microRNAs (miRNAs) are small non-coding RNA molecules (of ~ 22 nucleotides), which regulate the expression of up to 30% protein coding genes, usually binding to specific sites within the 3′ untranslated regions (3’-UTRs) of the mRNA targets [
1]. The expression of miRNAs is cell- and tissue-specific, indicating that miRNAs are closely associated with cell differentiation and development [
2]. In addition, miRNAs exert a regulatory role in several pathophysiological processes, including many types of tumor [
2‐
5]. In this regard, it should be mentioned that a distinct miRNA may be found up-regulated in certain carcinomas and down-regulated in others, suggesting, therefore, a potential oncogenic and tumor suppressor function, respectively, depending on the cell context. Breast cancer, which represents the most common female malignancy in western countries [
6], is one of the first solid tumors investigated for miRNA expression [
7]. Among the most significant miRNAs overexpressed in breast carcinoma, miR-21 has been shown to mediate cell survival and invasion [
7,
8]. Likewise, miR-144 was shown to induce stimulatory effects in breast cancer cells [
9] and miR-103/miR-107 were associated with a poor outcome in patients affected by triple-negative breast cancer [
10]. Next, the involvement of miR-221/miR-222 has been recently shown in many tumors [
11]. For instance, miR-222 was implicated in the progression [
12] and the drug-resistance [
13] of breast cancer, whereas miR-221 elicited stimulatory effects in diverse types of malignancies down-regulating certain onco-suppressor genes [
14,
15]. In addition, in bone marrow-derived macrophages, miR-221 was reported to down-regulate the expression of the ubiquitin-editing A20 [
16] enzyme, which acts toward the maintenance of tissue homeostasis and the prevention of inflammatory disorders [
17]. In this vein, it was demonstrated that A20 inhibits the activity of the nuclear factor kappa B (NF-kB) [
18], which is largely involved in the development of many types of tumors [
19,
20]. A variety of mechanisms may regulate the expression of A20, like specific A20 binding proteins (ABINs), the ubiquitin binding protein TAX1BP1 and the histone methyltransferase Ash1l [
21‐
23]. In addition, miR-29c, miR-873 and let-7 have been reported to suppress A20 expression, therefore contributing to the activation of NF-kB signaling [
24‐
26]. miR-125a and miR-125b were shown to also target A20 and aberrantly activate the NF-kB transduction pathway in B-cell lymphoma [
27]. NF-kB embraces a family of transcription factors formed by hetero- or homo-dimers, including the subunits p65 (RelA) p50, p52, c-Rel or RelB [
28]. The NF-kB inhibitor alpha (IkB)α, one of the members of the IkB family, keeps the dimers in an inactive form within the cytoplasm. The release from the IkBα leads to nuclear translocation of the NF-kB dimers and their DNA binding, hence leading to the transcription of a variety of genes [
28]. Recently, it has been reported that A20 may inhibit the NF-kB activity by reducing the nuclear levels of c-Rel [
29]. c-Rel is a 587–amino acid protein, which contains a highly conserved N-terminal DNA-binding/dimerization domain named Rel homology domain (RHD) [
30]. The C-terminal half of c-Rel encompasses two C-terminal transactivation sequences (TAD1 and TAD2), separated from the RHD by a transactivation inhibitory domain (RID) [
30]. c-Rel exists either as a homodimer or a heterodimer with p50, however c-Rel can also form dimers with p65 and p52 [
30]. Dysregulated expression and activity of c-Rel have been demonstrated in various cancers [
30]. In agreement with these findings, the ectopic expression of c-Rel triggered the development of breast tumors in transgenic mice [
31]. It has been recently reported that NF-kB regulates the expression of the connective tissue growth factor (CTGF) [
32,
33]. CTGF is involved in many cellular processes including cell adhesion, matrix production, structural remodelling, angiogenesis, cell proliferation and differentiation [
34]. CTGF is mainly regulated by mechanical stresses as well as by a number of cytokines and growth factors [
34]. Previous studies have reported its involvement in various malignancies as breast and endometrial tumors [
35], melanoma [
36], gastric [
37], pancreatic [
38], prostate [
39], hepatic [
40] and colon cancer [
41].
On the basis of the aforementioned observations, we attempted to provide novel insights into the molecular mechanism through which miR-221 may induce oncogenic effects in breast tumor, using as experimental models crucial players of the tumor microenvironment as cancer-associated fibroblasts (CAFs) [
42], the MDA-MB 231 and SkBr3 breast cancer cells. We found that miR-221 down-regulates A20 expression and increases both c-Rel and CTGF, leading to cell growth and migration. Worthy, these effects were abrogated silencing c-Rel and CTGF expression and using the specific Locked Nucleic Acid (LNA)-Inhibitor of miR-221 (LNA-i-miR-221), which is a 13-mer oligonucleotide designed specifically to sequester the miR-221 [
43,
44]. In particular, the LNA-i-miR-221, recently approved for first-in-human clinical trial (EudraCT 201,700,261,533), takes advantage from both LNA technology and phosphorothioate backbone to increase the seed sequence binding and nuclease resistance in vivo, respectively [
45,
46]. Together, our data highlight the oncogenic action of miR-221 in CAFs and breast cancer cells, hence suggesting that its inhibition may represent a further preventive and therapeutic strategy in breast cancer.