| BACKGROUND AND OBJECTIVEBreast cancer is the most common malignancy in women in the world and seriously threatens the health of women. Radiotherapy is among the primary strategies for most patients receiving breast-conserving surgery and displays significant clinical benefits, such as decreasing the risk of local recurrence and reducing the risk of mortality due to breast cancer. However, for some subtypes, such as basal-like breast cancer, the local and regional control remains unsatisfactory. A major reason for this failure in treatment may due to its radioresistance. Therefore, understanding the molecular mechanisms involved in the radioresistance of breast tumors may lead to improved clinical outcomes.Macroautophagy, typically referred to as autophagy, is a homeostatic process involving self-degradation and recycling of macromolecules and cellular organelles and functions as a prosurvival mechanism under stressful conditions by degrading subcellular debris and regenerating metabolic precursors, therefore maintaining cellular integrity. During the last decade, the number of studies on the roles of autophagy in human diseases and its cellular mechanism has become extensive. Recent investigations have demonstrated that autophagy is a critical adaptive response for tumor cells to survive stressful conditions, such as hypoxia, nutrient deprivation, chemotherapy or radiotherapy, thereby providing therapeutic resistance. However, the molecular mechanisms by which autophagy mediates the radioresistance of breast tumors is less clear.MicroRNAs (miRNAs) have been shown to be key regulators of a variety of biological processes, including cell proliferation, differentiation and invasion. Dysregulation of miRNAs has been reported to contribute to many human diseases, including cancer. Recent studies indicated that miRNAs may function as key regulators of autophagy. Several miRNAs have also been reported to be involved in chemoresistance or radioresistance via modulation of autophagy. However, relatively little is known about the role of autophagy in miRNA-mediated radioresistance in breast cancer. The miR-200family is involved in cancer stem cells self-renewal, the epithelial to mesenchymal transition (EMT) and chemosensitivity. Recent studies indicated that miR-200c, the prevailing member of the miR-200family, can also sensitize cancer cells to radiation by targeting TBK1and VEGFR2. However, the relationship between miR-200c and the modulation of autophagy that induces radioresistance remains unclear.Here, we show that miR-200c can sensitize breast cancer cells to radiation via a mechanism associated with inhibition of irradiation-induced autophagy. We also identified the protein ubiquilin1(UBQLN1) as a direct target of miR-200c whose expression is positively correlated with radioresistance and irradiation-induced autophagy. Our results provide new insights into the molecular functions of miR-200c in radiosensitivity in breast cancer cells and imply a rationale for enhancing radiosensitivity via miR-200c for the treatment of breast cancer.METHODS1. Detection of miR-200c in breast cancer cellsThe normal breast epithelial cell line MCF-10A, the luminal phenotype breast cancer cell lines MCF-7and BT474and basal phenotype breast cancer cell lines MDA-MB-231and BT549were used in this study. Total RNA was extracted from the cells using TRIzol reagent according to the manufacturer’s protocol. The cDNA library was synthesized using the PrimeScript RT reagent kit following the manufacturer’s instruction. For mature miRNA quantification, cDNA was generated using specific stem-loop universal primers. RT-PCR for miR-200c in these five cell lines was performed using SYBR Premix Ex Taq II according to the manufacturer’s protocol and was measured using an ABI7500Sequence Detection System. U6was used as the internal control.2. Oligonucleotide and siRNA transfectionThe miRNA mimic, the inhibitors, the control oligonucleotides and UBQLN1siRNA were purchased from RiBoBio. Transfection of oligonucleotides and siRNA was performed using Lipofectamine2000reagent according to the manufacturer’s protocol. For efficient inhibition of the miR-200bc/429cluster, equivalent amounts of miR-200c and miR-429inhibitors were combined.3. Irradiation and clonogenic survival assaysIncreasing numbers of cells were seeded on6-well plates (previously transfected with oligonucleotides or siRNA for48hours) in triplicate and exposed to the indicated dose of radiation using6MV X-rays generated from linear accelerators at a dose rate of5Gy/min. After incubation at37℃for10to14days, the cells on the plates were fixed using100%methanol and stained using1%crystal violet. Colonies containing>50cells were counted via microscopic inspection. The surviving fraction was calculated according to the multi-target single-hit model.4. Western blot analysisWhole cell protein lysates were extracted using radio-immunoprecipitation assay (RBPA) buffer containing proteinase and phosphatase inhibitor cocktails and quantified using a bicinchoninic acid protein assay kit. Equivalent amounts of total protein were resolved via SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were then blocked using5%nonfat milk for1hour at room temperature, followed by incubation in the primary antibodies overnight at4℃. After washing three times, the membranes were incubated in HRP-conjugated secondary antibodies for1hour at room temperature. The immunoreactive bands were visualized using an ECL western blot substrate.5. Preparation of stable GFP-LC3-expressing cellsThe recombinant lentivirus containing GFP-LC3was purchased from Genepharma (Shanghai, China). MDA-MB-231cells were infected with lentivirus particles and isolated via fluorescence-activated cell sorting to generate populations stably expressing GFP-LC3.6. Bioinformatic analysis for predicting miR-200c target genes regulating autophagyTo investigate the molecular mechanism by which miR-200c suppressed irradiation-induced autophagy, we identified potential targets of miR-200c based on three publicly available databases (TargetScan, Pictar and miRBase) and then intersected the results from these databases, followed by searching in the Pubmed to analyze the relationship between these genes and autophagy.7. Analysis of the expression of UBQLN1mRNA and protein after ectopic expression of miR-200c in breast cancer cellsQuantitative real-time PCR (RT-PCR) and Western blot analysis were used to analyzed the expression of UBQLN1mRNA and protein after transfection with miR-200c mimic.8. Luciferase reporter assayThe3’ untranslated region (UTR) of UBQLN1and mutant3’UTR, which contains the putative miR-200c binding site, were amplified from genomic DNA via PCR and cloned into the Renilla luciferase gene (pLUC-REPORT vector, Promega, Madison, WI, USA). For the luciferase reporter assay, MDA-MB-231cells were co-transfected with a luciferase reporter vector (either pLUC-3’UTR-UBQLN1or pLUC-3’UTR-mut-UBQLN1) and negative control miRNA or the miR-200c mimic. Forty-eight hours after transfection, the cells were assayed for luciferase activity using the Dual-Luciferase Assay Kit (Promega) according to the manufacturer’s instructions. For each sample, the relative luciferase activity was normalized to firefly luciferase activity. Three independent experiments were performed in triplicate.9. Detection of the expression of UBQLN1and LC3in breast cancer tissue.Immunohistochemical staining (IHC) was performed on3-μm-thick sections excised from paraffin blocks. The sections were fixed using4%formaldehyde overnight and embedded in paraffin, followed by deparaffinization and hydration. Then, the sections were pretreated with sodium citrate buffer in a microwave for antigen retrieval and blocked using goat serum. The sections were stained using rabbit anti-UBQLN1and anti-LC3A/B overnight at4℃. Then, the sections were incubated in biotinylated goat anti-rabbit IgG secondary antibody for1hour, followed by staining with the avidin-biotin peroxidase complex according to the manufacturer’s instructions. Scoring of the sections was performed by two independent pathologists.10. Detection of the miR-200c level in breast cancer tissueFor detection of the expression of miR-200c in breast cancer tissue, in situ hybridization (ISH) was performed.11. Statistical analysisIn the experimental studies, the data are presented as the means±standard deviation. The samples were analyzed using Student’s t test or analysis of variance (ANOVA). The Pearson Chi-square test, Fisher’s Exact test or t test was used to analyze the relationship between miR-200c expression and the clinical characteristics. All statistical analysis was performed using SPSS13.0software, and a P value of<0.05was considered to be statistically significant. The qualitative data were representative of more than three independent experiments that were performed in triplicate.Results:1. The expression level of miR-200c in breast cancer cell linesIn this study, we first investigated miR-200c expression in four breast cancer cell lines and the normal breast epithelial cell line MCF-10A. The results of quantitative real-time PCR indicated that the expression of miR-200c was higher in luminal phenotype breast cancer cells (MCF-7and BT474) and lower in basal phenotype breast cancer cells (MDA-MB-231and BT549) compared to the normal breast epithelial cell line MCF-10A.2. MiR-200c sensitizes breast cancer cells to radiation treatment.Overexpression of miR-200c enhanced the radiosensitivity of the MDA-MB-231and BT549cell lines, while inhibition of the expression of miR-200c led to radioresistance in MCF-7cells. These results suggest that restoring the normal expression level of miR-200c can enhance the radiosensitivity of breast cancer cells.3. Autophagy is induced by irradiationAutophagy may contribute to either cytoprotective or cytotoxic effects, depending on the type of cell and stress. Western blot analysis indicated that, after receiving6Gy radiation at various time points, autophagy was activated in three human breast cancer cell lines, as demonstrated by the increased expression of LC3Ⅱ, accompanied by decreased expression of p62. However, the activity of autophagy in MCF-7cells was not significant different compared to the MDA-MB-231and BT549cell lines.4. Autophagy acts as a protective mechanism in breast cancer cells after irradiation.Western blot analysis indicated that,3-MA, an autophagy inhibitor that blocks autophagosome formation, can effective inhibited the activity of autophagy induced by irradiation. Clonogenic assays demonstrated that3-MA significantly decreased the survival fraction of three human breast cancer cell lines after irradiation. These results indicate that autophagy is activated due to an adaptive response to promote the survival of breast cancer cells after receiving irradiation.5. miR-200c inhibited radiation-induced autophagyTo determine whether miR-200c can inhibit irradiation-induced autophagy, we established a MDA-MB-231cell line stably expressing the GFP-LC3fusion protein. After24hours of irradiation, miR-200c overexpression significantly decreased the lipidation of LC3and increased p62protein expression, suggesting a decreased level of autophagy.6. miR-200c inhibited the formation of autophagosomeTo further investigate the specific stage of autophagy that was inhibited by miR-200c, we used bafllomycin Al, a fusion inhibitor of lysosomes and autophagosomes, which can inhibit the degradation of LC3Ⅱ. Twenty-four hours after irradiation, the bafilomycin Al-treated control group displayed markedly increased accumulation of LC3Ⅱ, whereas the miR-200c-treated group displayed only a small alteration. These data indicate that the inhibitory effect of miR-200c on autophagy resulted from the suppression of the early stage of autophagy, the inhibition of autophagosome formation, rather than from the suppression of autophagosome degradation.7. MiR-200c modulates irradiation-activated autophagy, which is associated with the radioresistance of human breast cancer cells.Next, we sought to determine whether miR-200c-induced radiosensitization was dependent on suppression of autophagy. Clonogenic survival assay indicated that3-MA significantly increased radiosensitivity, as miR-200c expression was suppressed in MCF-7cells. In MDA-MB-231cells,3-MA also enhanced radiosensitivity when miR-200c was overexpressed, but this effect was not significant when miR-200c expression was suppressed in MCF-7cells. Additionally, we found that miR-200c overexpression increased the irradiation-induced activation of caspase-3and PARP in MDA-MB-231cells. In MCF-7cells, suppression of miR-200c expression decreased the irradiation-induced activation of PARP. Because MCF-7cells are a caspase-3deficient breast tumor cell line, we failed to detect the expression of activation of caspase-3after treatment with irradiation,3-MA or both. Combined treatment of3-MA and radiation significantly increased the activation of apoptosis-related proteins in both groups. These results suggest that miR-200c enhanced the radiosensitivity of breast cancer cells in a manner that may be associated with the suppression of IR-induced autophagy.8. Identified the autophagy related target genes of miR-200c by bioinformatic analysisTo investigate the molecular mechanism by which miR-200c suppressed irradiation-induced autophagy, we identified potential target of miR-200c based on three publicly available databases (TargetScan, Pictar and miRBase) and then intersected the results from these databases. After this, we predicted11target genes of miR-200c. Then we searched in the Pubmed to analyze the relationship between these genes and autophagy, and screened four genes, RAC1(ras-related C3botulinum toxin substrate1), NPC1(Niemann-Pick disease, type C1), PPP2CA (Serine/threonine-protein phosphatase2A catalytic subunit alpha isoform) and UBQLN1(ubiquilin1). According to the mirSVR score and PhastCons score of prediction from microRNA.ORG database, we choose UBQLN1as the target gene of miR-200c in this study finally.9. miR-200c inhibit the expression of UBQLNlEctopic expression of miR-200c exhibited no effect on the mRNA expression level of UBQLN1, whereas overexpression of miR-200c significantly suppressed the protein expression of UBQLN1. In addition, the luciferase reporter assay revealed that miR-200c significantly suppressed the luciferase activity of the wild-type3’UTR of UBQLN1but not that of the mutant reporter gene, indicating the specificity of miR-200c to target the UBQLNl3’UTR. These results indicated that miR-200c suppressed UBQLN1expression at the post-transcriptional level via translational arrest.10. Silencing the expression of UBQLNl enhanced the radiosensitivity and suppressed the autophagy activity of breast cancer cellsTo confirm that miR-200c enhanced radiosensitivity due to the direct targeting of UBQLN1, we examined the effect of silencing the expression of UBQLN1on radiosensitivity. A clonogenic assay indicated that knockdown of the expression of UBQLN1significantly enhanced the radiosensitivity of MDA-MB-231and BT549cells. Furthermore, silencing of the expression of UBQLN1led to significant inhibition of irradiation-induced autophagy, consistent with the phenotype induced by the overexpression of miR-200c. These results demonstrate that miR-200c suppressed irradiation-induced autophagy by directly targeting UBQLN1.11. The expression of miR-200c, UBQLNl and LC3are inversely correlated in human breast cancer tissueTo investigate the correlation between the expression of miR-200c, UBQLN1and LC3in human breast cancer tissue, we measured the expression levels of miR-200c, UBQLN1and LC3in human breast cancer specimens via in situ hybridization and immunohistochemistry. We found decreased expression of miR-200c in human breast cancer tissues that exhibited elevated UBQLN1and LC3 expression, and increased miR-200c expression correlated with decreased UBQLN1and LC3expression. In matched adjacent noncancerous breast tissues, we detected moderate expression of miR-200c and did not detect any expression of UBQLN1and LC3. Furthermore, the expression of miR-200c, UBQLN1and LC3displayed little correlation with the clinical and pathological characteristics of breast cancer. However, the miR-200c expression level inversely correlated with lymph node metastasis, but the UBQLN1and LC3expression level displayed little correlation with this characteristic. Whether the expression of UBQLN1influences breast cancer metastasis requires further investigation. These results are consistent with the above finding that decreased expression of miR-200c correlated with increased UBQLN1expression.Conclusion:1. The expression of miR-200c was higher in luminal phenotype breast cancer cells and lower in basal phenotype breast cancer cells compared to the normal breast epithelial cell line MCF-10A.2. Ectopic expression of miR-200c enhanced the radiosensitivity of breast cancer cells.3. Overexpression of miR-200c suppressed the activity of radiation-induced autophagy, which may associated with radioresistance.4. UBQLN1is a directly functional target of miR-200c.5. The expression of miR-200c, UBQLN1and LC3are inversely correlated in human breast cancer tissue. |
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