MicroRNA-502-5p Targets ATG16L1 To Sensitize Breast Cancer Cells To Doxorubicin By Inhibiting Autophagy

Research background: Breast cancer(BC)is one of the most common malignant tumors in women and is the leading cause of death in women worldwide.Currently,chemotherapy is a common treatment for breast cancer.Doxorubicin(Adriamycin,ADR)is an anthracycline cytotoxic antibiotic that inhibits the synthesis of DNA and RNA in tumor cells.It is mainly used in the treatment of malignant tumors such as breast cancer,osteosarcoma and leukemia.However,it has been clinically found that long-term use of doxorubicin to treat breast cancer cells leads to resistance to doxorubicin in patients,and the efficacy is limited,resulting to chemotherapy failure.Therefore,solving the resistance of breast cancer cells to doxorubicin has become an urgent problem to be solved.Autophagy is a highly conserved process for the turnover of intracellular substances that is widely present in eukaryotic cells.The cells maintain their intracellular homeostasis by self-decomposing damaged proteins or organelles under stress conditions such as hypoxia,starvation,and chemotherapeutic stimulation,which is beneficial to the cells to obtain survival advantages under adverse conditions.It has been pointed out in the literature that protective autophagy of tumor cells may be a potential mechanism for resistance to anticancer drugs.Studies have shown that cytoprotective autophagy can overcome some of the stress caused by chemotherapy,leading to a decrease in chemotherapy.microRNAs(miRNAs)is widely present in eukaryotes and is a non-coding single-stranded RNA having a length of 19-25 nucleotides that binds to the 3'UTR region of the target gene by way of base complementary pairing Target mRNA degradation or post-transcriptional translation inhibition.Recently,it has been reported that miRNAs can regulate the role of autophagy in tumors by regulating the expression of autophagy-related proteins.We used a high-throughput miRNA expression analysis system to screen out miRNA expression profiles that are well-reacted to doxorubicin from the Balb/c nude mouse osteosarcoma xenograft model and microRNA-502-5p(miR-502)is one of the most significant miRNAs.The gene encoding hsa-miR-502 is located on the chromosome X.Several studies have reported that miR-502 is downregulated in the breast cancer,kidney cancer,liver cancer and colorectal cancer tissues,and plays an inhibitory role in tumor cell proliferation,invasion and metastasis.Moreover,miR-502 targets Rab1 B to inhibit colon cancer autophagy and tumor growth.However,the molecular mechanism by which miR-502 promotes doxorubicin sensitivity is unclear.ATG16L1(autophagy related 16 like 1)is a major autophagy-related protein that forms a complex with ATG5 and ATG12.The ATG16L1-ATG5-ATG12 system functions as novel E3-like enzyme to regulate the site at which microtubule-associated protein light chain 3(LC3)binds with phosphatidylethanolamine(PE),promoting the formation of autophagosome membranes.The miRNA analysis software Targetscan predicts that ATG16L1 may be one of the targets of miR-502.Therefore,we hypothesized that miR-502 targets ATG16L1 to sensitize breast cancer cells to doxorubicin by inhibiting autophagy.Objectives:(1)To determine whether ATG16L1 is a direct target of miR-502.(2)To explore the role and mechanism of miR-502/ATG16L1 axis in the sensitivity of doxorubicin in breast cancer cells.Methods:(1)The breast cancer doxorubicin-resistant MCF-7/ADR cells were induced by a small dose start,gradual addition and intermittent administration of MCF-7 cells.The breast cancer doxorubicin-resistant cell line MCF-7/ADR and its parental cell line MCF-7 were used as experimental subjects.IC50 of the two cell lines were investigated by CCK-8 assay.In miR-502 upregulation experiment,non-specific miRNA(NC)?miR-502 mimic(miR-502)and small interfering RNA against ATG16L1(siATG16L1)were transfected into doxorubicin resistance breast cancer MCF-7/ADR cells by lipofectamine 2000.In miR-502 downregulation experiment,non-specific miRNA(NCi)and miR-502 inhibitor(502i)were transfected into MCF-7 cells by lipofectamine 2000.(2)The expression level of miR-502 was detected by Taqman probe real-time qRT-PCR.The ATG16L1 mRNA level was measyred by SYBR Green qRT-PCR and the protein expression levels of ATG16L1 and the autophagy marker proteins LC3-II and P62 were detected by Western blot.(3)The binding site of miR-502 and ATG16L1 mRNA 3'UTR was predicted by miRNA analysis software Targetscan.Chemical synthesis of wild-type ATG16L1 mRNA containing the miR-502 binding site 3'UTR double-stranded oligonucleotides(WT)and miR-502 binding site mutated mutant ATG16L1 mRNA 3'UTR double-stranded oligonucleotides(Mut),artificially synthesized ATG16L1 The 3'UTR wild-type and mutant double-stranded oligonucleotides were inserted into the SacI and XhoI sites of the GP-miRGLO vector,and ATG16L1 was confirmed to be a direct target of miR-502 by dual luciferase reporter assay.(4)MCF-7/ADR cells were co-transfected with miR-502 mimic and GFP-LC3 plasmids(miR-502+GFP-LC3)by lipofectamine 2000,compared with co-transfection of non-specific miRNA and GFP-LC3 plasmids(NC+GFP-LC3)as control;MCF-7 cells were co-transfected with miR-502 inhibitor and GFP-LC3 plasmids(502i+GFP-LC3)by lipofectamine 2000,compared with co-transfection of non-specific miRNA and GFP-LC3 plasmids(NCi+GFP-LC3).After 24 hours of transfection,the corresponding concentration of doxorubicin was used to induce autophagy.After 12 hours of drug induction,observation and counting were performed under an inverted fluorescence microscope.At least 100 cells were counted and GFP-LC3 positive cells in which more than five bright green fluorescent spots were counted in the cells relative to the total cells under each condition.Results:(1)CCK-8 assay results showed successful construction of breast cancer doxorubicin-resistant cells MCF-7/ADR.The IC50 of MCF-7/ADR cells was 16.41 ± 0.07 ?g/ml,which was 75.98 ± 0.11 times higher than MCF-7 cells(0.22 ± 0.02 ?g/ml).Light microscopy showed that compared with the corresponding parental MCF-7 cells,the volume of MCF-7/ADR cells became larger,the edges were rounded,the cells were loosely arranged,and the trypsin digestion time was shortened.(2)Taqman probe real-time qRT-PCR showed that the expression level of endogenous miR-502 in MCF-7/ADR cells was significantly lower than that in the corresponding sensitive MCF-7 cells(P < 0.01).SYBR Green qRT-PCR and Western blot results showed that the mRNA and protein levels of endogenous ATG16L1 in MCF-7/ADR cells were dramatically higher compared to MCF-7 cells(P < 0.01).The LC3-II protein level of MCF-7/ADR cells was higher while the P62 level was significantly lower than that of MCF-7 cells.(3)The luciferase assay showed that the co-transfected of miR-502 mimic and ATG16L1 3'UTR vector(NC+WT)into breast cancer doxorubicin-resistant MCF-7/ADR cells significantly suppressed the luciferase activity related to co-transfection of negative miRNA and wild type 3'UTR of ATG16L1 mRNA(NC+WT)(P < 0.01).However,there was no significant difference in luciferase activity between the co-transfected of miR-502 mimic and mutant ATG16L1 3'UTR vector(miR-502+Mut)and the co-transfected of negative miRNA and ATG16L1 mutant 3'UTR vector(NC+Mut)to MCF-7/ADR cells(P > 0.05).(4)Real-time quantitative qRT-PCR and Western blot results showed that the ATG16L1 mRNA and protein levels in MCF-7/ADR cells transfected of miR-502 mimic(miR-502)was significantly lower than transfected of negative miRNA(NC)(P < 0.01),while in MCF-7 cells transfected of miR-502 inhibitor(502i)was significantly higher than transfected of negative miRNA(NCi)(P < 0.01).The results of CCK-8 showed that the doxorubicin IC50 of MCF-7/ADR cells in miR-502 group was significantly lower than that of NC group(P < 0.05),and the doxorubicin IC50 of siATG16L1 group was also significantly lower than that of NC group.The doxorubicin IC50 of MCF-7 cell 502 i group was significantly higher than that of NCi group(P < 0.05).(5)Western blot analysis showed that the LC3-II protein levels in MCF-7/ADR cells in miR-502 group and siATG16L1 group were lower than those in NC group,and P62 protein levels were higher than NC group.Conversely,the level of LC3-II protein in the 502 i group was higher than that in the NCi group,and the P62 protein level was lower than that in the NCi group.The results of GFP-LC3 analysis under showed that the percentage of GFP-LC3 positive cells in the MCF-7/ADR cells co-transfected with miR-502+GFP-LC3 group was significantly lower than that of co-transfected NC+GFP-LC3 group(P < 0.01),and the percentage of GFP-LC3 positive cells co-transfected into MCF-7 cells in the 502i+GFP-LC3 group was significantly higher than that in the co-transfected NCi+GFP-LC3 group(P < 0.05).Conclusions:(1)ATG16L1 is a direct target of miR-502 in breast cancer cells,and miR-502 inhibits ATG16L1 expression at the post-transcriptional level.(2)miR-502 inhibits autophagy by targeting ATG16L1 and increases the drug sensitivity of breast cancer cells to doxorubicin.As a result,miR-502 may be a potential candidate for reversing resistance to doxorubicin.