Tumor-promoting properties of miR-8084 in breast cancer through enhancing proliferation, suppressing apoptosis and inducing epithelial-mesenchymal transition

Abstract

Background: Breast cancer is one of the most frequent malignancies and the second leading cause of cancer-related mortality in women. MicroRNAs play a key role in breast cancer development and progression. microRNA(miR)-8084 has been observed an aberrant expression in breast cancer. However, the functions and regulatory axes of miR-8084, particularly in breast cancer, were not entirely clear.

Methods: miR-8084 expression in breast cancer were investigated in a GEO dataset by in silico analysis and in 42 paired tumor tissues by qPCR. The effects of deregulation of miR-8084 on breast cancer cell proliferation, migration and invasion in vitro and tumorigenicity in vivo were examined by colony-formation assay, wound healing assay, transwell assay and nude mouse subcutaneous tumor formation model. The target gene of miR-8084 were predicted by TargetScan and miRDB, and confirmed by luciferase reporter system. The roles of miR-8084 in the breast cancer cell proliferation, apoptosis and epithelial-mesenchymal transition (EMT) were investigated by MTS, FACS and associated-marker detection by western blot.

Results: miR-8084 is significantly up-regulated in both serum and malignant tissues from the source of breast cancer patients. miR-8084 promotes the proliferation of breast cancer cells by activating ERK1/2 and AKT. Meanwhile miR-8084 inhibits apoptosis by decreasing p53-BAX related pathway. miR-8084 also enhances migration and invasion by inducing EMT. Moreover, the tumor suppressor ING2 is a potential target of miR-8084, and miR-8084 regulatory axes contribute to pro-tumor effect, at least partially through regulating ING2.

Conclusion: Our results strongly suggest that miR-8084 functions as an oncogene that promotes the development and progression of breast cancer, and miR-8084 is a potential new diagnostic marker and therapeutic target of breast cancer.

Keywords: Breast cancer; ING2; Tumorigenesis; miR-8084.

Fig. 1

miR-8084 is up-regulated in breast cancer and promotes the clonogenicity of breast cancer cells. a miR-8084 in serum of breast cancer and that of non-cancer samples was analyzed using data from GEO (GSE73002) (**P < 0.01). b miR-8084 was detected in breast cancer tissues. The data is shown as ^−△△CT values. c Relative expression of miR-8084 in breast cancer tissues compared with normal tissues. Data is shown as Mean ± SEM (**P < 0.01). d The effect of miR-8084 on clonogenicity was evaluated by the plate colony formation assay. BT-549 cells were transfected with miR-8084 mimics or control, seeded onto six-well plates. The number of colonies was counted on the 14th day after seeding. e MDA-MB-231 cells were transfected with miR-8084 inhibitor or control then seeded onto six-well plates. The number of colonies was counted on the 14th day after seeding. f, g Colonies containing 50 or more cells were counted. Results are means of three independent experiments ± SD (*P < 0.05)

Fig. 2

miR-8084 enhances scratch wound healing ability, migration and invasion of breast cancer cells. a Movement ability of BT-549^NC, BT-549^miR-8084 cell lines was detected by scratch wound healing assays. b Cell migration was quantified as distance of wound-healed area. Data represent mean ± SD (**P < 0.01). c Movement ability of MDA-MB-231^NC or MDA-MB-231^{miR-8084 inhibitor} cell lines was detected by scratch wound healing assays. d Cell migration was quantified as distance of wound-healed area. Data represent mean ± SD (**P < 0.01). e Representative photographs of migratory or invasive BT-549^NC or BT-549^miR-8084 cells on the membrane (magnification, ×100). f, g Average number of migratory or invasive BT-549^NC or BT-549^miR-8084 cells. (**P < 0.01). h Representative photographs of migratory or invasive MDA-MB-231^NC or MDA-MB231^{miR-8084 inhibitor} cells on the membrane (magnification, ×100). i, j Average number of migratory or invasive MDA-MB-231^NC or MDA-MB231^{miR-8084 inhibitor} cells (*P < 0.05, **P < 0.01). The data represent the mean ± SD

Fig. 3

miR-8084 contributes to tumorigenicity of breast cancer cells in vivo. a–c Photographs of tumors derived for miR-8084 or control stably transfected BT-549 cells in nude mice. d Growth kinetics of tumors in nude mice. Tumor diameters were measured every 5 days (*P < 0.05). e Average weight of tumors in nude mice. Mean ± SD were shown (*P < 0.05)

Fig. 4

miR-8084 targets the 3′-UTR of ING2. a The putative binding sites of miR-8084 in ING2 3′-UTR region were predicted. The matched seed sequences were indicated by vertical lines. b Schematic graph of the putative binding sites of miR-8084 in the ING2 3′UTR and the mutation in miR-8084 binding sites. c miR-8084 mimics down-regulated luciferase activities controlled by wild-type ING2 3′UTR, but did not affect luciferase activity controlled by mutant ING2 3′UTR. The results are means of three independent experiments ± SD (*P < 0.05). d The Cancer Gemone Atlas online database was queried using cBioportal to analysis ING2 gene alteration in human cancers. e The survival curves was performed by Kaplan–Meier plotter (http://kmplot.com/analysis/; probe ID: 213544_at). Totally, 3951 patients were included, 1977 cases were in ING2 low-expression cohort, and 1974 cases were in ING2 high-expression cohort. The median survival for ING2 high-expression cohort is 228.85 months, and 185.16 months for ING2 low-expression cohort. f Survival analysis for breast cancer using data from TCGA. 1091 patients were included, 546 cases were in ING2 low-expression cohort, and 545 cases were in ING2 high-expression cohort. The median survival for ING2 high-expression cohort is 129.6 months, and 120.53 months for ING2 low-expression cohort. 449 and 478 cases are censored in ING2 low-expression and high-expression cohort respectively. g ING2 participates in the regulation of genes involved in cell proliferation, cell cycle, and apoptosis

Fig. 5

miR-8084 enhances the proliferation and epithelial-to-mesenchymal transition. a Protein levels of ING2 in BT-549^NC, BT-549^miR-8084, MDA-MB-231^NC and MDA-MB-231^{miR-8084 inhibitor} cells were detected by western blotting. b Relative protein levels of proliferation related markers (AKT, ERK1/2, PCNA and Ki-67) in BT-549^NC, BT-549^miR-8084, MDA-MB-231^NC and MDA-MB-231^{miR-8084 inhibitor} cells were analyzed by western blotting. c Cell proliferation was measured by the MTS assay. BT-549 cells were transfected with miR-8084 mimics or control, at 24 h post-transfection, the MTS assay was performed every 24 h for 4 days (*P < 0.05). d MDA-MB-231 cells were transfected with miR-8084 inhibitor or control, at 24 h post-transfection, the MTS assay was performed every 24 h for 4 days (*P < 0.05). e EMT-like morphology of BT-549 cells transfected with miR-8084 mimics or control. f EMT-like morphology of MDA-MB-231 cells transfected with miR-8084 inhibitor or control. g Relative protein levels of EMT markers (epithelial cell marker E-cadherin and the mesenchymal cell markers N-cadherin, vimentin and snail) in BT-549^NC, BT-549^miR-8084, MDA-MB-231^NC and MDA-MB-231^{miR-8084 inhibitor} cells were analyzed by western blotting

Fig. 6

miR-8084 inhibited apoptosis by decreasing p53-BAX related pathway. a Representative histograms depicting apoptosis of BT-549 transfected with miR-8084 mimics or control. b The percentage of apoptotic cells of three independent experiments ± SD (**P < 0.01). c Relative levels of apoptosis related markers (p53, BAX and PARP-1) in BT-549^NC or BT-549^miR-8084. d Representative histograms depicting apoptosis of MDA-MB-231 transfected with miR-8084 inhibitor or control. e The percentage of apoptotic cells of three independent experiments ± SD (**P < 0.01). f Relative levels of apoptosis related markers (p53, BAX and PARP-1) in MDA-MB-231^NC or MDA-MB-231^{miR-8084 inhibitor}

Fig. 7

Schematic graph depicting the function of miR-8084 in breast cancer. miR-8084 enhances proliferation and epithelial–mesenchymal transition, meanwhile inhibits apoptosis in breast cancer. The increasing of miR-8084 reinforces suppression of its target ING2, leading to promote the tumorigenicity in breast cancer