Introduction
Breast cancer is often treated successfully with surgery and conventional therapies; however, approximately 25% of patients eventually develop treatment resistance and recurrence of metastatic disease, which is a major cause of mortality in women worldwide [
1]. Studies have demonstrated that the cancer stem cells (CSCs) are typically seed cells of tumors that initiate and maintain tumor growth, and traditional chemotherapy is usually able to shrink the tumor mass, but often fails to completely eradicate CSCs [
2‐
4].
Accumulating evidence suggests that noncoding microRNA (miR-7) is a breast cancer suppressor because it inhibits Kruppel-like factor-4 (KLF-4), which is one of the oncogenes that enhance breast cancer growth [
5‐
7]. High expression of miR-7 significantly reduced both the capacity of self-renewal and invasion of breast cancer stem-like cells (BCSCs). Interestingly, the long noncoding RNA (lncRNA), terminal differentiation-induced lncRNA (TINCR), is required for normal epidermal differentiation by a posttranscriptional mechanism [
8]. Recently, a report showed that TINCR modulated KLF4 expression by competing with miR-7, to mediate its oncogenic function [
9,
10]. MiR-7 can downregulate SET domain bifurcated 1 (SETDB1), an oncogene that is overexpressed in breast cancer, resulting in the inhibition of tumor progression and metastasis in mice, accompanied by a decrease in the BCSC subset in tumor tissues, as in our previous report [
6]. The exact molecular mechanism, however, has yet to be elucidated.
Therefore, the present study aimed to further investigate the mechanisms of miR-7-mediated reduction of the BCSC subset and elucidate its molecular linkage in breast cancer cells and in breast cancer xenografts in mice.
We report that lncRNA XIST (X-inactive-specific transcript), which is involved in genomic imprinting and the transcriptional silencing of genes on the X chromosome and is highly expressed in tumor tissues [
11‐
15], is an epigenetic regulator targeted by miR-7 that directly bound and inhibited XIST expression in breast cancer by using the RNA in vivo precipitation (RIP) method, which underlies the attenuation of properties of BCSCs and the decrease of the BCSC subset. MiR-7 also modulated the miR-92b/Slug/ESA (epithelium-specific antigen) axis and reduced CD44 and Slug expression, resulting in a decrease in cancer cell stemness and the BCSC subset in breast cancer. Furthermore, miR-7 overexpression could suppress the tumorigenicity of CD44
+CD24
−ESA
+BCSCs and reduce the BCSC subset in breast cancer animal model. Our study characterizes the regulation of XIST by miR-7 and provides evidence that overexpression of miR-7 or downregulation of XIST underlies the attenuation of properties of BCSCs and the decrease of the BCSC subset, which may be a potential strategy for breast cancer therapy.
Discussion
Crucial concerns for the BCSC subset are nearly related with the chemoresistance and recurrence of breast cancer; we explored the mechanisms of decreasing the BCSC subset regulated by miR-7. Our findings in the present study revealed that miR-7 and miR-92b expression levels were markedly decreased in breast cancer surgery samples in contrast to the high expression of lncRNA XIST, RELA, CD44, slug, and ESA, suggesting a negative correlation between miR-7/miR-92b and the expression of BCSC stemness-related molecules in vivo. In Fig.
2 and Figure S
2 a to d, we found that the expression levels of XIST, RELA, CD44, Slug, and ESA were remarkably decreased in the miR-7 mimic transiently transfected cells, especially in the case of XIST, which may regulate the stemness characteristics and tumorigenicity of some types of cancer cells, resulting in cancer progression [
28‐
33]. To verify these claims, we attempted to identify whether XIST expression was regulated by miR-7/miR-92b. The results indicated that miR-7/miR-92b could significantly inhibit the reporter activity of XIST, whereas knockdown of XIST led to high miR-7/miR-92b expression, which suggested the reciprocal regulation between miR-7 and XIST in MDA-MB-231 and LD cells.
To further confirm that miR-7 directly targets XIST in breast cancer, we performed a RIP assay. The results in Fig.
4 g to j showed that miR-7 could directly bind to XIST in MDA-MB-231 cells, which fully demonstrated that miR-7 could inhibit XIST expression.
It is known that RELA is not only a member of the NF-κB family and participates in many biological processes but is also involved in the NF-κB signaling pathway, which correlates with BCSC proliferation activity [
28]. Thus, we evaluated whether RELA could directly regulate CD44 expression. As expected, ChIP-PCR results showed that RELA had increased occupancy in the promoter region of
CD44, and this was further confirmed by knockdown of RELA, which resulted in reduced CD44 expression. Next, we observed that miR-7 could directly regulate RELA expression, as confirmed by the luciferase reporter assay. Since CD44 is tightly associated with the BCSC phenotype [
10,
16,
34], we hypothesized that miR-7 decreased the BCSC subset mainly by targeting RELA to inhibit CD44 expression. Our findings agreed with a recent report by Cantley et al., which reported a targeted relationship between miR-7 and the NF-κB subunit RELA and miR-7 inhibition of RELA expression could impact the proliferation, invasion, and apoptosis of non-small cell lung cancer cells. However, the report did not involve the CSC subset [
35].
In the present study, we also observed low miR-92b and high XIST expression concurrently in breast cancer tissues and cell lines. These findings were in agreement with several studies on XIST and miR-92b expression in tumors [
13‐
15,
36‐
38].
In addition, we found that miR-7 negatively regulated XIST expression but positively regulated miR-92b expression and that high miR-92b expression enhanced the inhibition of transcription factor Slug activity, resulting in decreased ESA expression. The miR-7 mimic not only decreased ESA expression by indirectly increasing miR-92b expression to inhibit slug activity but also directly downregulated Slug to reduce ESA expression. These epigenetic regulatory effects finally led to a decrease in the BCSC subset percentage in breast cancer.
It is possible that XIST serves as a competing endogenous RNA to bind miR-7 to inhibit its function. Since while XIST was knocked down, the expression of miR-7 was clearly increased. Similarly, miR-92b exhibited an inverse relationship with XIST gene expression. Consequently, the results from the in vitro experiments demonstrated that the miR-7 mimic could exert multiple reciprocal regulatory actions to inhibit the expression of XIST, RELA, Slug, CD44, and ESA but increase miR-92b expression.
In vivo animal experiments demonstrated that the local treatment of the BCSC-driven xenograft with the miR-7 agomir had the same effects as applying the combined treatment of Ad plus Cy in a mouse model, which resulted in a reduction in tumor growth and improvement of tumor-bearing mouse survival as well as decrease in BCSC phenotypes (CD44, ESA). A combination of Ad plus Cy treatment of breast cancer is well known as one of the conventional therapies in the past few decades [
39,
40]. Our results suggested a potential value of miR-7 agomir as a local treatment reagent in the treatment of breast cancer.
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