| Backgroud:Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in females worldwide. Comprehensive treatment, including surgical resection, postoperative chemotherapy, is the most effective means to improve survival in patients with breast cancer. Currently, neoadjuvant chemotherapy has been widely used. However, drug resistance is a major factor leading to the failure of chemotherapy. miRNAs is one of the hotspots in recent cancer research. Emerging evidences have shown that miRNAs can directly target mRNA and inhibited the expression of drug-associated genes, and thus resulting in drug resistance. In this study, we detected the expression of miR-489in clinical samples and performed functional analysis in vitro and in vivo, to investigate the mechanism of miR-489to reverse drug resistance in breast cancer.Methods:Breast biopsy tissue samples and their resected tumor tissues were collected and divided into two groups (drug-resistant group and drug-sensitive group) according to the sensitivity of chemotherapy. The expression of miR-489in breast cancer tissues and breast cancer cells was quantified by real-time PCR. After transfection with miR-489precursor or miR-489inhibitor, MTS and EdU cell proliferation assays, flow cytometry, cell migration and invasion assays were performed to test the effects of miR-489on cell biological behaviours in breast cancer cell lines. A tumor-burdened mice model was constructed to mimic the process of human breast cancer chemotherapy treatment course, and to observe the effects of miR-489on chemotherapy sensitivity. TargetScan and Pictar and Miranda were used to predict the target genes of miR-489, and then, luciferase assay, immunohistochemistry and Western blot were used to confirm SPIN1as a direct target of miR-489. The expression of SPIN1in breast cancer was detected by immunohistochemistry and the correlation between SPIN1and clinicopathological parameters was analyzed.Results:1. Expression of miR-489in breast cancer tissues and cells:miR-489expression in drug-resistant group was significantly decreased than that of drug-sensitive group. Compared with MCF-7, miR-489expression in adriamycin-resistant breast cancer cell MCF-7/ADM was dramatically reduced. Moreover, the expression of miR-489in metastatic breast cancer cell MDA-MB-231was even lower than that in non-metastatic breast cancer cell MCF-7and T47D.2. Cell functional experiments in vitro:MTS, EdU, migration and invasion assays showed that miR-489could inhibit the proliferation, invasion and migration ability of breast cancer cells. Flow cytometry results showed that miR-489could promote apoptosis of breast cancer cells.3. Tumor-bearing mice experiments:Mice injected with miR-489-expressing cells were more sensitive than those in the negative control group. And the tumor volume and growth speed of mice injected with miR-489-expressing cells was significantly smaller than that of the negative group. Our data slso showed that miR-489could inhibit tumor invasion in vivo.4. Prediction and validation of target genes:Using Pictar, TargetScan and Miranda forecasting software, we chose8potential target genes for further investigation. The luciferase assay showed that miR-489transfection significantly reduced the luciferase activity compared to the negative control group. Immunohistochemistry and western blot was used to further validate that miR-489could significantly inhibit the expression of SPIN1protein. These data suggest that SPIN1is a direct target gene of miR-489.5. Expression of SPIN1in breast cancer:SPIN1was significantly expressed in drug-resistant breast cancer tissues. And SPIN1was associated with tumor grade and lymph node metastasis.Conclusion:MiR-489could inhibit the expression SPIN1to suppress cell growth, invasion and metastasis, and promote cell apoptosis, and subsequently increase the sensitivity to chemotherapy in breast cancer. |
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