Silver nanoparticles achieve cytotoxicity against breast cancer by regulating long-chain noncoding RNA XLOC_006390-mediated pathway

Abstract

The specific cytotoxic effect of nanoparticles on tumor cells may be used in future antitumor clinical applications. Silver nanoparticles (AgNPs) have been reported to have potent cytotoxic effect, but the mechanism is unclear. Here, AgNPs were synthesized, and the particle average size was 63.1 ± 8.3 nm and showed a nearly circular shape, which were determined by transmission electron microscopy and field emission scanning electron microscopy. The selected area electron diffraction patterns showed that the nanoparticles were crystalline. The energy-dispersive X-ray spectrum proved that silver is the main component of nanoparticles. The AgNPs showed potent cytotoxicity in breast cancer cells, no matter whether they were tamoxifen sensitive or resistant. Next, we found that a long noncoding RNA, XLOC_006390, was decreased in AgNPs-treated breast cancer cells, coupled to inhibited cell proliferation, altered cell cycle and apoptotic phenotype. Downstream of AgNPs, XLOC_006390 was recognized to target miR-338-3p and modulate the SOX4 expression. This signaling pathway also mediates the AgNPs function of sensitizing tamoxifen-resistant breast cancer cells to tamoxifen. These results provide a new clue for the antitumor mechanism of AgNPs, and a new way for drug development by using AgNPs.

Keywords: breast cancer; cytotoxicity; lncRNA; microarray; silver nanoparticles.

Figure 1

Identification of AgNPs. (A) TEM images of synthesized AgNPs. Up: scale is 200 nm; middle: scale is 100 nm; down: scale is 20 nm. (B) The selected area electron diffraction (SAED) patterns of synthesized AgNPs. (C) Field emission scanning electron microscopy image of synthesized AgNPs. (D) EDS pattern of synthesized AgNPs.

Figure 2

Cytotoxic effects of AgNPs against different cancer cells in vitro. (A) Five breast cancer cells were treated with AgNPs at concentration ranges from 50 to 200 μg/ml for 24 h, and then the percent cell survival (%) were determined in comparing with the control cells without AgNPs treatment. (B) Patients-derived breast cancer cells and the adjacent normal cells were treated with AgNPs at concentration ranges from 50 to 200 μg/ml for 24 h, and then the percent cell survival (%) were determined in comparing with the control cells without AgNPs treatment. (C) Comparing the percent cell survival (%) described in (B) between tamoxifen resistant and sensitive breast cancer cells. (D) Comparing the percent cell survival (%) described in (B) between metastatic and in situ breast cancer cells. (E and F) MCF7 were treated with AgNPs at 150 μg/ml or not for 24 h, and the (E) cell cycle analysis and (F) apoptosis assay were performed. (G) MCF7-Re cells (tamoxifen-resistant breast cancer cells) were treated with 4-hydroxytamoxifen (TAM) at 10 μM or not, together with AgNPs at concentration ranges from 50 to 200 μg/ml or not for 24 h, and then the percent cell survival (%) were determined. (H) Determination of the mRNA expressions of the targets involved in the cell cycles (P21 and cyclin D1), apoptosis progressions (Bax and BCL-2) and DNA damages (γH2AX) in the cells described in (E and F) by qRT-PCR assay.

Figure 3

Microarray analyses reveal a role of AgNPs in lncRNAs expressions. (A) MCF7 cells were treated with AgNPs at 150 μg/ml for 24 h, and then the total RNA was then collected for microarray analyses. The differentially expressed lncRNAs between AgNPs treatment group and control group were clustered by heatmap (P < 0.001 included). (B) Fold change of the differentially expressed lncRNAs between AgNPs treatment group and control group described in (A). (C) Confirmation of XLOC_006390 expression in five breast cancer cell lines after 150 μg/ml AgNPs treatment for 24 h by qRT-PCR analysis. (D) Confirmation of XLOC_006390 expression in patients-derived breast cancer cells after 150 μg/ml AgNPs treatment for 24 h by qRT-PCR analysis. (E) Confirmation of XLOC_006390 expression in tamoxifen resistant and sensitive breast cancer cells described in (D). (F) Confirmation of XLOC_006390 expression in metastatic and in situ breast cancer cells described in (D).

Figure 4

AgNPs-regulated miR-338-3p/SOX4 axis through decreasing the XLOC_006390 expressions in breast cancer cells. (A and B) The MCF7 cells were transfected with or without XLOC_006390 overexpressing plasmid (XLOC_006390 OV), together with 150 μg/ml AgNPs treatment for 24 h, and then the (A) XLOC_006390 and (B) miR-338-3p expressions were determined by qRT-PCR assay. (C and D) Luciferase reporter assay demonstrated that SOX4 is a direct target of 338-3p. (C) Sequences of the binding sites between miR-338-3p and 3′UTR of SOX4. (D) Bars show the ratio of Renilla activity/Firefly activity in HEK293 cells 24 h after cotransfection of luciferase vectors containing wild-type or mutant 3′UTR of SOX4 and miR-338-3p mimics, inhibitor or mimics negative control, respectively. (E and F) The (E) MCF7 cells and (F) MCF7-Re cells were transfected with or without XLOC_006390 OV plasmid, or with or without miR-338-3p inhibitor, together with 150 μg/ml AgNPs treatment for 24 h, and then the SOX4 mRNA expressions were determined by qRT-PCR assay.

Figure 5

XLOC_006390/miR-338-3p/SOX4 axis-mediated cytotoxicity of AgNPs and its function of sensitizing tamoxifen-resistant breast cancer cells to tamoxifen. (A) MCF7 cells were transfected with or without XLOC_006390 OV plasmid, or with or without miR-338-3p inhibitor, together with 150 μg/ml AgNPs treatment for 24 h, and then the mRNA expressions of the targets involved in the cell cycles (P21 and cyclin D1), apoptosis progressions (Bax and BCL-2) and DNA damages (γH2AX) were determined by qRT-PCR assay. (B) The MCF7 cells were transfected with or without XLOC_006390 OV plasmid, or with or without miR-338-3p inhibitor, and then were treated with AgNPs at concentration ranges from 50 to 200 μg/ml for 24 h, and then the percent cell survival (%) were determined in comparing with the control cells without AgNPs treatment. (C and D) The (C) cell cycles and (D) apoptosis rate in cells described in (A) were determined. (E) MCF7-Re cells (tamoxifen-resistant breast cancer cells) were transfected with or without XLOC_006390 OV plasmid, or with or without miR-338-3p inhibitor, and then treated with 4-hydroxytamoxifen (TAM) at 10 μM or not, together with AgNPs at concentration ranges from 50 to 200 μg/ml or not for 24 h, and then the percent cell survival (%) were determined.