MiR-218-5p promotes breast cancer progression via LRIG1

Background: MiR-218-5p is a small non-coding RNA acting as either oncogenes or tumor suppressor genes in human cancer. The expression levels of some miRNAs in human breast cancer plays a potential role in disease pathogenesis. Methods: Thirty pairs of invasive ductal carcinoma and adjacent specimens were included in the study. Breast tissues cell lines MCF-7 and MDA-MB-231 were identified as a breast cancer research cell line. MiR-218-5p mimics, miR-218-5p inhibitor, or negative controls were transfected. Specific antibodies were probed with LRIG1, ErbB2, and EGFR. Proliferation, migration, cell cycle and apoptosis, dual-luciferase reporter assay and immunohistochemistry were used to analyze miR-218-5p 、 LRIG1 and so on. Results: It was shown that miR-218-5p expression was higher in 30 breast cancer specimens than adjacent normal breast tissues. In human breast cancer cells MCF-7 and MDA-MB-231, restoring miR-218-5p promoted cell proliferation and migration and inhibited cell apoptosis and cell cycle arrest in the G1 stage. Luciferase assays indicated miR-218-5p could bind with its putative target site in the 3'-untranslated region (3'-UTR) of LRIG1. RT-qPCR, western blot, and immunocytochemistry analyses all indicated miR-218-5p overexpression results in LRIG1 downregulation at the mRNA and protein levels. ErbB2 and EGFR were found to be downstream effectors of miR-218-5p. Conclusion: MiR-218-5p promotes ErbB2 and EGFR expression by inhibiting LRIG1 in breast cancer cells, which suggests miR-218-5p and LRIG1 may act as an oncogene in breast cancer and it could be used as a therapeutic target for breast cancer treatments.


Introduction
MicroRNAs (MiRNAs) are a class of 19-24-nucleotide-long non-coding RNAs that repress gene expression in one of two manners: by inhibiting mRNA translation or promoting mRNA degradation. During the last few years, increasing evidence has indicated miRNAs are involved in a wide range of biological processes, including cell proliferation, apoptosis, and migration [1][2][3] .
Regarding cancers, miRNAs have also been found to play important tumor suppressor or oncogene roles according to their expression levels and the involved downstream targets [4][5][6] .
However, the precise molecular mechanism through which miR-218-5p influences breast cancer progression remains largely unknown, indicating further investigation is required.
LRIG1, a member of the LRIG family of transmembrane leucine-rich repeat proteins, is a negative regulator of several oncogenic receptor tyrosine kinases, including all members of the ErbB family [12][13][14] as well as the Met [15] and Ret receptors [16] . LRIG1 is broadly expressed in healthy tissue [17] , but its expression decreases in cancers such as renal cell carcinoma [18] , cervical cancer [19] , and breast cancer [14] . Relieving LRIG1-mediated negative regulation in LRIG1 knock-out mice results in ErbB [20,21] and Met [21] receptor up-regulation in the intestinal epithelium, underscoring the physiological significance of the receptor negative regulation performed by LRIG1 [21] .
This study first demonstrated that miR-218-5p expression was significantly elevated in breast cancer specimens relative to normal tissues. This overexpression promoted MCF-7 and MDA-MB-231 breast cancer cell proliferation and migration, as well as inhibiting cell apoptosis and disrupting the cell cycle by targeting LRIG1. These results indicated that miR-218-5p functions as a tumor promoter gene whose dysregulation may be involved in the development of human breast cancer.

Materials and Methods
Human breast cancer specimens 30 paired breast cancer specimens and adjacent normal breast tissues were collected from the Department of General Surgery of the Shanghai Tenth People's Hospital (Shanghai, China). One of these samples was immediately snap-frozen in liquid nitrogen. Other tissues were formalin-fixed and paraffin-embedded. All samples were confirmed as invasive ductal breast cancer by trained pathologists. No patients had received chemotherapy or radiotherapy prior to surgery. The investigation was approved by the ethics committee at Shanghai Tenth People's Hospital, and informed consent for the use of the postsurgery samples was obtained from the donors who were patients with breast invasive ductal carcinoma.
Cells were incubated at 37 ℃ in a humidified chamber with 5% supplemental CO2.
For transfections, 2×10 5 cells were added to each well of a 6-well plate and cultured with DMEM medium without either serum or antibiotics. When MCF-7 and MDA-MB-231 breast cancer cells reached 30-40% density, miR-218-5p mimics/negative control (NC), miR-218-5p inhibitor/inhibitor negative control (GenePharma Co., Ltd., Shanghai, China), and Lipofectamine transfection reagent (Invitrogen, USA) were each diluted in 500 μl DMEM medium at a ratio of 1 μg : 3 μl and incubated for 5 min at room temperature (RT). The two mixtures were then gently combined and incubated for a further 20-30 min at RT. Subsequently, 1,000 μl of the complexes were added to each well. After 5-6 h of incubation, the DMEM medium was replaced by DMEM with 10% FBS. Cells were incubated at 37 ℃ in a CO2 incubator for 48 h prior to further testing.

RNA extraction and RT-qPCR assay
Breast cancer specimens and adjacent normal breast tissues were prepared for miRNA extraction using a miRNA rapid extraction kit (Tiangen, Beijing, China). Total RNA was isolated from harvested cells of the selected cell lines using Trizol reagent (Invitrogen, USA). Reverse transcription PCR and quantitative PCR (RT-qPCR) kits (TaKaRa, Japan) were used according to the manufacturer's instructions to detect the relative quantity of RNA. GAPDH was used as an endogenous control.

Western-blot assay
Whole cell proteins were extracted using a protein lysis buffer (Sigma-Aldrich, USA) and quantified via bicinchoninic acid assay (Pierce, USA). Protein samples were then electrophoresed in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (PVDF, EMD Millipore, MA, USA), which was probed with LRIG1-, ErbB2-, and EGFR-specific antibodies. Blots were subsequently detected and visualized using an enhanced chemiluminescence detection kit (Millipore, Billerica, MA, USA) according to protocols provided by the manufacturer. A Bio-RAD scanning system was used to detect immunoreactive protein bands.

Proliferation assay
Cell proliferation assays were monitored using cell-counting kit 8 (Invitrogen, Shanghai, China) according to the instructions provided by the manufacturer. Approximately 4-5 h after miR-218-5p mimics, miR-218-5p inhibitor, or negative controls were transfected, cells from each condition were plated (3,000/well) onto 96-well plates (BD Biosciences, USA) and incubated at 37 ℃ in a humidified chamber with 5% supplemental CO2. Cell proliferation was assessed at 0, 24, 48, and 72 h. The optical density (OD) of each well was measured using a microplate spectrophotometer at 450 nm. All experiments were performed in biological triplicate.

Migration assay
Cell migration assays were conducted in transwell chambers with polycarbonate membrane inserts (Corning, NY, USA). Cells (6×10 4 per well) were suspended in 180 μl of serum-free DMEM, and 600 μl of the same medium (containing 10% FBS) were placed in the lower chambers to stimulate cell migration. After 16 h incubation, cells adhering to the transwells were fixed with 3% paraformaldehyde (30 min), then stained with 0.1% crystal violet (15 min). Cells in the upper chambers were wiped with a cotton ball, leaving migrating cells adhered to the bases of the chambers. Five random fields were picked at 200× by the camera of an inverted microscope (Thermo Fisher Scientific).

Cell cycle and apoptosis assay
Thirty-six hours after miR-218-5p mimic, miR-218-5p inhibitor, or negative control transfection, cells were trypsinized and centrifuged at 1,000 rpm for 5 min, then washed twice in cold PBS. Subsequently, 3 ml ice-cold ethanol was added in a dropwise fashion and cells were allowed to affix for ≥30 min. A total of 250 μl 0.05 g/l propidium iodide (PI) staining solution was added into each sample, after which samples were incubated for 30 min at RT. Cells were then analyzed using a flow cytometer (Beckman coulter, KBB, CA, USA).
Cell apoptosis was performed using an Annexin V-FITC apoptosis detection kit (BestBio, Shanghai, China) according to protocols provided by the manufacturer: cells were seeded onto 6-well plates at a 1.5×10 5 cells-per-well density, then, 48 h after transfection, cells were digested by 0.08% EDTA-free trypsin and washed twice with ice-cold PBS. Porpodium iodide (PI) and Annexin V-fluresceinisot hiocyanate (FITC) stainings were applied determined the percentage of cells undergoing apoptosis or necrocytosis. Cell apoptosis was measured via flow cytometry (Beckman coulter, KBB, CA, USA), and data were analyzed using FlowJo software.

Statistical analysis
Statistical analyses were performed using GraphPad Prism 5.0 software (GraphPad Software Inc., La Jolla, CA, USA). Comparisons between two groups were subject to t-testing, whereas those between multiple groups underwent an analysis of variance (ANOVA). All experiments were performed in triple replicate, and the data below are presented in the mean±standard deviation (S.D) format. A p-value < 0.05 was considered statistically significant. According to the modified guideline recommendations for LRIG1 testing in breast cancer, immunoreactivity was graded by scoring the percentage of positive membrane staining:

miR-218-5p expression increased in human breast cancer tissues and cell lines
To investigate the expression level of miR-218-5p in breast cancer, miR-218-5p expression was investigated in 30 pairs of breast cancer and normal adjacent tissues using RT-qPCR. As depicted in Fig. 1A, the 2 -ΔΔCt value of miR-218-5p was significantly increased in breast cancer tissues relative to that of normal adjacent tissues (p<0.05). This finding was consistent with an in  Figures 2C-2F). Overall, these results suggest miR-218-5p exerts proliferative and migratory effects on breast cancer cells and therefore may act as a tumor promoter in breast cancer.

miR-218-5p disrupts the cell-cycle progression of breast cancer cells in different phases
As cell growth inhibition can result from cell cycle arrest, the possibility that miR-218-5p affects cell-cycle progression was examined. First, miR-218-5p mimics and inhibitor were transfected into MCF-7 and MDA-MB-231 cel respectively. These cells were then analyzed via flow cytometry. Results showed that miR-218-5p mimic transfection arrested significantly more MCF-7 and MDA-MB-231 cells in the G2/M-phase than transfection with NC did (Figures 3A to   3D). Furthermore, transfecting MCF-7 cells with miR-218-5p mimics resulted in a significant increase in the percentage of S-phase cells relative to NC. However, miR-218-5p inhibitor transfection arrested less MCF-7 and MDA-MB-231 cells at G2/M-phase than NC transfection ( Figures 3A to 3D). These findings suggest miR-218-5p can initiate S phase arrest, and upregulating miR-218-5p expression could lead to an increase in S-and G2/M-phase cells.  (Figures 4A to 4D). Taken together, these data indicate that miR-218-5p could inhibit breast cancer cell apoptosis.

ERBB2, EGFR in LRIG1-mediated signaling pathway were downstream effectors of miR-218-5p
ERBB2 and EGFR have been reported as primary downstream messengers in LRIG1 signaling.
To elucidate whether miR-218-5p could regulate ERBB2 and EGFR via LRIG1 inhibition, the protein levels of LRIG1, ERBB2, and EGFR in MCF-7 cells (transfected with quantified miR-218-5p mimics or miR-218-5p inhibitor and the corresponding NC) via western blot. Results demonstrated that miR-218-5p overexpression could suppress the mRNA expression of LRIG1 ( Figures 6A and 6B). Furthermore, miR-218-5p was proven to increase the protein expression of ERBB2 and EGFR ( Figures 6A, 6C and 6D). Western blot and immunocytochemistry assay results showed that miR-218-5p significantly reduced the protein expression of LRIG1 ( Figure 6E and 6F). As LRIG1 is a direct target gene of miR-218-5p, all data demonstrated that ERBB2 and EGFR were downstream effectors of miR-218-5p, at least in part induced by LRIG1 targeting ( Figure 6G).

Discussion
Recently, miRNAs have been revealed to play an important role in the genesis and progression of human cancers has emerged. Many researchers have reported the extensive alteration of miRNA expression in the initial and developmental stages of human cancers, as well as the effects of miRNAs in tumor suppression and promotion [22][23][24] . The importance of miRNA function and dysfunction in various human cancers suggests that modulating miRNA expression may serve as a novel therapeutic modality for such diseases. To date, three main approaches have been used in potential miRNA-targeting therapy: expression vectors (miRNA sponges), small-molecule inhibitors, and antisense oligonucleotides. Chemically synthesized miRNAs and oligonucleotides that target miRNAs have already been proven to efficiently inhibit cancer development [25,26] . At this time, several preclinical and clinical miRNA-targeting therapy trials which may pave the way for cancer therapy are in-progress [27,28] .
Several miRNAs are essential for tumor development in breast cancer, including miR-9, let-7, and miR-193a-3p [29][30][31] . This study examined the expression levels of miR-218-5p in human breast cancer and its potential role in disease pathogenesis. First, miR-218-5p expression levels in human breast cancer specimens were detected via RT-qPCR. These results showed miR-218-5p was significantly downregulated in breast cancer tissues relative to normal breast tissues. Similar findings have been reported in other cancer types [32,33] , indicating decreases in miR-218-5p expression are common in human cancer specimens and cell lines. Next, miR-218-5p mimics were transfected into MCF-7 cells, simulating overexpression. This led to a significant inhibition of cellular proliferation, measured by CCK8, as well as a reduction in the colony number, determined by clone formation assay. Both experiments indicated miR-218-5p repressed the growth of breast cancer cells. Using a transwell migration assay, it was discovered that overexpressing miR-218-5p in breast cancer cells could suppress their migratory ability. miR-218-5p was definitively found to arrests cancer cells in the G1 phase relative to the cell cycle of NC groups. However, no significant differences in apoptosis were found between the miR-218-5p and NC groups in this study. The strong vitality of cancer cells was speculated to be one possible reason why miR-218-5p could not promote apoptosis.
To investigate the downstream targets of miR-218-5p that may play a role in mediating its cell function, putative targets were searched for in the miRanda, targetscan, and miRBase databases.
Through luciferase assays, LRIG1 was predicated as a direct targe t of miR-218-5p in MDA-MB-231 cells.
Additionally, both the mRNA and protein levels of LRIG1 were found to be significantly lower in miR-218-5p than in the NC groups. These findings support the assumption that LRIG1 is a downstream target of miR-218-5p.
According to reports, LRIG1 plays an important role in regulating cell surface levels of ErbB family RTKs [34] . In tamoxifen-treated luminal breast cancers, up-regulation of LRIG1 suppresses ErbB RTK family expression and signaling in luminal breast cancers, including EGFR, ErbB2, ErbB3 and ErbB4 [35] . Our study found that miR-218-5p could up-regulate the protein expression of ErbB2 and EGFR by targeting LRIG1, suggesting that miR-218-5p may promotes the biological function of breast cancer through LRIG1-mediated signaling pathway.

Conclusions
Collectively, these findings suggest that miR-218-5p can disrupt the cell cycle by targeting