Long non-coding RNASNHG17 promotes the progression of breast cancer by sponging miR-124-3p

A total of 58 pairs of BC tissues and paired non-tumor tissues were collected from the First Hospital of Jilin University from Jan 2013 to Jan 2014. All the experiments concerning the use of patient samples in this stud were conducted in accordance with the Declaration of Helsinki and were approved by the Medical Ethics Committee of Jilin University. Written informed consents were acquired from all patients. All tissues were stored at in liquid nitrogen. The clinical features of the patients with BC were listed in Table 1.

Human normal human breast epithelial cell (MCF-10A) and four breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-468 and BT-474) were purchased from the ATCC (Manassas, VA, USA). L-15 medium (Gibco, CA, USA) supplemented with 10% FBS (fetal bovine serum, Gibco) was used to culture the cells in a high humidity incubator (37 °C; 5% CO₂).

Recombinant lentiviruses expressing the sh-SNHG17 (shRNA directly targeting SNHG17; 5′-GAUUGUCAGCUGACCUCUGUCCUGU-3′) and sh-NC (negative control shRNA; 5′-UUCUCCGUUCGUGUCACGUUU-3′) were bought from Sangon Biotech Company (Shanghai, China). MCF-7 and MDA-MB-231 were transfected with sh-NC or sh-SNHG17 lentiviral transduction particles (MOI = 20) using 5 μg/mL polybrene (Genechem). Stably transfected cells was chosen with 1 μg/ml puromycin (Calbiochem, USA) for 2 weeks. The mimics and inhibitor of miR-124-3p and their controls (miR-NC) were bought by GenePharma (Shanghai, China), and were transfected with MCF-7 and MDA-MB-231 cells using Lipofectamine 2000 (Invitrogen) in 6-well plates when cells grown to 70% confluent.

Trizol reagent kit (Invitrogen) was applied to isolate total RNA from cultured cells or tissues. The concentration and purify of RNA was determined using Nano-Drop 2000 spectrophotometer (Termo Scientific, USA). The PrimeScript™ RT reagent Kit (Takara, Japan) was used to synthesis cDNA. The expression of miR-124-3p was determined using miRNA qRT-PCR Starter kit (Riobobio, USA) using primers of U6 and miR-124-3p (Riobobio). SYBR^® Green Real time PCR Msater Mix (Toyobo, Japan) was used to detect SNHG17 expression. Expression levels of miR-124-3p and SNHG17 were normalized by U6 and GADPH, respectively. The relative expression of miR-124-3p and SNHG17 was calculated by using 2^− ΔΔCt method.

The NE-PER™ Nuclear and Cytoplasmic Extraction Reagents Kit (Thermo Scientific, USA) was used to isolate and collect cytosolic and nuclear fractions according to manufacturer’s protocol. The expression levels of SNHG17, U6 (nuclear control transcript) and GAPDH (cytoplasmic control transcript) were examined in cytoplasmic and nuclear fractions using qRT-PCR as described above-mentioned.

Cell Counting Kit-8 (CCK-8; Dojindo, Japan) and colony formation assays were used to determine cell proliferation. In CCK-8 assay, stable transfected cells were collected and seeded into 96-well plates at a den-sity of 1 × 10⁴ cells per well. After 24, 48 or 72 h (hs), cells was treated using 10 μl CCK-8 assay for 2 h. The proliferation ability was determined by measuring absorbance at 450 nm using an enzyme immunoassay analyzer (Biorad, Hercules, CA, USA).

For colony formation assay, 1000 stable transfected cells were added into six-well plates and cultured for 10 days. After washed with PBS, colonies were fixed with methanol and stained with 0.1% crystal violet (Sigma-Aldrich, StLouis, MO, USA) for 15 min. The visible colonies were counted.

Wound healing assay used to assess cell migration ability as described previously [19]. Stable transfected cells were seeded in six-well plates until full confluence. The monolayer cells were manually scraped to create wound area using a 100 µl sterile pipette tip. The wounding area at 0 h and 24 h was measured and imaged a light micro-scope (Olympus, Tokyo, Japan).

Transwell invasion assay was performed to assess cell invasion. Briefly, stably transfected cells were suspended in serum-free medium and added to upper transwell chambers (BD Biosciences, Sparks, MD, USA) pre-coated with BD BioCoatMatrigel. After incubation 24 h, invasive cells were fixed with methanol, stained with 0.1% crystal violet, and counted at five randomly selected fields in each well using a light micro-scope (Olympus).

Oligonucleotides that encode SNHG17 cDNA encompassing miR-124-3p-binding sites were synthesized and inserted into pGL3 vector (WT-SNHG17).We also constructed a pGL3-Report plasmid that carried the mutant SNHG17 binding region (MUT-SNHG17). For the luciferase reporter assay, MCF-7 and MDA-MB-231cells were co-transfected with luciferase reporter plasmids and negative control (miR-NC) or miR-124-3p mimics using Lipofectamine 2000. A double-luciferase assay system (Beyotime, Wuhan, China) was used to determine the luciferase activity at 24 h post-transfection.

MCF-7 and MDA-MB-231 cells transfected with miR-124-3p mimics or miR-NC were harvested and subjected to RIP assay using an anti-Ago2 antibody and a RIP kit (Millipore Inc., Bedford, MA, USA) according to the manufacturer’s instructions. Mouse anti-human immunoglobulinG (IgG) antibody was used as control. Finally, RNA was extracted and quantified by qRT-PCR to examine the expression of SNHG17 as described above-mentioned.

Female athymic BALB/c nude mice (5 weeks old, 18–20 g) were bought from Laboratory Animal center of Jilin University. All of the animal experiments were performed based on experimental protocols and approved by the Institutional Animal Care and Use Committee of the Jilin University.

To initiate tumor xenografts, 2 × 10⁶ MCF-7 cells that were stably transfected with sh-NC or sh-SNHG17 were subcutaneously injected into the flank of mice. Tumor growth was measured by caliper measurements every week and was calculated based on following formula: volume = 0.5 × length × width × width. After 5 weeks, all mice were euthanized by intraperitoneal injection of 200 mg/kg pentobarbital. Tumor tissues were removed and weighed. A part of tumor was used for immunohistochemical staining to detect the expression of Ki-67 (a proliferation marker) as described previously [20].

To evaluate the expression pattern of SNHG17 in BC, we first examined the expression of SNHG17 in the BC tissues and adjacent normal tissues. As shown in Fig. 1a, the expression of SNHG17 was upregulated in BC tissues compared with non-tumor breast tissues. To assess the association with clinical characteristic of BC patients and SNHG17 expression, we divided the patients to high-expression group (n = 32) and low-expression group (n = 26) based on the median level of SNHG17 expression. Table 1 displayed that increasedSNHG17 expression was positively associated with advanced TNM stages (III–IV stages) and lymph node metastasis. Kaplan–Meier analyses revealed that high SNHG17 expression group has poorer survival than in low SNHG17 expression group (Fig. 1b). In addition, we found that SNHG17 expression was increased in four BC cell lines compared to MCF-10A cells (Fig. 1c).

To investigate the role of SNHG17 in BC cells, loss-of function experiments were performed in MCF-7 and MDA-MB-231 cells by transfection with shRNA against SNHG17 plasmid (sh-SNHG17). We found that transfection of sh-SNHG17 was able to decrease SNHG17 expression in MCF-7 and MDA-MB-231 cells (Fig. 2a). CCK8 assay revealed that knockdown of SNHG17 could result in a significant decrease of cell proliferation in MCF-7 and MDA-MB-231 cells (Fig. 2b). Consistent with this result, silence of SNHG17 inhibited colony formation of MCF-7 and MDA-MB-231 cells (Fig. 2c).

To investigate whether SNHG17 depletion affect the tumor growth in vivo, the xenograft tumor were established. We found that xenografts grew more slowly in SNHG17 depletion group than in sh-NC group (Fig. 2d). Moreover, the weight and size of tumors was decreased in the SNHG17 depletion group compared with those in the sh-NC group (Fig. 2e). More importantly, the Ki-67 expression was reduced in SNHG17 depletion group compared with sh-NC group (Fig. 2f). These results suggested that SNHG17 depletion inhibited BC growth in vitro and in vivo.

Next, we checked the effects of SNHG17 knockdown on cell invasion and migration using the transwell assay and wound healing assay, respectively. The migration and migration capabilities were significantly decreased in MCF-7 and MDA-MB-231 cells transfection with sh-SNHG17 compared with cells transfected with sh-NC (Fig. 3a, b).

To elucidate whether SNHG17 functioned as a ceRNA in regulating BC progression, Starbse2.0 software (http://​starbase.​sysu.​edu.​cn/​starbase2/​index.​php) was used to predict potential target miRNAs of SNHG17. Among the candidates, miR-124-3p, a tumor suppressor miRNA that is frequently observed to be downregulated in BC [15‐18], was selected and further study. As illustrated in Fig. 4a, there is a predicted targeting site of miR-124-3p on SNHG17. To test this predication, luciferase activity was performed. Our result demonstrated that miR-124-3p mimics could inhibit the luciferase activity in WT-SNHG17 group, with no effect in MT-SNHG17 group in MCF-7 and MDA-MB-231 cells (Fig. 4b). Moreover,we ascertain the subcellular localization of SNHG17 in MCF-7 and MDA-MB-231 cells. As illustrated in Fig. 4c, SNHG17 was mainly expressed in the cytoplasm of MCF-7 and MDA-MB-231 cells.RIP assays further demonstrated that miR-124-3p and SNHG17 were all enriched in Ago2-immunoprecipitation (Ago2-IP) relative to the control in MCF-7 and MDA-MB-231 cells (Fig. 4d). In addition, we found that knockdown of SNHG17 significantly increased miR-124-3p expression in MCF-7 and MDA-MB-231 cells (Fig. 4e), while overexpression of miR-124-3p led to a significant decrease in both MCF-7 and MDA-MB-231 cells (Fig. 4f). We also found that SNHG17 levels were negatively correlated with miR-124-3p levels in BC tissues (Fig. 4g). These results suggested that miR-124-3p bound to the transcript position of SNGH17.

Considering the close correlation between miR-124-3p and SNHG17, we next evaluated whether the miR-124-3p expression implicates in biological effects by SNHG17 in BC cells. To this end, MCF-7 and MDA-MB-231 were transfected with sh-NC, sh-SNHG17 and sh-SNHG1 + miR-124-3p inhibitor. We found that transfection with sh-SNHG17 obviously increased miR-124-3p expression in MCF-7 and MDA-MB-231 cells, while transfection of miR-124-3p inhibitor partially reversed this trend (Fig. 5a). In addition,miR-124-3p inhibitor partially reversed the inhibitory effect on cell proliferation, colony formation, invasion and migration caused by SNHG17 knockdown in BC cells (Fig. 5b–e). In summary, these findings suggested that SNHG17 promoted BC growth and metastasis via modulation of miR-124-3p (Fig. 5f).