To investigate the role of metadherin (MTDH) in regulating stem cell characteristics of triple negative breast cancer and its mechanism and explore the regulation of MTDH by miR-30a-5p.

(1) The tumor tissue specimens of 37 triple-negative breast cancer (TNBC) patients who underwent surgery and 37 non-TNBC tissue specimens in Taihe Hospital Affiliated to Hubei Medical College from May 2017 to September 2018 were collected for a retrospective study. Immunohistochemistry was used to detect the expression of MTDH in all specimens. (2) The mRNA and protein expression of MTDH in four cell lines (MCF-10A, MCF-7, MDA-MB-231 and MDA-MB-468) by quantitative RT-PCR and Western blot. (3)To explore the effect of MTDH on the stemness and proliferation of TNBC cells and the Wnt/ β-catenin signaling pathway, TNBC cell lines MDA-MB-231 and MDA-MB-468 were transfected with MTDH siRNA (siMTDH group), and the cells transfected with invalid interfering RNA served as the control group. The relative expression of stem cell markers sox2, Nanog and CD133 was determined in MDA-MB-231 and MDA-MB-468 cell lines by Western blot. Cell cloning experiments were used to observe the number of cell clones and Western blot was used to detect the expression of the Wnt/β-catenin signaling pathway proteins (β-catenin, Myc and cyclin D1). (4)To investigate the targeted regulation of MTDH by miRNA-30a-5p, the luciferase reporter assay, 3′-UTR of MTDH and miRNA-30a-5p were used to synthesize wild-type and mutant luciferase reporter plasmids containing 3′-UTR of MTDH, and then the HepG2 cells were transfected with wild-type and mutant plasmids, respectively. The HepG2 cells transfected with pGL3 empty vector served as a blank control group. The fluorescence of cells was observed 24 h later. The protein expression of MTDH was compared between TNBC tissues and non-TNBC tissues using t test. The mRNA and protein expression of MTDH was compared among four cell lines using analysis of variance. The protein expression of sox2, Nanog, CD133, β-catenin, Myc, cyclin D1 and the number of cell clones were compared between the control group and the siMTDH group using analysis of variance. The relative fluorescence ratio of cells was compared using analysis of variance. The LSD method was used for pairwise comparison.

The relative expression of MTDH was 2.82±1.37 in TNBC tissue, and 5.65±2.02 in non-TNBC tissue, indicating a significant difference (t=-7.046, P<0.001). MTDH mRNA expression presented a significant difference among the four cell lines (MCF-7, MDA-MB-231, MDA-MB-468 and MCF-10A) (F=7 155.320, P<0.001). Pairwise comparison showed that compared with normal breast cells (MCF-10A), the expression of MTDH mRNA in breast cancer cells (MCF-7, MDA-MB-231, MDA-MB-468) was significantly increased (all P<0.001); compared with MCF-7, the expression of MTDH mRNA in TNBC cell lines MDA-MB-231 and MDA-MB-468 was significantly higher (both P<0.001); the expression of MTDH mRNA in MDA-MB-468 cells was significantly lower than that in MDA-MB-231 cells (P<0.001). Among the above-mentioned four cell lines, the protein expression of MTDH presented a significant difference (F=90.053, P<0.001). Pairwise comparison showed that compared with normal breast cells (MCF-10A), the protein expression of MTDH in breast cancer cells (MCF-7, MDA-MB-231, MDA-MB-468) was significantly increased (all P<0.050); compared with MCF-7, the protein expression of MTDH in TNBC cell lines MDA-MB-231 and MDA-MB-468 was significantly higher (both P<0.050); the protein expression of MTDH in MDA-MB-468 cells was significantly lower than that in MDA-MB-231 cells (P<0.050). In the TNBC cell lines MDA-MB-231 and MDA-MB-468, the relative expression of stem cell markers sox2, Nanog and CD133 showed a significant difference between the control group and the siMTDH group (MDA-MB-231: 1.19±0.10 vs 0.52±0.05, 1.16±0.13 vs 0.34±0.03, 1.19±0.06 vs 0.54±0.08, t=10.304, 10.959, 11.700, P=0.001, 0.005, P<0.001; MDA-MB-468: 1.26±0.05 vs 0.34±0.02, 1.19±0.07 vs 0.52±0.04, 1.21±0.02 vs 0.37±0.01, t=31.185, 15.584, 75.001, all P<0.001). The number of cell clones showed a significant difference between the control group and the siMTDH group (MDA-MB-231: 87.33±9.02 vs 33.33±3.51, t=9.664, P=0.001; MDA-MB-468: 70.67±4.73 vs 24.33±3.21, t=14.041, P<0.001). The expression of β-catenin, Myc and cyclin D1 also showed a significant difference between the control group and the siMTDH group (MDA-MB-231: 0.26±0.06 vs 0.08±0.01, 0.74±0.03 vs 0.32±0.00, 0.72±0.01 vs 0.26±0.04, t=5.115, 21.222, 21.690, all P<0.050; MDA-MB-468: 0.33±0.03 vs 0.15±0.06, 0.56±0.04 vs 0.22±0.02, 0.65±0.02 vs 0.31±0.02, t=4.973, 13.969, 21.897, all P<0.001). The results of the luciferase reporter assay showed that there was a significant difference in relative fluorescence ratios among the three groups (F=174.189, P<0.001); the relative fluorescence ratio in the empty vector group and the mutant plasmid transfection group were significantly higher than that in the wild-type plasmid transfection group (both P<0.001).

With the targeted regulation on MTDH, miR-30a-5p can be involved in regulating the stemness of TNBC. Therefore, miR-30a-5p/MTDH pathway may be a new therapeutic target.