Downregulation of hypermethylated in cancer-1 by miR- 4532 promotes adriamycin resistance in breast cancer cells

Abstract

Background: MicroRNAs are small RNAs (~ 22 nt) that modulate the expression of thousands of genes in tumors and play important roles in the formation of multidrug resistance. In this study, we firstly investigated that miR-4532 involved in the multidrug resistance formation of breast cancer by targeting hypermethylated in cancer 1 (HIC-1), a tumor-suppressor gene.

Methods: To identify and characterize the possible miRNAs in regulating multidrug resistance, we employed the transcriptome sequencing approach to profile the changes in the expression of miRNAs and their target mRNAs were obtained by bioinformatics prediction. Then the molecular biology experiments were conducted to confirm miR-4532 involved in multidrug resistance formation of breast cancer.

Results: The luciferase reporter assay experiment was employed to confirm that HIC-1 was the target of miR-4532. Transfection with an miR-4532 mimic indicated miR-4532 mimic significantly increased breast cancer cell resistance to adriamycin. Cell proliferation and invasion assay experiments showed overexpression of HIC-1 inhibited the invasion and metastasis of breast cancer cells. Meanwhile, the interleukin (IL)-6/signal transducer and activator of transcription 3 (STAT3) signaling pathway was confirmed to be involving in multidrug resistance by western blotting experiments.

Conclusions: These results suggest that downregulation of hypermethylated in cancer-1 by miR-4532 could promote adriamycin resistance in breast cancer cells, in which the IL-6/STAT3 pathway was regulated by the HIC-1. This finding might contribute to new therapeutic target for reversal of tumor resistance.

Keywords: Breast cancer; Hypermethylated in cancer-1; Interleukin-6/signal transducer and activator of transcription 3 pathway; Multidrug resistance; miR-4532.

Fig. 1

GO analysis of target genes predicted according to differential expression of miRNAs. The X-axis indicates the function of each GO annotation, and the Y-axis represents the relative expression of proteins for every GO annotation

Fig. 2

qPCR analysis of differentially expressed miRNAs in MDA-MB-231 cells. NS not significant; ***p < 0.01

Fig. 3

Overexpression of miR-4532 increased adriamycin resistance in MCF-7 cells. a After transfection with miR-4532 negative control (red), the cells were washed three times with PBS and then observed under a fluorescence microscope with a ×400 lens. b qPCR results showed that the expression of miR-4532 was significantly increased in MCF-7 cells transfected with the miR-4532 mimic (***p < 0.01, Student’s t-tests). c After transfection with miR-4532 mimic or negative control (NC) for 48 h, MCF-7 cells were subsequently treated with various concentrations of adriamycin for 48 h. Cell viability was determined using CCK-8 assays, and the calculation of IC₅₀ was obtained by using Probit regression analysis in SPSS 18.0 software (IC₅₀ for mimic and negative control group was 3.457 ± 0.274 and 0.603 ± 0.108 μg/ml, respectively, p < 0.05). d Overexpression of miR-4532 in MCF-7 cells rescued adriamycin-induced apoptosis after 48 h of treatment with 1 μM adriamycin, the rate of early apoptosis of NC group and mimic group was 31.21% and 10.09%, respectively, p < 0.01

Fig. 4

miR-4532 suppressed the expression of the HIC-1 gene. a Sequence alignment between miR-4532 and the 3′-UTR of HIC-1. b Effects of miR-4532 on HIC-1 were assessed with the luciferase reporter system. The miR-4532 mimic, together with the luciferase reporter vector or control vector, was transfected into MCF-7 cells (NS: not significant, Student’s t-test). c qPCR was used to measure the expression levels of HIC-1 mRNA in MCF-7 cells and MCF-7 cells transfected with the miR-4532 mimic or negative control (***p < 0.01, Student’s t-test). d Western blot analysis of HIC-1 protein expression in MCF-7 cells and MCF-7 cells transfected with the miR-4532 mimic or negative control

Fig. 5

HIC-1 inhibited breast cancer cell invasion and metastasis in vitro. a In vitro transwell assays for MCF-7^HIC−1 and MCF-7^NC cells with or without IL-6 stimulation. b In vitro transwell assays for MCF-7^sh-HIC−1 and MCF-7^NC cells with or without IL-6 stimulation. c In vitro transwell assay for MCF-7 and MCF-7^sh-HIC−1 cells treated with the STAT3 inhibitor AG490 (100 μM), and western blot analysis of proteins in the STAT3 signaling pathway. The invading cells were observed and analyzed using a microscope with a ×10 objective. Student’s t-tests: NS: p > 0.05, **p < 0.05, ***p < 0.01, SPSS

Fig. 6

HIC-1 was downregulated in human breast cancer tissue specimens, predicting poor survival and prognosis. a Western blot analysis of HIC-1 (90 kDa) in breast cancer tissue specimens. b Kaplan–Meier plots of recurrence-free survival (RFS) from data in the dataset GSE1456, stratified by HIC-1 expression. The p value was calculated using a log-rank test