A total of 60 BCa (46 males and 14 females; age range, 37–61 years old; median age, 51 years old) and adjacent normal tissue samples were obtained from The Tiantai People's Hospital of Zhejiang Province (Taizhou, China) between September 2014 and November 2016. Written informed consent was obtained from all enrolled patients. Patients treated by radiotherapy or chemotherapy before surgery were excluded. All other patients were included. The present study was approved by The Ethics Committee of Tiantai People's Hospital of Zhejiang Province (approval no. 2016020342). A total of four BCa cell lines (T24, J82, UMUC3 and 5637) and a normal bladder cell line (SV-HUC-1) were purchased from The Cell Bank of The Chinese Academy of Sciences (Shanghai, China). Cells were cultured in the Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and maintained at 37°C in a cell incubator with a humidified atmosphere containing 5% CO₂.

A small interfering RNA against LINC00978 (siLINC00978; 5′-GGACAGAGCAGAAGACAAA-3′), siRNA negative control (siNC; 5′-AAUUCUCCGAACGUGUCAC-3′), miR-4288 mimics (5′-UUGUCUGCUGAGUUUCC-3′), miR-4288 inhibitors (5′-GGAAACUCAGCAGACAA-3′) and controls (5′-UCACAACCUCCUAGAAAGAGUAGA-3′) were purchased from GeneCopoeia, Inc. (Rockville, MD, USA). To overexpress LINC00978, its coding sequence was constructed into pcDNA3 vector (Invitrogen; Thermo Fisher Scientific, Inc.) to generate pcDNA3-LINC00978. The siRNAs (100 nM), mimics (100 nM), inhibitor (100 nM) r, pcDNA3-LINC00978 (1 µg) and their corresponding negative controls (100 nM or 1 µg) were transfected into T24 cells using Lipofectamine^® 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. After 48 h, the transfection efficiency was confirmed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) as described below.

Cell proliferation was measured by Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). A total of 5×10³ cells were seeded in 96-well plates and incubated for 1, 2 and 3 days. Subsequently, 10 µl CCK-8 reagent was added to the 96-well plates. Following 2 h incubation at 37°C, the absorbance at 450 nm was measured to detect the number of viable cells using a SUNRISE microplate reader (Tecan Group, Ltd., Männedorf, Switzerland).

A total of 5×10² T24 cells transfected with siLINC00978 or siNC were seeded in 6-well plates. The culture medium was replaced every other day. Following incubation for 10 days, the colonies from three groups were fixed with ice-cold 100% methanol for 2 h at 4°C and stained with 0.1% crystal violet for 1 h at room temperature. Subsequently, the number of colonies was determined using a light microscope (magnification, ×100; Nikon Corporation, Tokyo, Japan).

Total RNA was extracted from cultured cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol and cDNA was synthesized from 1 µg total RNA using a PrimerScript RT reagent kit (Takara Bio, Inc., Otsu, Japan). The RT conditions were: Incubation for 1 h at 42°C and inactivation at 70°C for 15 min. miRNA from total RNA was reverse transcribed using the PrimeScript miRNA cDNA synthesis kit (Takara Bio, Inc.). RT-qPCR was performed with the SYBR-Green Premix Ex Taq II (Takara Bio, Inc.) using the StepOnePlus RT PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The PCR thermocycling conditions were: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 34 sec. U6 was used as the endogenous control for miRNA and LINC00978 expression analysis. The relative gene expression level was calculated using the 2^−ΔΔCq method (16). The sequences of the primers were: LINC00978 (Forward, 5′-TAGGAGACACAGGCAGAGCC-3′ and reverse, 5′-CTGACTAGAGCTTGTCTCA-3′), miR-4288 (Forward, 5′-AACGAGACGACGACAGAC-3′ and reverse, 5′-TTGTCTGCTGAGTTTCC-3′) and U6 (Forward, 5′-AACGAGACGACGACAGAC-3′ and reverse, 5′-GCAAATTCGTGAAGCGTTCCATA-3′).

For Transwell migration, following transfection of siLINC00978 or siNC, 5×10³ BCa cells were plated in the upper chamber (Corning, Inc., Corning, NY, USA) containing 200 µl serum-free DMEM. A total of 600 µl DMEM containing 10% FBS was added to the lower chamber. Following an incubation of 24 h, the cells migrated into the lower chamber were fixed with 100% methanol at 4°C and stained with 1% crystal violet for 30 min at room temperature. Cells were imaged using a light microscope at magnification, ×100. For the Matrigel assay, the upper chamber was pre-coated with Matrigel (1:3; Becton, Dickinson and Company, Franklin, Lakes, NJ, USA). The invasion assay was performed following the protocol used for the migration assay.

The potential binding site for miR-4288 in LINC00978 were predicted by the miRDB tool (http://mirdb.org/miRDB/index.html). Then the sequence containing the wild-type or mutant binding site was constructed into pMIR-Report vector (Promega Corporation, Madison, WI, USA). For luciferase reporter assay, miR-4288 mimics and wild-type or mutant LINC00978 reporter were co-transfected into the cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Following transfection, cells were incubated for 24 h, and luciferase activity was determined with a luciferase assay system kit (Promega Corporation). Renilla luciferase activity was used for normalization.

Statistical analysis was conducted using SPSS 20.0 (IBM Corp., Armonk, NY, USA). Data from three independent experiments were expressed as mean ± standard deviation. Significant differences were calculated using Student's t-test or one-way analysis of variance followed by Tukey's post hoc test. Pearson's correlation coefficient analysis was used to determine correlation. P<0.05 was considered to indicate a statistically significant difference.

To investigate the role of LINC00978 in BCa, the expression level of LINC00978 was analyzed by RT-qPCR. LINC00978 was significantly upregulated in BCa tissues compared with adjacent normal tissues (Fig. 1A). Furthermore, the expression level of LINC00978 was examined in BCa cell lines. The results suggested that LINC00978 was significantly upregulated in BCa cell lines, including J82, T24, 5637 and UMUC3 cells, compared with SV-HUC-1 cells (Fig. 1B). The present results suggested that LINC00978 may be involved in BCa progression.

To investigate the function of LINC00978 in BCa, a knockdown of LINC00978 was performed by transfecting cells with siLINC00978. Additionally, overexpression of LINC00978 was conducted by transfecting T24 cells with a plasmid expressing LINC00978. RT-qPCR analysis demonstrated that the LINC00978 expression level was significantly decreased upon knockdown and upregulated following overexpression (Fig. 2A and B). Subsequently, a CCK-8 assay was conducted. LINC00978 knockdown significantly inhibited the proliferation of T24 cells at 4 days (Fig. 2C). Conversely, LINC00978 overexpression promoted cell proliferation at 4 days (Fig. 2D). To test the effect of LINC00978 on BCa cell proliferation, a colony formation assay was additionally conducted. The results suggested that LINC00978 knockdown led to a significant decrease in the number of colonies (Fig. 2E), whereas, LINC00978 overexpression caused an increased number of colonies (Fig. 2F). Collectively, the present data suggested that LINC00978 promoted the proliferation of BCa cells.

Malignancies are frequently associated with tumor metastasis. To test whether LINC00978 had an effect on BCa metastasis, a Transwell assay was conducted, using T24 cells. LINC00978 knockdown significantly inhibited migration (Fig. 3A), whereas, overexpression of LINC00978 caused the opposite effect (Fig. 3B). Similarly, LINC00978 knockdown decreased the number of invasive cells (Fig. 3C), and overexpression of LINC00978 promoted invasion (Fig. 3D). The present results suggested that LINC00978 contributed to the metastasis of BCa.

To investigate the mechanism underlying LINC00978 function in BCa, a bioinformatics analysis was performed. It was identified that LINC00978 may serve as a ceRNA of miR-4288. In total, four predicted binding sites of miR-4288 were identified in LINC00978 (Fig. 4A). To test this prediction, a luciferase reporter assay was performed, using wild-type (WT) or mutant binding sites in a reporter plasmid. The results demonstrated that overexpression of miR-4288 increased the expression level of miR-4288 (Fig. 4B), and suppressed the luciferase activity of WT-LINC00978 (Fig. 4C). Notably, mutation of the predicted binding sites abrogated this interaction (Fig. 4C). Furthermore, using RT-qPCR, it was identified that miR-4288 overexpression significantly decreased the expression level of LINC00978 in T24 cells (Fig. 4D). Notably, LINC00978 knockdown increased the expression level of miR-4288 in T24 cells (Fig. 4E). Furthermore, the expression level of LINC00978 was inversely correlated with miR-4288 expression level in BCa tissues (Fig. 4F). Collectively, the present data suggested that LINC00978 binds to miR-4288, inhibiting its expression in BCa cells.

As the present study demonstrated that LINC00978 interacted with miR-4288, it was examined whether miR-4288 was involved in regulating the function of LINC00978 in BCa cells. miR-4288 was inhibited in LINC00978-silenced T24 cells using miR-4288 inhibitors (Fig. 5A). RT-qPCR analysis demonstrated that the miR-4288 expression level was restored in LINC00978-silenced T24 cells with miR-4288 inhibitors (Fig. 5A). Subsequently, CCK-8, Transwell and Matrigel assays were conducted. LINC00978 knockdown inhibited the proliferation, migration and invasion of T24 cells, whereas, inhibition of miR-4288 was able to abrogate the effects of LINC00978 knockdown (Fig. 5B-D). Collectively, the present data demonstrated that LINC00978 promoted the proliferation, migration and invasion of BCa cells by inhibiting miR-4288.