MiR-30c suppresses the proliferation, metastasis and polarity reversal of tumor cell clusters by targeting MTDH in invasive micropapillary carcinoma of the breast

Abstract

Purpose: Invasive micropapillary carcinoma (IMPC) of the breast has a high propensity for lymphovascular invasion and axillary lymph node metastasis and displays an 'inside-out' growth pattern, but the molecular mechanism of invasion, metastasis and cell polarity reversal in IMPC is unclear.

Methods: and Patients: Cell growth curves, tumor sphere formation assays, transwell assays, mouse xenograft model and immunofluorescence were evaluated to investigate the effects of miR-30c and MTDH. Dual luciferase reporter assays was performed to confirm that the MTDH (metadherin) 3'UTR bound to miR-30c. MiRNA in situ hybridization (ISH) and immunohistochemistry (IHC) were carried out on IMPC patient tissues for miR-30c and MTDH expression, respectively.

Results: We found miR-30c as a tumor suppressor gene in cell proliferation, metastasis and polarity reversal of IMPC. Overexpression of miR-30c inhibited cell growth and metastasis in vitro and in vivo. MiR-30c could directly target the MTDH 3'UTR. MiR-30c overexpression inhibited breast cancer cell proliferation, invasion and metastasis by targeting MTDH. Moreover, miR-30c/MTDH axis could also regulate cell polarity reversal of IMPC. By ISH and IHC analyses, miR-30c and MTDH were significantly correlated with tumor size, lymph nodule status and tumor grade, the 'inside-out' growth pattern, overall survival (OS) and disease-free survival (DFS) in IMPC patients.

Conclusions: Overall, miR-30c/MTDH axis was responsible for tumor proliferation, metastasis and polarity reversal. It may provide promising therapeutic targets and prognostic biomarkers for patients with IMPC.

Keywords: Breast cancer; Invasive micropapillary carcinoma (IMPC); Metastasis; MiRNA; Polarity reversal; Proliferation.

Fig. 1

MiR-30c overexpression inhibits the cell proliferation, migration and invasion in vitro. (A) Relative miR-30c expression levels in the normal breast cell line and six breast cancer cell lines were examined by quantitative real-time PCR (qPCR) assay, while U6 was used as an endogenous control for miRNA expression analysis. (B) The overexpression of miR-30c in MCF-7, T47D and MDA-MB-231 cells were validated by qPCR. (C)The down expression of miR-30c in MDA-MB-231and T47D cells was validated by qPCR. (D) Growth curves are presented after MCF-7 and T47D were transfected with miR-30c mimics or negative control. Cell growth was quantified and normalized to that at day 1. (E) Growth curves are presented after MDA-MB-231and T47D cells were transfected with miR-30c inhibitors or negative control. The growth of cells was quantified and normalized to that at day 1. (F) Transwell assays were performed to examine the migration and invasion abilities of MCF-7 and T47D cells transfected with miR-30c mimics or negative control. The relative migration and invasion abilities of MCF-7 and T47D cells were analyzed quantitatively. (G) Transwell assays were performed to examine the migration and invasion ability of MDA-MB-231 cells and T47D cells transfected with miR-30c inhibitors or negative control. The relative migration abilities of MDA-MB-231 cells and T47D cells were analyzed quantitatively. Columns, average of at least three biological repeats; bars represent s. d.; *p < 0.05; **p < 0.01.

Fig. 2

MiR-30c inhibits tumorigenesis and metastasis in vivo. (A) MCF-7 cells transfected with lenti-miR-30c or lenti-empty were inoculated subcutaneously into the flanks of BALB/c female mice (n = 12), as described in the Materials and Methods. (B) Sixty days after injection, the mice were sacrificed, necropsies were performed, and the tumors were weighed. (C) The size of tumors in the two groups was measured and calculated and compared every week. (D, E) Histological examination of the tumor xenografts. Immunohistochemical analysis revealed Ki67 expression in xenografts (magnification at 400 × ). (F) MDA-MB-231 cells with lenti-miR-30c or lenti-empty were injected into the tail veins of nude mice (n = 7 per group). Four weeks after injection, mice were sacrificed. The numbers of pulmonary metastatic nodules in the lung were counted and compared with Student's t-test. Representative images of lung metastasis in each group (left). The micrometastases of the two groups in the lung per H&E stained section from individual mice were present (right). (G) The numbers of pulmonary metastatic nodules in each group were calculated and compared. (H) IHC analysis shows the expression of MTDH, E-cadherin, N-cadherin, Vimentin in xenografts (magnification at 400 × ). Columns, average of at least three biological repeats; bars represent s. d.; *p < 0.05; **p < 0.01.

Fig. 3

MiR-30c inhibits MTDH expression by directly targeting its 3′UTR. (A) MiR-30c target genes were predicted by TargetScan, PicTar and DIANA algorithms. The overlapping genes were MTDH, LHX8, and EED. (B) The alignment of miR-30c target sequences in the MTDH 3′UTR from five mammals. The evolutionarily conserved nucleotides are highlighted in red. (C) Two predicted miR-30c target sites reside at nucleotides 287–294 and 1569–1576 of the MTDH 3′UTR; Wt: wild type, Mut: mutant. (D) Relative luciferase activity was analyzed after the Wt or Mut reporter plasmids were cotransfected into HEK293 cells with miR-30c mimics or negative control. (E) Western blot analysis of MTDH protein levels in MCF-7, T47D, MDA-MB-231 cells transfected with miR-30c mimics (20 μM, 5 μl) or negative control. Relative expression level was defined as the ratio between the gray value of MTDH and β-actin. (F) Relative MTDH mRNA levels were determined by quantitative real-time PCR (qPCR). GAPDH was used as the endogenous control for MTDH mRNA level analysis. Columns, average of at least three biological repeats; bars, s. d.; *p < 0.05; **p < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

MiR-30c inhibits breast cancer cell proliferation, migration and invasion by targeting MTDH. (A) Western blotting assay of MTDH are presented after MCF-7 and T47D cells were transfected with miR-30c mimics or negative control together with either LV6-MTDH or LV6-vector. β-actin was used as the endogenous control. (B) Growth curves are presented after MCF-7 and T47D cells were transfected with miR-30c mimics or negative control together with either LV6-MTDH or LV6-vector. Cell growth was quantified and normalized to that at day 1. (C, D) Transwell assays were performed to examine the migration and invasion abilities of MCF-7 and T47D cells transfected with miR-30c mimics or negative control together with either LV6-MTDH or LV6-vector. The relative migration and invasion abilities of MCF-7 and T47D cells were analyzed quantitatively. Columns, average of at least three biological repeats; bars, s. d. *p < 0.05; **p < 0.01.

Fig. 5

MiR-30c regulates the polarity reversal of tumor cell clusters in IMPC by targeting MTDH. (A) The overexpression of miR-30c in IMPC primary cells was validated by qPCR. (B) Western blot analysis of MTDH protein levels in IMPC primary cells transfected with miR-30c mimics (20 μM, 5 μl) or negative control. Relative expression level was defined as the ratio between the gray value of MTDH and β-actin. (C) Immunofluorescence image of the inside-out staining pattern of MUC1 in LV6-vector and LV6-MTDH IMPC primary cells transduced by negative control or miR-30c mimics. (D) The spheroids of the inside-out staining pattern of MUC1 were analyzed quantitatively in LV6-vector and LV6-MTDH IMPC primary cells transduced by negative control or miR-30c mimics. Columns, average of at least three biological repeats; bars represent s. d.; *p < 0.05; **p < 0.01.

Fig. 6

MiR-30c/MTDH is correlated with the clinicopathologic characteristics and prognosis of IMPC. The miR-30c expression levels were analyzed in IMPC and IDC-NST tissues by in situ hybridization (ISH). MTDH and MUC1 expression levels were analyzed in IMPC and IDC-NST tissues by immunohistochemistry (IHC). (A, B) Representative images of miR-30c, MTDH and MUC1 expression in IMPC and IDC-NST tissues. (C) QPCR analysis shows the expression levels of miR-30c in 12 pairs of tissues. Expression for the gene was normalized to U6 expression. *p < 0.05. (D, E) The overall survival and disease-free survival of patients with high or low miR-30c expression in IMPC patients. (F, G) The overall survival and disease-free survival of patients with high or low MTDH expression in IMPC patients. (H) A schematic showing how the signaling pathway of miR-30c regulates tumor cell invasion, metastasis and polarity reversal in invasive micropapillary carcinoma of the breast.

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