| BackgroundThe new and death cases of breast cancer is about 1.3 million and 0.4 million.The health burden of cancer is increasing in China, breast cancer is now the most common cancer in Chinese women; cases in China account for 12.2% of all newly diagnosed breast cancers and 9.6% of all deaths from breast cancer worldwide. Breast cancer is now the most frequently diagnosed cancer and is the sixth leading cause of cancer-related death in Chinese women.Recent studies have made great progression with understanding the mechanism of breast cancer tumorigenesis and progression, and several clinical drugs had developed for target therapy based on these researches, But it is far away to fully understand the mechanism of breast cancer tumorigenesis and progression.MicroRNAs regulate expression at the posttranscriptional level through complementary pairing with the 3’untranslated region (UTR) of target gene mRNAs that are involved in physiological processes, such as cell proliferation, apoptosis, differentiation and metabolism, and pathological processes, including development of cardiovascular diseases, neurological diseases and tumors. Multiple miRNAs including miR-31, miR-205, miR-335, miR-200 family, miR-124, miR-340, and miR-196s have been shown to be downregulated and function as tumor suppressor genes in BC; however, miR-10b, miR-373, miR-520c, miR-9, and miR-221/222 play oncogenic roles in BC carcinogenesis and progression. miR-409-3p, located on 14q32.31, is involved in tumor formation or progression. Previous findings have shown that miR-409-3p regulates cell proliferation and apoptosis and suppresses cell invasion and metastasis in gastric cancer. Similar results have been reported for bladder cancer, lung adenocarcinoma and colorectal cancer, but its functional mechanisms in breast cancer are yet to be established.ObjectiveThe aim of this study is to examine the expression of miR-409-3p, identify its possible role in breast cancer pathogenesis, and elucidate the molecular mechanisms of its suppressive or oncogenic activities on breast cancer. We hope that this study will improve the better understanding of breast cancer pathogenesis and the development of novel effective therapies for breast cancer.Methods1. Cell culture and tissue samplesThirty human BC tissues and adjacent non-tumor samples were collected. All samples were frozen in liquid nitrogen. Human BC cell lines MCF-7, T47D, MDA-MB-468, MDA-MB-231, and the breast epithelial cell line, HBL-100 were cultured.2. Lentivirus transduction and oligonucleotide transfectionLentiviral vectors overexpressing miR-409-3p were constructed according to a previously described method. MDA-MB-231 and MDA-MB-468 cells were infected with recombinant lentivirus-transducing units. Target cells were transfected with miR-409-3p antagomir and negative control.3. RNA extraction and quantitative RT-PCRTotal RNA was extracted from all samples and cells using TRIzol Reagent. RNA samples were mixed with miRNA-specific stem-loop RT primers, and reverse-transcribed to cDNA. cDNA samples were subjected to qPCR to detect expression of miR-409-3p or Aktl mRNA. U6 or β-actin were used as the controls for normalization. Relative gene expression data were analyzed using the 2-△△Ct method.4. Western blottingTotal protein was separated via 10% SDS-PAGE, transferred to PVDF membrane, and blocked with 5% non-fat milk. Membranes were probed with Aktl or phospho-Akt primary antibodies at 4℃ overnight, followed by incubation with HRP-conjugated secondary antibody, using GAPDH as the internal control. Proteins were visualized with enhanced chemiluminescence reagents.5. Plasmid construction and transfectionThe open reading frame (ORF without 3’UTR) of Aktl was cloned into the expression vector, pcDNA3.1, for ’rescue’experiments. The 3’UTR fragment of Akt1 containing the predicted miR-409-3p-binding site was inserted into the pGL3 control luciferase reporter vector. Mutants of the 3’UTR miR-409-3p binding sites were generated using the QuikChange Site-Directed Mutagenesis kit. Transfection of plasmids was performed using the Lipofectamine 2000 reagent.6. Cell proliferation and colony formation assaysCell viability was detected using the MTT assay. Absorbance was measured at 490 nm. For the colony formation assay, cells were seeded in 6-well plates and cultured for 14 days. Colonies were fixed with 30% formaldehyde and stained with crystal violet. Colonies were counted under an optical microscope.7. Cell migration and invasion assaysCell migration and invasion were examined using a Transwell chamber assay coated without or with Matrigel, respectively, on the upper surface of the membrane, according to the manufacturer’s protocol. After 24 h, cells migrating or invading through the lower surface were fixed, stained and counted using a light microscope. Five random fields of view were analyzed for each chamber.8. Tumor xenograft modelMDA-MB-231 expressing miR-409-3p and NC cells were injected. Tumor volumes were measured every four days. Mice were killed 28 days after injection, final tumor volumes measured, and the tumors weighed. A portion of each tumor was selected for western blotting for Aktl and immunohistochemical staining for Ki67.9. Luciferase reporter assayFor the luciferase assay, MDA-MB-231 and MDA-MB-468 cells were seeded in 96-well plates 24 h before transfection and co-transfected with the Aktl wild-type (Wt) or mutant (Mt) 3’UTR reporter vector, hU6-miR-409-3p, or NC and Renilla plasmid using Lipofectamine 2000. Luciferase activities were determined with the Dual-Luciferase Reporter System.10. Statistical analysisAll data were presented as means ±SD. Each experiment was repeated at least three times. Student’s t test (two tailed) was used for comparisons between groups. All statistical analyses were performed using SPSS 13.0, and data considered statistically significant at P<0.05.Results1.Expression of miR-409-3p in human breast cancer tissues and cell linesThe miR-409-3p level was lower in all the tumor cell lines tested, compared with HBL-100. miR-409-3p was downregulated to a significant extent in tumor samples, relative to the corresponding non-tumor tissues.2. miR-409-3p inhibits proliferation, migration and invasion of breast cancer cellsThe MTT assay performed to determine its effects on cell growth. Overexpression of miR-409-3p markedly inhibited the growth of both cell lines, compared to their corresponding controls and decreased colony formation efficiency. Furthermore, overexpression of miR-409-3p significantly suppressed cell migration in a Transwell assay without Matrigel and reduced cell invasion in a Transwell assay with Matrigel. On the other hand, the proliferation, migration and invasion of T47D cells were increased after antagomiR-409-3p transfection in comparison with the cells treated with antagomiR-NC.3. miR-409-3p suppresses tumor growth in nude miceTumor growth in the miR-409-3p-expressing group was significantly slower than that of the control groups. Moreover, the average tumor volume and weight of the miR-409-3p-expressing group was lower than that of the control groups. Ki-67 staining revealed a significant decrease in the Ki-67-positive cell population of the miR-409-3p-expressing group, compared with that of the control groups. These findings support a strong ability of miR-409-3p to suppress tumor cell growth.4. Aktl is a direct downstream target of miR-409-3pWe used TargetScan and miRanda to explore target searches and the results suggested that miR-409-3p is highly likely to interact with the 3’UTR of Aktl. The 3’UTR sequence of Aktl contains one miR-409-3p binding site. Accordingly, a reporter construct harboring Aktl 3’UTR flanking the entire putative target sequence was generated and the luciferase assay performed. Ectopic expression of miR-409-3p resulted in significant reduction of luciferase activity in MDA-MB-231 and MDA-MB-468 cells in the presence of the Aktl plasmid containing wild-type 3’UTR whereas luciferase activity was not reduced to a significant extent in cells expressing the mutant 3’UTR. qRT-PCR and western blot assays were additionally performed to examine the effects of miR-409-3p on endogenous expression of Akt1. miR-409-3p overexpression failed to affect Aktl mRNA, but induced a significant decrease in the Aktl protein level in MDA-MB-231 and MDA-MB-468 cells, whereas inhibition of miR-409-3p by antagomiR-409-3p resulted in upregulated expression of Aktl in T47D cells. Similar results were obtained in tumor tissues isolated from nude mice injected subcutaneously with MDA-MB-231 cells.5. Ectopic expression of Aktl reverses the effects of miR-409-3p on cell proliferation, migration and invasionWe performed gain-of-function analyses by transfecting Aktl plasmids (lacking 3’UTR) into miR-409-3p-expressing MDA-MB-231 cells. Functional studies revealed that re-introduction of Aktl significantly reversed miR-409-3p-induced inhibition of cell proliferation and colony formation. Moreover, suppression of migration and invasion by miR-409-3p was reversed following Aktl overexpression.Conclusions1. The expression of miR-409-3p decreases in breast cancer tissues and cell lines, indicating that miR-409-3p may be a tumor suppresser in breast cancer.2. The over-expression of miR-409-3p can significantly inhibit breast cancer cell proliferation and invision, suggesting that miR-409-3p could be a tumor suppresser in breast cancer.3. miR-409-3p suppresses tumor growth in nude mice suggesting that miR-409-3p could be a tumor suppresser in vivio.4. miR-409-3p suppresses breast cancer cell growth and invasion by targeting Aktl.5. Ectopic expression of Aktl reverses the effects of miR-409-3p on cell proliferation, migration and invasion. |
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