LncRNA MIR503HG inhibits cell migration and invasion via miR‐103/OLFM4 axis in triple negative breast cancer

Abstract Long non‐coding RNA MIR503 host gene (MIR503HG) is located on chromosome Xq26.3, and has been found to be deregulated in many types of human malignancy and function as tumour suppressor or promoter based on cancer types. The role of MIR503HG in breast cancer was still unknown. In our study, we found MIR503HG expression was significantly decreased in triple‐negative breast cancer tissues and cell lines. Furthermore, we observed low MIR503HG expression was correlated with late clinical stage, lymph node metastasis and distant metastasis. In the survival analysis, we observed that triple‐negative breast cancer patients with low MIR503HG expression had a statistically significant worse prognosis compared with those with high MIR503HG expression, and low MIR503HG expression was a poor independent prognostic factor for overall survival in triple‐negative breast cancer patients. The study in vitro suggested MIR503HG inhibits cell migration and invasion via miR‐103/OLFM4 axis in triple negative breast cancer. In conclusion, MIR503HG functions as a tumour suppressive long non‐coding RNA in triple negative breast cancer.


| INTRODUC TI ON
As the most common types of cancer among women in the world with an estimated 2,088,849 new cases at 2018, breast cancer is responsible for 15.0% of deaths in females. 1 At present, breast cancer has been recognized as a heterogeneous disease which can be categorized into different pathological subtypes according to the status of estrogen receptor (ER) and progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). 2,3 Triple negative breast cancer is the most aggressive subtype accounting for 10%-20% of all breast cancer cases, and characterized by lacking expression of ER, PR and HER2. 4,5 Due to the lack of effective treatment including target therapy and endocrine therapy, it is useful to elucidate the mechanisms about triple negative breast cancer progression for developing novel therapies.
In our previous study, we identified olfactomedin 4 (OLFM4) as tumour-suppressing gene to suppress cell migration and invasion through controlling MMP9 in triple negative breast cancer. 6 Meanwhile, down-regulation of OLFM4 expression was correlated with present lymph node metastasis, distant metastasis, advanced clinical stage and poor prognosis in triple-negative breast cancer patients. 6 Furthermore, we found miR-103 functioned as oncogenic microRNA to modulate triple negative breast cancer cell migration and invasion through targeting OLFM4 expression. 7 Long non-coding RNAs are a subgroup of non-coding RNAs composed of more than 200 nucleotides. 8,9 Recently, lncRNA MIR503 host gene (MIR503HG) has been suggested to be dysregulated and function as tumour-suppressing ln-cRNA in a variety of human cancers. 10 Interestingly, we analysed the online prediction tool (Starbase v2.0; http://starbase.sysu.edu.cn/) to search for lncRNAs paired with miR-103, and found MIR503HG has potential binding sites for miR-103. In addition, we further observed there was a negative correlation between MIR503HG expression and miR-103 expression in triple negative breast cancer. Therefore, we had a hypothesis that MIR503HG inhibits cell migration and invasion via miR-103/OLFM4 axis in triple negative breast cancer. So, the aim of this research is to investigate the role of MIR503HG in regulating triple negative breast cancer cell migration and invasion.

| Cell lines
Two human triple negative breast cancer cell lines (MDA-MB-231 and TB549) and a human normal breast epithelial cell line (MCF-10A) were cultured according to our previous description. 6

| Western blot
Western blotting was performed with a SDS-PAGE Electrophoresis System according to previous report 6 with the following antibodies: OLFM4 (Billerica, MA), MMP 9 (Cell Signalling Technology, Beverly, MA) and β-actin antibody (CWBIO, Jiangsu, China).

| Cell transfection
The full-length sequence MIR503HG cDNA was amplified by PCR, and cloned into pcDNA3.1 express vector to make a MIR503HG overexpression vector (pcDNA-MIR503HG). The siRNA for down-regulating MIR503HG expression (siRNA-MIR503HG) and non-targeting siRNA (siRNA-NC) were obtained from GenePharma. siRNA and plasmid transfections were conducted by using Lipofectamine 3000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The miR-103 inhibitor and miR-103 mimics transfections were conducted in accordance with previous description. 7

| Bioinformatics analysis and Luciferase reporter assay
The binding sites of lncRNA and miRNA were predicted on star-Base (http://starbase.sysu.edu.cn/). The MIR503HG wild-type or mutant binding miR-103 was inserted into the pmiR-RB-REPORT luciferase reporter plasmid, and named as MIR503HG-wt and MIR503HG-mut, respectively. MIR503HG-wt or MIR503HGmut was co-transfected with miR-103 inhibitor or miR-103 mimics in triple-negative breast cancer cells with Lipofectamine 3000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The determination of luciferase activity was performed according to previous report. 7

| Cell migration and invasion assays
Cell migration and invasion experiments were conducted by transwell chambers with 8 mm pores (Corning, Cambridge, MA) according to previous description. 6

| Statistical analysis
SPSS statistics 17.0 software (SPSS Inc, Chicago, IL) was used to perform the statistical analysis. The significance of difference between two group quantitative variables was estimated by using Student's t tests. The significance of relationship between two categorical variables was evaluated by using Chi-squared test. Survival analysis was performed through Kaplan-Meier method and Cox regression models. Spearman's correlation analysis was performed to determine the correlation between MIR503HG expression and miR-103 expression. A P < 0.05 was considered statistically significant.

| MIR503HG is down-regulated in triplenegative breast cancer tissues and cell lines
The relative expression of MIR503HG in primary triple-negative breast cancer tissue samples and corresponding adjacent normal mammary tissue samples was measured using qRT-PCR. Compared with adjacent normal mammary tissue samples, MIR503HG expression was markedly decreased in primary triple-negative breast cancer tissue samples (P < 0.001, Figure 1A). Moreover, we also measured the relative ex-

| MIR503HG is associated with poor prognosis in triple-negative breast cancer patients
For exploring the prognostic value of MIR503HG in triple-negative breast cancer patient, we estimated the relationship of MIR503HG expression and overall survival by Kaplan-Meier method. We found triple-negative breast cancer patients with low MIR503HG expression had a statistically significant worse prognosis compared with those with high MIR503HG expression (P < 0.001, Figure 2). Furthermore, we evaluated the prognostic factors for overall survival in triple-negative breast cancer patients, and found clinical stage (P = 0.004), T classification (P = 0.006), N classification (P = 0.016), M classification (P < 0.001) and MIR503HG expression (P < 0.001) were identified as prognostic factors through univariate Cox regression models (Table 2). Then, low MIR503HG expression was further identified as a poor independent prognostic factor for triple-negative breast cancer patients through multivariate Cox regression models (P = 0.014, Table 2).

| MIR503HG directly interacts with miR-103 in triple-negative breast cancer
The bioinformatics analysis was conducted using online prediction tool (Starbase, http://starbase.sysu.edu.cn/) to search for lncRNAs paired with miR-103. The data suggested that there were putative binding sites between MIR503HG and miR-103 ( Figure 3A). Furthermore, luciferase reporter gene assay revealed that miR-103 directly targeted of MIR503HG-wt to negatively regulate the luciferase activity of MIR503HG-wt, rather than MIR503HG-mut (P < 0.001, Figure 3B). The above findings suggested that miR-103 was a target of MIR503HG in triple-negative breast cancer cells.

| Reciprocal modulation of MIR503HG and miR-103 in triple-negative breast cancer
For further explore the relationship between MIR503HG and miR-103, the correlation between of them in triple-negative breast cancer tissues was evaluated by Spearman's correlation analysis.
We observed that the expression of MIR503HG was negatively correlated with miR-103 expression in triple-negative breast cancer tissues (P < 0.001, Table 3). Furthermore, we investigated the effect of MIR503HG on miR-103 expression in triple-negative breast cancer cells, and found up-regulation of MIR503HG expression significantly elevated miR-103 expression (P = 0.001, Figure 4A) and down-regulation of MIR503HG expression greatly reduced miR-103 expression (P = 0.007, Figure 4A). Moreover, we investigated the impact of miR-103 on MIR503HG expression in triple-negative breast cancer cells, and suggested miR-103 mimics or miR-103 inhibitor had no significant effect on MIR503HG expression (P > 0.05, Figure 4B).

| MIR503HG interacts with miR-103 to regulate cell migration and invasion in triple-negative breast cancer
To explore the effects of MIR503HG on triple-negative breast

| OLFM4 as the target gene of miR-103 is modulated by MIR503HG in triple-negative breast cancer
Our previous report suggested miR-103 functioned as oncogenic microRNA to modulate triple negative breast cancer cell migration and invasion through targeting OLFM4 expression. 7