| Breast cancer is the most common cancer-related cause of death in females worldwide.The incidence of the breast cancer is increasing year by year.Though Taxol has improved the survival of cancer patients as a first-line chemotherapeutic agent,drug resistance to Taxol remains an important factor limiting the clinical efficacy.Thus,it is strongly important to enhance the sensitivity of breast cancer to Taxol.With the development of miRNA research,miRNA has attracted the researchers in the study of multidrug resistance(MDR)by targeting its downstream genes.MiR-129-5p is a recently discovered miRNA and downregulates in various cancers including breast cancer which has been validated to inhibit cancer cells migration,proliferation,differentiation,apoptosis,autophagy and epithelial-mesenchymal transition.It also has involved in MDR.Through target gene prediction online,we found that HMGB1 may be the target gene of miR-129-5p.MiR-129-5p also has been reported to directly target HMGB1 in breast cancer MCF-7 cells.High mobility group box 1(HMGB1)is a regulator of autophagy,which is highly expressed in tumor cells.It can participate in the development of tumor and modulate autophagy to affect the sensitivity of tumor cells to chemotherapeutic drugs.Autophagy is a well-known important protective mechanism which can help cells against adverse growth environment.Under the adverse conditions of ischemia,hypoxia and chemotherapeutic drug stimulation,autophagy can degrade the cellular organelle or denatured protein in cells into energy for cell reutilization to maintain cell growth and recovery.In the process of cancer treatment,some of the tumor cells are using autophagy to protect the cells and resist the damage of chemotherapeutic drugs,so this can reduce the sensitivity of tumor cells to drugs,make tumor cells escape the killing effect of drugs and eventually lead to the occurrence of drug resistance.Therefore,it is important to study whether miR-129-5p can regulate autophagy by targeting HMGB1 and then affect the sensitivity of breast cancer cells to Taxol.It may provide new ideas and experimental basis for clinical treatment of breast cancer.Objective In this study,we used breast cancer MCF-7 cells as the research object.After MCF-7 cells was overexpression of miR-129-5p and interfered with HMGB1,the expression of HMGB1,LC3 B,p62 was detected,as well as apoptosis and proliferation.In order to investigate whether miR-129-5p can regulate autophagy and affect the sensitivity of breast cancer MCF-7 cells to Taxol through regulating HMGB1.Materials and Methods 1 Object In this study,breast cancer MCF-7 cells as the research object,the cells were cultured in RPMI 1640 complete medium containing 10% inactivated fetal bovine serum,placed in 37 °C,5% CO2 incubator,until the cells confluent Degrees of 80% to 90% are passed or used in subsequent experiments.2 Methods 2.1 Experiment grouping This study was carried out the following three kinds of groups according to the purpose of the study.(1)The experiment was divided into four groups which explored the sensitivity of MCF-7 cells to Taxol after autophagy inhibited—control group(without treatment factors),Taxol group,3-MA group and 3-MA+Taxol group.(2)The effect of miR-129-5p overexpression on the sensitivity of MCF-7 cells to Taxol was divided into 4 groups—miR-NC group(transfection negative control),miR-129-5p group(transfection of miR-129-5p mimics),miR-NC+Taxol group and miR-129-5p+Taxol group.(3)The effects of HMGB1 interference on the sensitivity of MCF-7 cells to Taxol was divided into 4 groups—si-NC group(transfection negative control),si-HMGB1 group,si-NC + Taxol group and si-HMGB1 + Taxol group.2.2 Cell transfection miR-129-5p mimics or si-HMGB1 was transfected into MCF-7 cells by liposome transfection technique,respectively,for subsequent experiments.2.3 CCK-8 CCK-8 assay was used to detect the inhibition rate of proliferation in MCF-7 cells which were treated with or without 3-MA inhibited autophagy,transfected with miR-129-5p mimcs,or interfered with si-HMGB1.2.4 q RT-PCR The expression of HMGB1 m RNA before and after transfection of miR-129-5p was detected by q RT-PCR.2.5 Immunofluorescence The fluorescence intensity of HMGB1 in MCF-7 cells before and after transfection with miR-129-5p was detected by immunofluorescence.2.6 Western Blot Western Blot was used to detect the expression levels of HMGB1,LC3 B,and p62 proteins after autophagy inhibition,transfection with miR-129-5p,and interference with HMGB1 expression.2.7 Flow cytometry Flow cytometry was used to detect the apoptosis of MCF-7 cells induced by autophagy,transfection of miR-129-5p and interference of HMGB1 with 3-MA.3 Statistics analysis SPSS21.0 statistical software was used.All the result were presented as mean ± standard deviation(SD).The two groups of quantitative data were tested by t-test or corrected by t-test.The comparison of multiple quantitative data was compared with One-way ANOVA and LSD-t test.Take the alpha =0.05 as the test level.Results 1 The sensitivity of MCF-7 cells to Taxol was increased effects by autophagy.First,MCF-7 cells were stimulated by 31.2n M Taxol at different time(0h,6h,12h training,24h),the ratio of LC3B-II/LC3B-I was decreased with the increasing of time.Then MCF-7 cells were pretreated by 3-MA before using 31.2n M Taxol for 24 h.Compared with the control group,the ratio of LC3B-II/LC3B-I was significantly decreased in 3-MA group(P<0.05),the expression of p62 was significantly increased(P<0.05),the total rate of cell apoptosis was significantly increased(P<0.001);Compared with the Taxol group,the ratio of LC3B-II/LC3B-I was significantly decreased in group 3-MA+Taxol(P<0.05),the expression of p62 was significantly increased(P<0.05),the total rate of cell apoptosis was significantly increased(P<0.05),and the sensitivity of MCF-7 cells to Taxol was increased(P<0.05).2 miR-129-5p enhanced chemosensitivity of Taxol by inhibiting autophagy and promoting apoptosis in MCF-7 cells.MCF-7 cells were transfected with miR-129-5p mimics and then treated them with 31.2nm of Taxol for 24 h.Compared with miR-NC group,the expression of miR-129-5p in miR-129-5p group was significantly increased(P<0.001),the ratio of LC3B-II/LC3B-I in miR-129-5p group was significantly decreased(P<0.05),the expression of p62 in miR-129-5p group was significantly increased(P<0.05),the total rate of cell apoptosis in miR-129-5p group was obviously increased(P<0.05);Compared with miR-NC+Taxol group,the ratio of LC3B-II/LC3B-I in the miR-129-5p+Taxol group was significantly decreased(P<0.05),the expression of p62 in the miR-129-5p+Taxol group was significantly increased(P<0.05),the total rate of total cell apoptosis in the miR-129-5p+Taxol group was obviously increased(P<0.05);the sensitivity of MCF-7 cells to Taxol was increased(P<0.05).3 Results of bioinformatics analysis.The potential target genes of miR-129-5p were searched from Target Scan,miRDB and miRanda online analysis tools,and there were target genes of miR-129-5p in 3 sites.The online database indicated that there were two possible binding sites in the 3 'UTR area of miR-129-5p and HMGB1.The tumor database of Oncomine and The Human Protein Atlas showed that the expression of HMGB1 in breast cancer tissues was higher than that of normal para-cancerous tissue.4 HMGB1 was downregulated by miR-129-5p.After MCF-7 cells was transfected with miR-129-5p mimics,compared with miR-NC group,the expression of HMGB1 m RNA and protein in miR-129-5p group was significantly decreased(P<0.001),the fluorescence density of intracellular HMGB1 in miR-129-5p group was significantly decreased(P<0.05);Compared with miR-NC+Taxol group,the expression of HMGB1 m RNA and protein in miR-129-5p+Taxol group was decreased(P<0.001),the fluorescence density of HMGB1 in miR-129-5p+Taxol group was significantly decreased(P<0.05).5 Downregulation of HMGB1 enhanced the chemosensitivity of Taxol by inhibiting autophagy and promoting apoptosis in MCF-7 cells MCF-7 cells was transfected with miR-129-5p mimics and then treated them with 31.2nm of Taxol for 24 h.Compared with si-NC group,the expression of HMGB1 m RNA and protein in si-HMGB1 group was significantly decreased(P<0.001),the fluorescence density of intracellular HMGB1 in si-HMGB1 group was significantly decreased(P<0.05),the ratio of LC3B-II/LC3B-I in si-HMGB1 group was significantly decreased(P<0.05),the expression of p62 in si-HMGB1 group was significantly increased(P<0.05),the total rate of cell apoptosis in si-HMGB1 group was significantly increased(P<0.05);Compared with si-NC+Taxol group,the fluorescence density of intracellular HMGB1 in si-HMGB1+Taxol group was significantly decreased(P<0.05),the ratio of LC3B-II/LC3B-I in si-HMGB1+Taxol group was significantly decreased(P<0.05),the expression of p62 in si-HMGB1+Taxol group was significantly increased(P<0.05),the total rate of cell apoptosis in si-HMGB1+Taxol group was significantly increased(P<0.05),the sensitivity of MCF-7 cells to Taxol in si-HMGB1+Taxol group was increased(P<0.05).Conclusion MiR-129-5p can inhibit autophagy by inhibiting the expression of HMGB1,thereby enhancing the sensitivity of breast cancer MCF-7 cells to Taxol. |
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