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. 2019 Sep 27;12:7993-8002.
doi: 10.2147/OTT.S206180. eCollection 2019.

MicroRNA-155-3p Promotes Breast Cancer Progression Through Down-Regulating CADM1

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Free PMC article

MicroRNA-155-3p Promotes Breast Cancer Progression Through Down-Regulating CADM1

Guochao Zhang et al. Onco Targets Ther. .
Free PMC article

Abstract

Background/purpose: Cell adhesion molecule 1 (CADM1) functions as a tumor suppressor and has been identified to be frequently inactivated in breast cancer, and closely associated with patients' poor prognosis and advanced TNM stage. However, the mechanisms underlying CADM1 in breast cancer progression remains incompletely clear. miR-155, a predicted modulator of CADM1 was reported to be overexpressed in breast cancer, and its high expression level was closely related to the malignant progression of breast cancer. The present study aimed to explore whether miR-155-3p could modulate CADM1 expression and then involved in the progression of breast cancer.

Methods: The expression patterns of miR-155-3p in breast cancer tissues and cell lines were determined by RT-PCR technology. The relationship between CADM1 and miR-155-3p were determined by the luciferase gene reporter and Western Blot (WB) assays. Cell proliferation, apoptosis rates and tumorigenesis were determined by CCK-8, flow cytometry and in vivo xenotransplanation experiments, respectively.

Results: miR-155-3p was up-regulated in breast cancer tissues and cells when compared to the adjacent normal tissues and normal breast MCF 10A cells. The mRNA and protein levels of CADM1 showed opposite expression patterns to that of miR-155-3p expression detected, and miR-155-3p could negatively regulate CADM1 expression in breast cancer MCF-7 cells. Moreover, gain-of function assay showed that overexpression of miR-155-3p promoted cell proliferation, tumorigenesis and repressed cell apoptosis, but these effects were all significantly impaired when the cells were simultaneously transfected with OE-CADM1, the overexpressing vector of CADM1.

Conclusion: This study revealed that miR-155-3p could accelerate the progression of breast cancer via down-regulation of CADM1 expression.

Keywords: CADM1; breast cancer; miR-155-3p.

Conflict of interest statement

The authors have declared that no competing interest exists in this work.

Figures

Figure 1
Figure 1
miR-155-3p expression was elevated in breast cancer tissues and cells. (A) RT-PCR was performed to evaluate miR-155-3p expression in 40 matched breast cancer tissues and its adjacent normal tissues (n=40; P<0.05, tumor group vs control group). (B) RT-PCR analysis of the mRNA level of miR-155-3p in normal breast cell line MCF 10A and breast cancer HCC1973, MCF-7 and MDA-MB-231 cell lines (n=3; ***P<0.001, HCC1973/MCF-7/MDA-MB-231 group vs MCF 10A group). (C) OS was used to evaluate the association between miR-155-3p expression levels with breast cancer patients’ prognosis.
Figure 2
Figure 2
Up-regulation of miR-155-3p promoted cell proliferation and inhibited cell apoptosis in breast cancer MCF-7 cells. (A) RT-PCR analysis of the transfected efficiencies after MCF-7 cells were transfected with miR-155-3p mimic, inhibitor or their controls. (B) CCK-8 analysis the proliferation of MCF-78 cells after the cells were transfected with miR-155-3p mimic, inhibitor or their controls. (C–D) Flow cytometry with Annexin V (FITC)/PI staining used to determine cell apoptosis after MCF-7 cells were treated with miR-155-3p mimic, inhibitor or their controls (right lower quadrant represent early apoptosis cells and right upper represent late apoptosis cells). (n=3, **P<0.01, ***P<0.001, mimic group vs mimic-NC group; #P<0.05, ###P<0.001, inhibitor group vs inhibitor-NC group).
Figure 3
Figure 3
miR-155-3p down-regulated CADM1 expression in breast cancer cells. (AB) RT-PCR and WB analysis of the mRNA and protein levels of CADM1 in 30 paired breast cancer tissues and adjacent non-tumor tissues, and the repressive WB figure was shown in 3B (n=30; *P<0.05, **P<0.01, tumor group vs control group). (C–D) RT-PCR and WB analysis of the mRNA and protein levels of CADM1 in breast cancer HCC1973, MCF-7 and MDA-MB-231 cell lines (n=3; **P<0.01, ***P<0.001, HCC1973/MCF-7/MDA-MB-231 group vs MCF 10A group). (E–F) RT-PCR and WB analysis of the mRNA and protein levels of CADM1 after MCF-7 cells were transfected with miR-155-3p mimics, inhibitors or their controls (n=3, ***P<0.001, mimics group vs mimic-NC group; ###P<0.001, inhibitors group vs inhibitor-NC group). (G) The luciferase activity of CADM1 promoter after transfection in MCF-7 cells with miR-155-3p MT (mutation) or WT (wild type) (n=3, *P<0.05, mimics groups vs mimic-NC group; #P<0.05, inhibitors group vs inhibitor-NC group).
Figure 4
Figure 4
Detection of the effects of miR-155-3p/CADM1 on the proliferation and apoptosis of MCF-7 cells. (A–B) The transfection efficiency of siRNAs and over-expressing plasmid targeting to human CADM1 gene was detected by RT-PCR and WB (n=3, *P<0.05, ***P<0.001, si-1/si-2 group vs si-NC group; ##P<0.01, P<0.001, OE-CADM1 group vs OE-NC group). (C) CCK-8 was used to assess cell proliferation after MCF-7 cells were transfected with inhibitor-NC + si-NC, inhibitors + si-NC, inhibitor-NC + si-CADM1 and inhibitors + si-CADM1, respectively (n=3, *P<0.05, ***P<0.001, inhibitors + si-NC group or inhibitor-NC + si-CADM1group vs inhibitor-NC + si-NC group; ##P<0.01, inhibitor + si-CADM1 group vs inhibitors + si-NC group). (D) CCK-8 was used to assess cell proliferation after MCF-7 cells were transfected with mimic-NC + OE-NC, mimics + OE-NC, mimic-NC + OE-CADM1 and mimics + OE-CADM1 (n=3, *P<0.05, ***P<0.001, mimics + OE-NC group or mimic-NC + OE-CADM1 group vs mimic-NC + OE-NC group; ##P<0.01, mimics + OE-CADM1 group vs mimics + OE-NC group). (E) Flow cytometry was used to assess cell apoptosis after MCF-7 cells were transfected with inhibitor-NC + si-NC, inhibitors + si-NC, inhibitor-NC + si-CADM1 and inhibitors + si-CADM1, respectively (n=3, *P<0.05, inhibitors + si-NC group or inhibitor-NC + si-CADM1group vs inhibitor-NC + si-NC group; #P<0.05, inhibitor + si-CADM1 group vs inhibitors + si-NC group). (F) Flow cytometry was used to assess cell apoptosis after MCF-7 cells were transfected with mimic-NC + OE-NC, mimics + OE-NC, mimic-NC + OE-CADM1 and mimics + OE-CADM1 (n=3, **P<0.01, mimics + OE-NC group or mimic-NC + OE-CADM1 group vs mimic-NC + OE-NC group; ##P<0.01, mimics + OE-CADM1 group vs mimics + OE-NC group). (G–J) WB technology was applied to detect the expressions of cleaved caspase3, total caspase3, Bcl-2 and Bax after cells were transfected with different vectors (n=3, *P<0.05, #P<0.05).
Figure 5
Figure 5
In vivo xenograft assay analysis of the tumor-forming potential. MCF-7 cells were stably transfected control, miR-155-3p mimics, miR-155-3p mimics + OE-CADM1, miR-155-3p inhibitors and miR-155-3p inhibitors + sh-CADM1, then the cells were subcutaneously injected into the flanks of mice (n=10/group). (**P<0.01, ***P<0.001, mimic/inhibitor group vs control group; ##P<0.01, mimics+CADM1 group vs mimics group; ++P<0.01, inhibitors + sh-CADM1 group vs inhibitors group).

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