To construct the lncRNA MALAT1 knockdown plasmid, two primers containing a sequence targeting the 5′-GGAAGATAGAAACAAGATA-3′ in the MALAT1 transcript were synthesized. The two primers were annealed and inserted into the pRNAH1.1 vector (Genscript, Nanjing, Jiangsu, China) at BamHI and HindIII sites. The sequence containing a negative control (NC) segment was inserted into the pRNAH1.1 vector and served as the control. The sequence information of the primers is presented in Table I.

To establish the overexpression plasmid of coronin 1C (CORO1C), the coding sequence (CDS) was amplified with human cDNA as the template, in the presence of CORO1C CDS primers. Following sequence analysis, the amplification segment was inserted into pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with BamHI and XhoI sites. The MALAT1 sequence targeted by miR-1-3p was also cloned into the pcDNA3.1 vector with BamHI and XhoI sites, in the presence of amplification PCR primers. The information regarding the CORO1C CDS and MALAT1 amplification PCR primers is presented in Table I.

Prostatic epithelial cell line RWPE2 was purchased from Shanghai Zhongqiaoxinzhou Biotech (Shanghai, China) and cultured with prostatic epithelial cell medium (Shanghai Zhongqiaoxinzhou Biotech) at 37°C in 5% CO₂. Prostate cancer cell lines DU145 and PC-3 were purchased from Procell Life Science & Technology Co., Ltd., (Wuhan, China), and cultured with minimum essential medium (MEM) or Ham's F-12K medium (Procell Life Science & Technology Co., Ltd.) supplemented with 10% fetal bovine serum (FBS; Biological Industries, Kibbutz Beit Haemek, Israel). Prostate cancer cell lines LNCaP and 22RV1 were purchased from Shanghai Zhongqiaoxinzhou Biotech and cultured with RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% FBS. The cells were seeded in 6-well plates and transfected with short hairpin RNA (sh)MALAT1 plasmid (~6.8 kb) with Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific Inc.). Following 24 h, the cells were treated with 100 µg/ml G418 for 2 weeks and returned to normal medium. Subsequent to culturing for ~2 weeks, the monoclonal cell masses were selected. The MALAT1-knocked down cells were obtained.

After culturing for 24 h, the LNCaP and 22RV1 cells were transfected with miR-1-3p mimics or inhibitor in serum-free medium, in the presence of Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). A total of 4 h later, the transfection medium was replaced with normal medium and the cells were cultured for other experiments.

The total RNA was extracted using a TRIpure RNA extraction kit (BioTek China, Beijing, China) and the concentration was measured with an ultraviolet spectrophotometer (Thermo Fisher Scientific Inc.). The RNA was reverse transcribed into cDNA with M-MLV reverse transcriptase (BioTek China) and 2×Power Taq PCR Master Mix buffer (BioTeke), in the presence of oligo(dT) and random primers, or specific miRNA RT primers (Sangon Biotech Co., Ltd., Shanghai, China): Denaturation at 70°C for 2 min, annealing at 25°C for 10 min, and extension at 42°C for 50 min. Subsequently, the cDNA was used for RT-qPCR with SYBR Green (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) to detect the expression levels of MALAT1 and miR-1-3p, with GAPDH or U6 as the respective internal control. The procedure was as follows: 94°C for 5 min 10 sec, 60°C for 20 sec, 72°C for 30 sec, and 40 cycles of 72°C for 2 min 30 sec, 40°C for 1 min 30 sec, melting from 60–94°C each 1°C for 1 sec, and finally incubation at 25°C for several minutes. The data were calculated using the 2^−ΔΔCq method (17). The information on primers is presented in Table I.

The protein was extracted with radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) and the concentration was determined with a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Following protein denaturation by boiling, the proteins were separated with SDS-PAGE (5, 8, 10 and 14% separation gel for different sized proteins) and transferred onto a polyvinylidene difluoride (PVDF) membrane (EMD Millipore, Billerica, MA, USA). Following blocking with 5% skimmed milk at room temperature for 1 h, the PVDF membrane was incubated with one of the following antibodies at 4°C overnight: Rabbit anti-CORO1C (1:500; cat. no. 14749-1-AP; Wuhan Sanying Biotechnology, Wuhan, China), mouse anti-epithelial (E)-cadherin (1:1,000; cat. no. 14472), rabbit anti-neural (N)-cadherin (1:1,000; cat. no. 4061), rabbit anti-vimentin (1:500; cat. no. 5741; all Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit anti-Slug (1:500; cat. no. ab27568; Abcam, Cambridge, UK), rabbit anti-Snail (1:1,000; cat. no. 3879; Cell Signaling Technology, Inc.). Following rinsing with TBST (TBS buffer with 0.15% Tween-20), the PVDF membrane was incubated with the corresponding secondary antibody labeled with IgG at 37°C for 45 min. The PVDF membrane was reacted with enhanced chemiluminescent reagent (Beyotime Institute of Biotechnology) for 5 min, followed by signal exposure. The immunoblotting bands were analyzed with Gel-Pro-Analyzer 4 software (Media Cybernetics, Rockville, MD, USA). GAPDH served as the internal control. The GAPDH level was detected via incubation with rabbit anti-GAPDH (1:500; cat. no. bs-2188R; Bioss, Beijing, China) and corresponding secondary antibody as described above.

StarBase v2.0 (http://starbase.sysu.edu.cn/) was used to predict the candidate miRNAs of MALAT1. TargetScan 7.2 (http://www.targetscan.org/vert_72/) was subsequently used to predict the targeted genes of miR-1-3p.

To demonstrate the binding of MALAT1 to miR-1-3p, the DNA segment containing the sequence miR-1-3p, was cloned into the dual-luciferase reporter, pmirGLO (Promega Cooperation, Madison, WI, USA). The 293T cells (Procell Life Science & Technology Co., Ltd.) were transfected with the dual-luciferase reporter containing the MALAT1 segment or its mutant sequence and miR-1-3p mimics, in presence of Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific Inc.). Following 48 h, the cells were treated with dual-luciferase reporter assay system (Promega Cooperation, Madison, WI, USA) and the firefly and the Renilla luciferase activities were detected.

Similarly, the DNA segment containing CORO1C 3′UTR sequence targeted by miR-1-3p, was cloned into dual-luciferase reporter constructs. The firefly and Renilla luciferase activities were detected.

The LNCaP and 22RV1 cells were cultured until they reached 80% confluence, and treated with 1 µg/ml mitomycin C (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 1 h. The wound was made with a 200 µl pipette tip and the cells were subsequently cultured. At 0 and 24 h, the wound size was viewed under an inverted light microscope (magnification ×100; Olympus Corporation, Tokyo, Japan).

The Transwell assay was performed and membrane precoated with Matrigel to detect the invasive ability of LNCaP and 22RV1 cells. The Transwell chamber with an 8.0 µm pore polycarbonate membrane (Corning Inc., Corning, NY, USA) was pre-coated with Matrigel (Becton, Dickinson and Company; BD Biosciences, Franklin Lakers, NJ, USA) at 37°C. A total of 200 µl cell suspension containing 2×10⁴ cells was added into the upper chamber and 800 µl medium containing 30% FBS was added into the lower chamber. After 24 h culture at 37°C, the cells on the reverse surface of the polycarbonate membrane were fixed with 4% paraformaldehyde at room temperature for 20 min, stained with 0.5% cresol violet at room temperature for 5 min and images were captured under an inverted light microscope (×200 magnification).

The data in the present study were presented as the mean ± standard deviation replicated in triplicate. Results were analyzed with one-way or two-way analysis of variance with Bonferroni's post hoc multiple comparisons. GraphPad Prism 5.01 (GraphPad Software, Inc., La Jolla, CA, USA) was used for analysis of data in this study. P<0.05 was considered to indicate a statistically significant difference.

The expression levels of MALAT1 and miR-1-3p were detected in the normal prostatic epithelial cell line, RWPE2 and prostate cancer cell lines, PC-3, DU145, LNCaP and 22RV1. As indicated in Fig. 1A and B, the expression of MALAT1 was variably upregulated in prostate cancer cells, compared with RWPE2 cells, while miR-1-3p was downregulated in prostate cancer cells. LNCaP and 22RV1 cells were selected to investigate the role of MALAT1, since the expression levels of MALAT1 and miR-1-3p differed between LNCaP and 22RV1 cells. In addition, LNCaP cells are androgen-dependent, whereas 22RV1 cells are androgen-independent (18). MALAT1 was significantly silenced in LNCaP and 22RV1 cells, and its effectiveness was verified by RT-qPCR (P<0.001; Fig. 1C). The expression level of miR-1-3p was significantly increased following MALAT1 knockdown (P<0.001; Fig. 1D). At the same time, CORO1C was decreased in MALAT1-silenced cells (Fig. 1E and F), suggesting that MALAT1 served a regulatory role in miR-1-3p and CORO1C expression levels.

Phenotypic experiments were performed to detect the effect of MALAT1 on prostate cancer cell malignancy. As indicated in Fig. 2, the knockdown of MALAT1 significantly decreased the migratory ability of prostate cancer cells (P<0.01; Fig. 2A-C). In addition, the MALAT1-silenced LNCaP and 22RV1 cells indicated a significant decrease in invasive ability (P<0.01; Fig. 2D and E). Taking into consideration the important role of EMT on cell migration and invasion in cancer cells, a number of EMT-associated proteins were detected. The results on the expression levels of E-cadherin indicated a >3-fold increase and the expression levels of N-cadherin, vimentin, Slug and Snail were decreased by ≥50% in MALAT1-silenced prostate cancer cells compared with NC (Fig. 2F and H).

Bioinformatics analysis was conducted using starBase v2.0 (http://starbase.sysu.edu.cn/) to predict the candidate miRNAs of MALAT1 and miR-1-3p was identified (Fig. 3A). TargetScan 7.2 (http://www.targetscan.org/vert_72/) was subsequently used and it was indicated that miR-1-3p seed sequence directly binds to 3′UTR of CORO1C mRNA (Fig. 3B). Following confirmation of the efficacy of the miR-1-3p mimic and inhibitor (Fig. 3C), a dual-luciferase reporter assay results demonstrated that the firefly/Renilla value of the wild type MALAT1 was decreased by 51% by the miR-1-3p mimic, and the firefly/Renilla value of CORO1C 3′UTR was decreased by 43% by the miR-1-3p mimic in 293T cells (Fig. 3D and F), demonstrating the binding of miR-1-3p to MALAT1 and 3′UTR of the CORO1C mRNA. As indicated in Fig. 3E, the expression levels of MALAT1 were decreased following transfection of with the miR-1-3p mimic, while they decreased following miR-1-3p inhibition, suggesting that MALAT1 may be a degradable sponge. The CORO1C expression level decreased following miR-1-3p overexpression and increased following miR-1-3p inhibition (Fig. 3G and H), in addition, the MALAT1 silencing-induced decrease of the CORO1C expression level was rescued by the inhibition of miR-1-3p (Fig. 3I and J). In addition, the dual-luciferase reporter assay demonstrated that MALAT1 attenuated the binding of miR-1-3p to the CORO1C 3′UTR (the luciferase activity of CORO1C 3′UTR + miR-1-3p mimics + MALAT1 group was compared with that of CORO1C 3′UTR + miR-1-3p mimics + pcDNA3.1 group; Fig. 3K), suggesting that MALAT1 additionally competes with CORO1C for the binding sites of miR-1-3p.

To confirm the underlying mechanism of action of MALAT1, phenotypic experiments were performed in MALAT1-silenced cells following miR-1-3p inhibition. The wound healing assay results revealed that the suppression of the migratory ability of MALAT1 knockdown-induced cells was significantly recovered by miR-1-3p in prostate cancer cells (P<0.01; Fig. 4A-C). Similar results were observed in the Transwell assay (Fig. 4D and E). Additionally, the expression level of E-cadherin was decreased by ≥70% and the expression levels of N-cadherin, vimentin, Slug and Snail indicated a 2-fold increase following transfection with the miR-1-3p inhibitor (Fig. 4F and H), suggesting enhanced EMT.

In order to confirm the competitive association between MALAT1 and CORO1C, CORO1C was overexpressed in MALAT1-silenced cells. CORO1C expression was significantly increased following transfection with a CORO1C overexpression plasmid (Fig. 5A and B), which proved the effectiveness of the CORO1C ectopic expression plasmid. The wound healing assay revealed that the shMALAT1-induced decline in migratory ability was rescued by forced expression of CORO1C (Fig. 5C-E). The Transwell assay demonstrated similar results for the invasion of prostate cancer cells (Fig. 5F and G). As presented in Fig. 5H-J, E-cadherin expression level was significantly decreased (P<0.01) and N-cadherin, vimentin, Slug and Snail was significantly increased following CORO1C overexpression, compared with the MALAT1-silenced cells (P<0.001), suggesting that shMALAT1-induced EMT inhibition was ceased by forced expression of CORO1C.