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. 2020 Oct;44(4):1479-1488.
doi: 10.3892/or.2020.7731. Epub 2020 Aug 12.

MicroRNA‑188‑5p inhibits the progression of breast cancer by targeting zinc finger protein 91

Zhiguang Yang  1 , Zhaoyu Liu  1 , Zhihui Chang  1 , Guangxin Yan  1
Affiliations

MicroRNA‑188‑5p inhibits the progression of breast cancer by targeting zinc finger protein 91

Zhiguang Yang et al. Oncol Rep. 2020 Oct.

Abstract

Breast cancer (BC) is the most commonly diagnosed malignant cancer in women. BC is the main cause of cancer‑related death in women and seriously threatens the life and health of women worldwide. MicroRNAs (miRNAs/miRs) have been reported to regulate the development and progression of different types of cancer. However, the regulatory functions of miR‑188‑5p in BC have not been thoroughly demonstrated. In this present research, we identified that miR‑188‑5p was downregulated in BC tissues and several BC cell lines. Downregulation of miR‑188‑5p was significantly associated with advanced TNM stage. Moreover, we identified that miR‑188‑5p mimics significantly inhibited proliferation using CCK‑8 assay, colony formation and xenograft animal model, suppressed invasion and migration detected by Transwell invasion assay, and increased the cellular apoptosis of BC cells as determined by cell apoptosis assay. Moreover miR‑188‑5p mimics also reduced the expression of NF‑κB p65(Rel). To further investigate its regulatory mechanism, transcription factor zinc finger protein 91 (ZFP91) was predicted as the targeted protein of miR‑188‑5p by bioinformatic method. We confirmed their specific binding by dual luciferase (DLR) assay. We demonstrated that the overexpression of miR‑188‑5p significantly inhibited the expression of ZFP91 in BC cell lines and reduced the expression of NF‑κB p65(Rel). An inverse correlation was found between the expression of miR‑188‑5p and ZFP91 in BC tissues. Importantly, we demonstrated that the restoration of ZFP91 was able to block the effect of miR‑188‑5p on the progression of MDA‑MB‑231 cells. Therefore, our study showed that miR‑188‑5p may be one of the important indicators and could inhibit the progression of human BC via targeting the ZFP91/NF‑κB p65(Rel) signaling pathway, suggesting that miR‑188‑5p may be a promising future target for BC treatment.

Keywords: breast cancer; miR-188-5p; zinc finger protein 91; ZFP91; proliferation; apoptosis.

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Figures

Figure 1.
Figure 1.
miR-188-5p is downregulated in BC tissue and BC cell lines. (A) RT-qPCR results showed that the level of miR-188-5p in BC tissues was downregulated compared with counterpart BC tissues. The comparisons between groups were evaluated by Student's t-test. (B) Relative expression level of miR-188-5p in patients at different clinical stages. (C) Relative expression level of miR-188-5p in BC cell lines MDA-MB-231, BT-549 and MCF-7 relative to the normal human breast epithelial cell line MCF-10A. One-way ANOVA followed by Tukey's test was used to evaluate the difference for multiple comparisons in B and C. All data are presented as means ± SD (n=3). BC, breast cancer.
Figure 2.
Figure 2.
Effect of miR-188-5p on BC cell line MDA-MB-231. MDA-MB-231 cells were transfected with miR-188-5p mimics and miR-NC. (A) The mRNA levels of miR-188-5p in MDA-MB-231 cells were determined by RT-qPCR. *P<0.05. The proliferation of MDA-MB-231 cells was examined by (B) CCK-8 Kit post transfection and (C) colony formation. *P<0.05, compared to control group; #P<0.05, compared to miR-NC group. (D) The apoptotic MDA-MB-231 cells were analyzed using Annexin V/PI staining and FACs. *P<0.05. (E) The invasion and migration capability of MDA-MB-231 cells were detected by Transwell assay. *P<0.05. (F) Expression of E-cadherin, N-cadherin, vimentin, MMP2, MMP9 and NF-κB p65(Rel) were detected by western blotting. The data are represented as the mean ± SD (n=3). *P<0.05. One-way ANOVA followed by Tukey's test was used to evaluate the difference for multiple comparisons. BC, breast cancer; MMP, matrix metalloproteinase; NC, negative control.
Figure 3.
Figure 3.
ZFP91 is a target of miR-188-5p. (A) ZFP91 provides binding sites with miR-188-5p. (B) RT-qPCR and western blotting were used for analysis of transcription and translation of ZFP91 in vitro and in vivo. *P<0.05. (C) The binding between miR-188-5p and ZFP91 was confirmed by luciferase assay. *P<0.05. Relative gene expression was normalized by GAPDH expression. The data are represented as the mean ± SD (n=3). One-way ANOVA followed by Tukey's test was used to evaluate the difference for multiple comparisons in B. The comparisons between two groups were evaluated by t-test in C. ZFP91, zinc finger protein 91.
Figure 4.
Figure 4.
MDA-MB-231 cell proliferation and apoptosis are regulated by miR-188-5p/ZFP91. pCMV-Tag2B vector (PC) was transfected into BC MDA-MB-231 cells with miR-NC (NC+PC) or miR-188-5p mimics (PC+miR-188-5p); pCMV-Tag2B-ZFP91 was transfected into MDA-MB-231 cells with miR-188-5p mimics (miR-188-5p+ZFP91). (A) The mRNA levels of miR-188-5p in the co-transfected overexpressing ZFP91/MDA-MB-231 cells were quantified by RT-qPCR. *P<0.05. Cell proliferation was measured by (B) the CCK-8 Kit and (C) colony formation assay. *P<0.05, compared to control group; #P<0.05, compared to miR-NC group. (D) Cell apoptosis was detected by Annexin V/PI staining and FACs. *P<0.05. (E) The invasion and migration capability of MDA-MB-231 cells were detected by Transwell assay. *P<0.05. (F) Expression of E-cadherin, N-cadherin, Vimentin, MMP2, MMP9 and NF-κB p65(Rel) were detected by western blotting. Relative genes expression was normalized by GAPDH expression. The data are represented as the mean ± SD (n=3). *P<0.05. One-way ANOVA followed by Tukey's test was used to evaluate the difference for multiple comparisons. BC, breast cancer; ZFP91, zinc finger protein 91; MMP, matrix metalloproteinase; NC, negative control.
Figure 5.
Figure 5.
mRNA and protein levels of ZPF91 in BC tissues. (A) The mRNA and protein levels of ZPF91 in BC tissue and corresponding non-tumor normal tissue were quantified using RT-qPCR and western blot analysis. The comparisons between two groups were evaluated by t-test. N, non-cancer tissue; C, cancer tissue. *P<0.05. (B) Correlation between ZPF91 mRNA and miR-188-5p was analyzed using Pearson's correlation coefficient. The data are represented as the mean ± SD (n=100). BC, breast cancer; ZFP91, zinc finger protein 91.
Figure 6.
Figure 6.
The regulatory role of miR-188-5p in a BC xenograft mouse model. BC MDA-MB-231 cells were transfected with miR-188-5p mimics and miR-NC, and then injected into nude mice. (A) The tumor volume and weight in the tumor xenograft mouse were compared among the miR-188-5p mimics group, miR-NC group and control group. *P<0.05, compared to control group; #P<0.05, compared to miR-NC group. (B) The protein expression and mRNA level of ZPF91 were detected by (C) western blotting and RT-qPCR. The data are represented as the mean ± SD (n=3). *P<0.05. One-way ANOVA followed by Tukey's test was used to evaluate the difference for multiple comparisons. BC, breast cancer; ZFP91, zinc finger protein 91; NC, negative control.
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