transmembrane regions in the molecular structure of ABCB1 are used as transmembrane transport channels;and the ATP binding site can utilize the energy released by the hydrolysis of ATP to pump out the chemotherapeutic drugs such as alkaloids,anthracyclines and taxanes against concentration gradient,and further to reduce the drug toxicity.It has been confirmed that multi-drug resistance?MDR?induced by high expression of ABCB1 is an important bottleneck in chemotherapy for malignant tumors.MDR means that tumor cells are resistant to a variety of chemotherapeutic agents with different structures,functions and killing mechanisms when they are exposed to a single chemotherapy drug for a long time.A number of experimental studies have shown that the expression of small RNAs?MicroRNAs,miRNAs?in the corresponding tumor resistant cell lines is significantly different from the corresponding parental cell lines,suggesting that miRNAs may be closely related to the formation of drug resistance in tumor cells.Ectopic expression of miRNAs can reverse the tumor resistance and increase the sensitivity of tumor cells to chemotherapeutic drugs.Studies have shown that a variety of miRNAs can affect the sensitivity of tumor cells to chemotherapeutic drugs by regulating the expression of members of the ABC transporter family.miR-129-5p is a common transcript of miR-129-1 and miR-129-2 in miR-129 family,and is the primary executor of biological function.miR-129-5p is low expression in breast cancer cells and belongs to tumor suppressor miRNA.It is closely related to the development,pathological classification,clinical stage,drug resistance and prognosis of breast cancer.Our study predicted ABCB1 was one of the target genes of miR-129-5p by bioinformatic website?http://www.targetscan.org/vert₇1/?,and the evidence has showed thatoverexpression of miR-129-5p could improve the drug sensitivity of gastric cancer cells to vincristine?VCR?and adriamycin?ADM?by targeting down regulation of ABCB1,but the role of mi R-129-5p in reversing the resistance of breast tumors,especially the regulation of miR-129-5p on drug-resistance related proteins,needs to be further studied.ObjectiveIn this study,the effect of miR-129-5p on the proliferation and apoptosis of MCF-7 cells under the condition of Taxol stimulation and the changes of ABCB1,Bcl-2 and Bax expression levels were observed to investigate whether miR-129-5p can regulate the sensitivity of MCF-7 cells to Taxol by regulating the expression of ABCB1,in order to provide a new direction and experimental basis for clarify complex mechanisms of chemotherapy resistance and screening more effective anticancer strategies and therapeutic targets.Materials and methods1 MaterialsThe human breast cancer MCF-7 cell lines?purchased from the ATCC cell bank of the United States?were selected and maintained with RPMI1640 complete medium at 37°C under the moist atmosphere of air with 5%CO₂.2 Methods2.1 Experimental group:The following two types of groups were carried out based on the different purposes of the experiment.?1?To select the concentration and time of Taxol:the experiment was divided into drug-free group and different concentrations of Taxol?1.95nM,3.9nM,7.81nM,15.63nM,31.25nM,62.5nM,250nM and 1000nM?group,the time for the stimulation of Taxo laws 24h,48h and 72h,respectively.?2?To investigate the effect of Taxol on MCF-7 cells when miR-129-5p was overexpressed:the experiment was divided into blank control group,transfection reagent group,miR-NC group,miR-NC+Taxol group,miR-129-5p mimics group and miR-129-5p mimics+Taxol group.2.2 Cell transfection:when the cell growth density reached 70%⁸0%,miR-129-5p mimics and its negative control?miR-NC?were transfected into MCF-7cells by liposome transfection.2.3 CCK-8:CCK-8 method was used to detect the effect of Taxol on MCF-7cells proliferation inhibition rate before and after miR-129-5p mimics transfection.2.4 Flow Cytometry:Flow cytometry was used to detect the effect of Taxol on apoptosis of MCF-7 cells before and after miR-129-5p mimics transfection.2.5 Fluorescence staining of cells:the effect of Taxol on apoptosis of MCF-7cells before and after mi R-129-5p mimics transfection was observed by acridine orange/ethidium bromide double fluorescence staining.2.6 qRT-PCR:the effect of Taxol on the relative expression of miR-129-5p and ABCB1,Bcl-2 and Bax mRNA in MCF-7 cells before and after miR-129-5p mimics transfection was detected by qRT-PCR.2.7 Western Blot:Western blot was used to detect the effect of Taxol on the relative expression of ABCB1,Bcl-2,and Bax proteins in MCF-7 cells before and after miR-129-5p transfection.3 Statistical analysisThe data obtained in this study were all presented as mean±standard deviation?SD?.Statistical analysis was performed by SPSS23.0 software package.Independent sample t-test or adjusted t-test was used to compare the statistical data from cells proliferation inhibition between two groups.The statistical significance from cell apoptosis,the mRNA and protein expression of related genes among several groups was determined by One-way ANOVA,then pairwise comparison was analyzed using LSD-t test.The test level was set to?=0.05.Results1 The optimal concentration and time of Taxol on MCF-7 cellsMCF-7 cells were cultured in different doses of Taxol for 24h,48h,72h and growth inhibition was determined.MCF-7 cells were treated with 7.81nM(IC₂₅^IC₃₀)Taxol displayed<30%cell death at 48h and were used for the subsequent studies.2 Overexpression of miR-129-5p affects the proliferation of MCF-7cells on TaxolThe results of CCK8 detection showed that the proliferation inhibition rate of MCF-7 cells in miR-129-5p mimics+Taxol group was significantly higher than that of that of miR-NC+Taxol group,and the difference was statistically significant?P<0.05?.3 Overexpression of miR-129-5p affects the apoptosis of MCF-7cells on TaxolFlow cytometry analysis demonstrated that compared with the miR-NC+Taxol group,the cell apoptotic rate in miR-129-5p mimics+Taxol group was increased,and the difference was statistically significant?P<0.05?.Under the fluorescence microscope,most of the nuclear chromatin in miR-129-5p mimics+Taxol group were orange-red and appeared to be pyknotic or bead-like;The nuclear chromatin in the miR-NC+Taxol were mostly green in the form of constricted or fragmented,with a few of the nuclear chromatin of the orange-red.4 Overexpression of miR-129-5p affects the relative expression of ABCB1 mRNA and protein of MCF-7 cells on TaxolThe results of qRT-PCR and Western Blot revealed that the relative expression of ABCB1 mRNA and protein of MCF-7 cells in miR-129-5p mimics+Taxol were lower than that of mi R-NC+Taxol group,and the differences were statistically significant?P<0.05?.5 Overexpression of miR-129-5p affects the relative expression of Bcl-2,Bax mRNA and protein of MCF-7 cells on TaxolThe results of qRT-PCR and Western Blot revealed that compared with the miR-NC+Taxol group,the relative expression levels of Bcl-2 mRNA and protein of MCF-7 cells in the miR-129-5p mimics+Taxol group were decreased,while the Bax mRNA and protein were increased,and the difference were statistically significant?P<0.05?.Conclusionmi R-129-5p can enhance the sensitivity of breast cancer cells to Taxol by inhibiting the expression of ABCB1.