Hypoxia‐responsive miR‐141–3p is involved in the progression of breast cancer via mediating the HMGB1/HIF‐1α signaling pathway

Abstract Background Hypoxia‐responsive miRs have been frequently reported in the growth of various malignant tumors. The present study aimed to investigate whether hypoxia‐responsive miR‐141–3p was implicated in the pathogenesis of breast cancer via mediating the high‐mobility group box protein 1 (HMGB1)/hypoxia‐inducible factor (HIF)‐1α signaling pathway. Materials and methods miRs expression profiling was filtrated by miR microarray assays. Gene and protein expression levels, respectively, were examined by a quantitative reverse transcriptase‐polymerase chaion reaction and western blotting. Cell migration and invasion were analyzed using a transwell assay. Cell growth was determined using nude‐mouse transplanted tumor experiments. Results miR‐141–3p was observed as a hypoxia‐responsive miR in breast cancer. miR‐141–3p was down‐regulated in breast cancer specimens and could serve as an independent prognostic factor for predicting overall survival in breast cancer patients. In addition, the overexpression of miR‐141–3p could inhibit hypoxia‐induced cell migration and impede human breast cancer MDA‐MB‐231 cell growth in vivo. Mechanistically, the hypoxia‐related HMGB1/HIF‐1α signaling pathway might be a possible target of miR‐141–3p with respect to preventing the development of breast cancer. Conclusions Our finding provides a new mechanism by which miR‐141–3p could prevent hypoxia‐induced breast tumorigenesis via post‐transcriptional repression of the HMGB1/HIF‐1α signaling pathway.


| INTRODUCTION
Breast cancer (BC) is the most commonly diagnosed cancer both in China and worldwide among females, contributing to approximately 15.1% or 24.2% of cancer incidence in China or worldwide, respectively. [1][2][3] Understanding the basic molecular mechanisms of the initiation and development of BC will be advantageous for optimizing clinical management strategies.
As a low oxygen condition, hypoxia may occur in both normal physiological processes and pathological conditions. 4 Hypoxia has frequently been reported as a stressor in the tumor microenvironment to confer chemotherapeutic resistance and associate with the poor prognosis of cancer patients. 5 In conditions of hypoxia, numerous pathways and transcription factors are deregulated, which play a crucial role in aggressive tumor phenotypes, such as angiogenesis, migration, invasion and metastasis. 6,7 Comprising the most crucial transcription factor, hypoxia-inducible factor (HIF)-1, which consists of an inducible α subunit and a constitutively expressed β subunit, is activated by oxygen deprivation. 4,8 In normal oxygen conditions, the α-subunit is hydroxylated at proline residues, resulting in its ubiquitination and proteasomal degradation, whereas it evades degradation and assembles with the β-subunit in conditions of hypoxia, aggregating a heterodimeric transcription factor that can modulate downstream gene expression by binding to target genes with their 5'-TACGTG-3' recognition sequence in the nucleus. 4 Previous investigations corroborate that HIF-1α can be regulated by high-mobility group box protein 1 (HMGB1), a non-histone nuclear protein implicated in various pathologic processes, including angiogenesis, rheumatoid arthritis and carcinogenesis. 9,10 Some previous studies report that hypoxic microenvironment can disturb the expression of microRNAs, which represent a class of small non-coding RNAs and function as post-transcriptional regulators by binding to the 3'-untranslated region (3'-UTR) of target genes to weaken the protein translation, 11 changing the behavior of the tumor cells, including glycolysis, radioresistance, autophagy and epithelial to mesenchymal transition. [12][13][14][15] In BC, hypoxia triggers miR-153 to manipulate angiogenesis via modulating the HIF1α/vascular endothelial growth factor axis. 16 Hypoxia induces the up-regulation of miR-210 to increase BC stem cell metastasis and proliferation by targeting E-cadherin. 17 In the present study, we aimed to investigate whether the hypoxia-responsive miR-141-3p was implicated in the pathogenesis of BC via mediating the HMGB1/HIF-1α signaling pathway. Our preliminary findings revealed that the expression of miR-141-3p was blocked by hypoxia in human BC. Moreover, the overexpression of miR-141-3p could counteract hypoxia-induced cell proliferation and migration through the suppression of the HMGB1/ HIF-1α axis. 2.2 | Analysis of cell viability using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay Cell (2 × 10 5 per well) viability was measured by use of an MTT Cell Proliferation/Viability Assay kit (R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturer's instructions.

| Cell transfection
Small interfering RNA was designed to silence HMGB1 expression.

| Overexpression of miR-141-3p attenuates hypoxia-induced cell migration
Previous studies highlight that hypoxia is a key stimulant of BC cell proliferation, migration and invasion. 16  HIF-1α signaling pathway can promote angiogenesis and tumor migration. 10,25 The present study aimed to investigate whether miR-141-3p could mediate HMGB1/HIF-1α signaling pathway in hypoxia-exposed BC cells. Our findings showed that protein expression of HMGB1 and HIF-1α was significantly increased in hypoxia-

| HMGB1 is a direct target of miR-141-3p
According to the above conclusions, our results indicate that miR-141-3p can regulate HMGB1 protein expression, yet the post-F I G U R E 2 miR-141-3p is an independent prognostic factor for predicting the overall survival of BC patients. The expression of miR-141-3p was detected in 74 pairs of BC tissues and adjacent non-tumor tissues using qRT-PCR assays (A) (n = 74), and miR-141-3p was down-regulated in 67 of 74 (90.5%) BC specimens (B) (n = 74). We defined that miR-141-3p low expression was a log 2 (fold change) ≤ −1 (n = 33), and high expression of miR-141-3p was a log 2 (fold change) > −1 (n = 41). Patients with low expression of miR-141-3p showed a shorter overall survival than those of patients with miR-141-3p high expression (C). * p < 0.05   Figure 5D). Moreover, IHC staining indicated a significant increase in HMGB1 protein in BC tissues ( Figure 5E), and a significant inverse correlation between HMGB1 protein level and miR-141-3p expression was observed in 74 BC tissues ( Figure 5F).  Figure 6A and 6B). We also found that overexpression of miR-141-3p could inhibit protein expression of HMGB1 and HIF-1α solid tumors ( Figure 6C and 6D).

| DISCUSSION
As a consequence of solid tumor growth, hypoxia has been frequently reported in various malignant tumors. 5,16,24 Meanwhile, the hypoxic condition is a key factor with respect to promoting tumor migration, F I G U R E 5 HMGB1 is a direct target of miR-141-3p. On-line bioinformatics algorithms (Targetscan; www.targetscan.org) revealed that a conserved sequence in the 3'-UTR of HMGB1 could be bonded with miR-141-3p (A). A direct interaction between miR-141-3p and the 3'-UTR of HMGB1 was analyzed using a luciferase reporter assay (B) (n = 3 in each group.). After transfection with miR-con or miR-141-3p mimics into MDA-MB-231 and MCF-7 for 48 hours, protein expression of HMGB1 (C) and HIF-1α (D) was measured using western blotting (n = 3 in each group.). Protein expression of HMGB1 was detected using IHC staining in 74 pairs of adjacent non-tumor tissues and BC tissues (E) (n = 74 in each group.). Pearson's correlation analysis showed that the expression of HMGB1 was significantly and inversely correlated with miR-141-3p in BC tissues (F) (n = 74 in each group.). * p < 0.05 compared to the corresponding control group invasion, metastasis and chemotherapy-resistance, leading to a poorer prognosis. 5,16,24 Indeed, hypoxia can stabilize HIF1α at the posttranslational level to stimulate malignant processes, including angiogenesis, migration and invasion, via up-regulating the expression of hypoxically regulated genes, such as the vascular endothelial growth factor and matrix metalloproteinases. 24,26 Intriguingly, some specific miRs can be regulated by hypoxia in the initiation and progression of cancers. 5 For example, overexpression of miR-210 is induced by hypoxia and exhibits a significant inverse correlation with disease-free and overall survival in BC patients. 5,16,27 As hypoxia-responsive miRs, miR-25 and miR-93 are implicated in hypoxia-induced immunosuppression in BC. 27 Hypoxia-induced miR-153 contributes to angiogenesis in BC. 16 These findings suggest that hypoxia-related miRs play an important role in cancer progression.
In the present study, miR-141-3p was observed as a hypoxia-responsive miR in BC. miR-141-3p was down-regulated in ZEB2. 29 In the present study, we extended the function of miR-141-3p, which could blunt BC cell migration under hypoxic conditions.
Prior studies have revealed that HMGB1 is persistently associated with hypoxia to promote tumor metastasis. 33 In the process of HCC, hypoxia exposure gives rise to HMGB1 expression to mediate mitochondrial biogenesis and stimulate macrophage-derived interleukin-6, which promotes tumor growth and enhances the invasiveness and metastasis of hepatoma cells. 33 In conclusion, the present study has study highlighted that miR-141-3p could act as an independent prognostic factor for BC and also revealed that overexpression of miR-141-3p plays an antineoplastic activity by suppressing hypoxia-induced cell migration in vitro and solid tumor growth in vivo via mediating the HMGB1/HIF-1α pathway. Our findings provide a new mechanism for understanding hypoxia-related BC progression and corroborate miR-141-3p as a potential therapeutic target for the treatment of BC.