MicroRNA-320a inhibits breast cancer metastasis by targeting metadherin

Dysregulated microRNAs play important pathological roles in carcinogenesis that are yet to be fully elucidated. This study was performed to investigate the biological functions of microRNA-320a (miR-320a) in breast cancer and the underlying mechanisms. Function analyses for cell proliferation, cell cycle, and cell invasion/migration, were conducted after miR-320a silencing and overexpression. The specific target genes of miR-320a were predicted by TargetScan algorithm and then determined by dual luciferase reporter assay and rescue experiment. The relationship between miR-320a and its target genes was explored in human breast cancer tissues. We found that miR-320a overexpression could inhibit breast cancer invasion and migration abilities in vitro, while miR-320a silencing could enhance that. In addition, miR-320a could suppress activity of 3′-untranslated region luciferase of metadherin (MTDH), a potent oncogene. The rescue experiment revealed that MTDH was a functional target of miR-320a. Moreover, we found that MTDH was negatively correlated with miR-320a expression, and it was related to clinical outcomes of breast cancer. Further xenograft experiment also showed that miR-320a could inhibit breast cancer metastasis in vivo. Our findings clearly demonstrate that miR-320a suppresses breast cancer metastasis by directly inhibiting MTDH expression. The present study provides a new insight into anti-oncogenic roles of miR-320a and suggests that miR-320a/MTDH pathway is a putative therapeutic target in breast cancer.


Quantitative real-time PCR (qRT-PCR)
Total RNAs were extracted from cells or tissues with Trizol reagent (Invitrogen, Carlsbad, CA USA). MiR-320a expression was determined by qRT-PCR using TaqMan miRNA Assay (Invitrogen), with RNU6B as an internal control. Target mRNA expression was determined by qRT-PCR with SYBR® Premix Ex Taq™ (Takara Bio, Dalian, China) with GAPDH as an internal reference. The primer sequences are listed in Supplementary Table S1. All reactions were run in ABI 7900 Fast Real Time PCR system. The Taqman miRNA assay thermal cycle condition was that one cycle at 95°C for 30 sec, followed by 40 cycles of amplification at 95°C for 5 sec, and then 60°C for 30 sec. Relative expression level was calculated by the 2 −ΔΔCT method [ΔCt = Ct (miR-320a) -Ct (GAPDH/RNU6B), ΔΔCt = ΔCt (cancer)-ΔCt (normal)], where Ct value represented the threshold cycle for each transcript.

Cell proliferation assay
Breast cancer cells were transfected with RNA oligonucleotide. Twenty-four hours after transfection, cells were seeded at a density of 5000-8000 cells per well in 96-well plates for cell proliferation assay. Cell growth was measured by the Cell Counting Kit-8 assay (Dojindo, Kumamoto, Japan) in accordance with the manufacturer's instructions. Absorbance values were tested at 450 nm on a spectrophotometer after incubating 2.5 hours.

Cell invasion and migration assay
Invasion and migration assays were performed using 24-well Transwell permeable supports with 8-μm pores (Corning Incorporated, Corning, NY USA). 1 × 10 5 or 5 × 10 4 cells in serum-free media were seeded into the upper chamber with or without Matrigel (BD Biosciences, San Jose, CA USA) and the bottom chamber was filled with complete media as chemo-attractant. After several hours of incubation, cells that had invaded or migrated through the membrane were stained with crystal violet, imaged and counted by Vectra Automated Quantitative Pathology Imaging System (PerkinElmer, Waltham, MA USA).

Immunohistochemistry
Immunohistochemical staining was performed on 5-μm FFPE sections. Briefly, slides were deparaffinized, rehydrated and treated with 0.3% H 2 O 2 , followed by antigen retrieval and staining according to standard guidelines. Tumor cells were considered positive for MTDH when they showed cytoplasm immunoreactivity. The MTDH staining pattern was examined and scored by two independent pathologists. The scores were determined by combining the proportion of positive staining and the staining intensity over 10 visual fields a by a microscope (400×, ZEISS, Oberkochen, Germany). The staining index (SI) was calculated as: SI = staining intensity x proportion of positively stained tumor cells. Analysis in detail was described previously. Cutoff value to define the high-and low-expression of MTDH was chosen by a measurement of heterogeneity with the logrank test statistic with respect to overall survival, and a score of 4 was chosen. Therefore, SI score ≥ 4 was taken to define tumors as high expression of MTDH and SI < 4 defined tumors as low expression.

MiR-320a is not involved in cell proliferation and cell cycle in breast cancer
MDA-MB-231 cells, in which miR-320a expression was relatively lower, were transfected with pre-miR-320a or pre-NC. T-47D cells with moderately higher expression of miR-320a were transfected with anti-miR-320a of anti-NC. Transfection with pre-miR-320a or anti-mir-320a led to significant increase or decrease in miR-320a expression when compared to their cognate NC as described before (Figure 2A). As is shown in Supplementary Figure S2, the cell proliferation and cell cycle analysis revealed that overexpression of miR-320a could not suppress cell proliferation or alter cell cycle distribution in MDA-MB-231 cells; meanwhile, suppression of miR-320a could not affect cell growth and cell cycle in T-47D cells. We consider miR-320a is not involved in breast cancer cell proliferation and cell cycle.