BC tissues and matched adjacent non-cancerous tissues were obtained from 17 patients at Zhujiang Hospital (Guangzhou, China) from January, 2013 to November, 2015. Matched adjacent normal tissues were obtained at a distance of 5 cm from the BC tissues. All clinical specimens were immediately frozen in liquid nitrogen and stored at −80°C for further analyses. This study was carried out in accordance with the guidelines by the Ethics and Scientific Committee of Southern Medical University (Guangzhou, China). Written informed consent was obtained from all patients enrolled in the current study.

One normal human breast epithelial cell line (MCF-10A) and four BC cell lines (MDA-MB-231, MDA-MB-468, MCF7 and MDA-MB-415) were obtained from the Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China). These cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL, Grand Island, New York, NY, USA) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA). All the cells were maintained at 37°C in a humidified incubator with 5% CO₂.

Total RNA and miRNAs were extracted from the tissues and cells using TRIzol reagent (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Following quantification with a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), the extracted total RNA was reverse transcribed using a Reverse Transcription kit (Takara, Dalian, China). Quantitative PCR (qPCR) was performed on an Applied Biosystems 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The specific primer sequences synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China) and were as follows: CASC9 forward, 5′-AGATGAAGCCGGTACCTCAGAT-3′ and reverse, 5′-TCA CTTTAAAGAGGGAGAGGAG-3′; checkpoint kinase 1 (CHK1) forward, 5′-CAGAATTTCAACCTTCGGTGTG-3′ and reverse, 5′-TCTTCACTGCGACTGCTTCTTC-3′; and GAPDH forward, 5′-ACAACTTTGGTATCGTGGAAGG-3′ and reverse, 5′-GCCATCACGCCACAGTTTC-3′. The PCR conditions were as follows: denaturation at 95℃ for 10 min, followed by 40 cycles of 95°C for 5 sec and 60℃ for 40 sec. GAPDH was used as an endogenous control to normalize the lncRNA CASC9 and CHK1 expression levels. The relative expression level was calculated using the 2^−ΔΔCq method (28). The experiments were performed in triplicate.

Cell transfection was performed using Lipofectamine 2000 (Invitrogen/Thermo Fisher Scientific) according to the manufacturer's instructions. The cells (5×10⁵ cells/well in 6-well plates, 70-80% confluency) were transfected with CASC9 mimics or si-CASC9. CASC9 mimics and small interfering RNA to knockdown CASC9 were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). Small interfering RNA sequences are listed as follows: si-CASC9 sense, 5′-TGTAAACTATAAAAATAAGC-3′ and si-CASC9 antisense, 5′-GCTTATTTTTATAGTTTACA-3′; and si-NC sense, ACAGGAGACAAATAAGCAT and anti-sense, ATGCTTATTTGTCTCCTGT. miR-195 inhibitor and miR-497 inhibitor were also designed by Shanghai GenePharma Co., Ltd. and used for the downregulation of miR-195 and miR-497, respectively. The sequences of miR-195 inhibitor and miR-497 inhibitor were as follows: miR-195 inhibitor, 5′-GCCAAUAUUUCUGUGCUGCUA-3′; miR-497 inhibitor, 5′-ACAAACCACAGUGUGCUGCUG-3′. The cells were harvested at 48 h post-transfection for use in further experiments.

Cell proliferation was assessed using the MTT Cell Proliferation and Cytotoxicity Assay kit (Sigma-Aldrich) according to the manufacturer's instructions. In brief, the cells were seeded in each well of a 96-well plate at a density of 1×10⁴ cells/well. Following incubation at 37°C for different periods of time (0, 24, 48 and 72 h), the culture medium was removed and MTT (20 µl; 5 mg/ml) was added to each well. Following incubation at 37°C for a further 4 h, the MTT solution was removed and replaced with dimethyl sulfoxide (DMSO; 150 µl, 4%; Sigma-Aldrich). The absorbance was measured at 560 nm using a microplate reader (BioTek Instruments GmbH, Bad Friedrichshall, Germany).

For apoptosis analysis, the cells were harvested at 48 h post-transfection. The cells were washed with ice-cold PBS and stained with Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kits (BD Biosciences, San Diego, CA, USA). The stained cells were measured using a flow cytometer (BD Biosciences).

RIP assays were performed using the EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation kit (Millipore, Billerica, MA, USA) according to the manufacturer's instructions. Briefly, RIP buffer containing magnetic beads conjugated with human anti-Ago2 antibody (1:500; 03-110; Millipore) or negative control IgG (1:500; ab172730; Abcam, Cambridge, MA, USA) was added to cell lysate and incubated overnight at 4℃. Proteinase K was used to digest the protein and the co-precipitated RNAs were then isolated. The purified RNAs were subjected to RT-qPCR analysis.

miRanda algorithms were used to predict the potential targets of lncRNA CASC9 and miR-197/495.

Wild-type lncRNA-CASC9 and mutant lncRNA CASC9 were inserted into pmirGLO reporter vectors (Promega, Madison, WI, USA), respectively. The MDA-MB-231 cells were co-transfected with miR-195 mimics and wild-type lncRNA CASC9 or mutant lncRNA CASC9 using Lipofectamine 2000 (Invitrogen/Thermo Fisher Scientific). The relative luciferase activity was measured on a dual-luciferase reporter assay system (Promega) at 48 h post-transfection. Data were expressed as the ratio of Renilla luciferase activity to Firefly luciferase activity. Luciferase reporter assays to validate the binding of miR-497 to lncRNA CASC9 were performed as described above.

Protein lysates were extracted from the cells using 500 µl radio immunoprecipitation assay (RIPA) buffer with 1 mM phenylmethane sulfonyl fluoride. The samples were subsequently sonicated for 2 min and centrifuged at 12,000 × g. The supernatants were collected and used for protein analysis. Protein concentrations were determined by BCA. Lysates (10 µl) were separated on 8% polyacrylamide gels and transferred onto PVDF membranes. The membranes were blocked with phosphate-buffered saline (PBS) containing 0.1% Tween-20 (PBST) and 5% non-fat milk (w/v) for 1 h at room temperature. After they were washed with PBST, the membranes were probed with the primary antibodies overnight at 4°C. The primary antibodies were as follows: Anti-CHK1 (ab40866), anti-cyclin D1 (CCND1; ab134175), anti-cyclin-dependent kinase 4 (CDK4; ab108357), anti-B-cell lymphoma 2 (Bcl2; ab32124), anti-caspase-3 (ab13585) and anti-GAPDH (ab181602) and were obtained from Abcam and used at the following dilutions: anti-CHK1 (1:1,000), anti-CCND1 (1:1,000), anti-CDK4 (1:1,000), anti-Bcl2 (1:1,000), anti-caspase-3 (1:1,000) and anti-GAPDH (1:3,000). The membranes were washed again with PBST, then horseradish peroxidase (HRP) labeled IgG (1:500; ab6728; Abcam) at a 1:5,000 dilution was added at room temperature for 1 h, and the blots were developed using ECL Western blotting reagents. Western blots were quantified by densitometry with Labworks 4.0 Software (UVP BioImaging Systems, Upland, CA, USA).

All animal experiments were performed in accordance with institutional and international animal regulations. The animal experimental protocol was approved by the Animal Care and Use Committee of Southern Medical University. Female BALB/c nude mice (6 weeks of age, weighing approximately 18 g) were purchased from Beijing HFK Bioscience Co., Ltd. (Beijing, China). Mice had ad libitum access to food and water. The MDA-MB-415 cells transfected with si-NC or si-CASC9 (1×10⁶ cells per mouse) were subcutaneously injected into the flanks of the BALB/c nude mice, respectively. The length and width of the tumors were measured using a caliper every 5 days. All the mice were euthanized using 4-5% isoflurane and sacrificed in a CO₂ chamber (flow rate of CO₂, 20% chamber volume per minute) at day 35 post-injection. The tumor nodules of the mice were then removed and weighed. The tumor volume was calculated according to the following equation: Tumor volume (mm³) = length (mm) x width (mm)²/2.

The samples were fixed in 10% neutral buffered formalin, embedded in paraffin, and sliced into thin sections (5 µm thickness). After being dewaxed and rehydrated, the sections were incubated with 3% H₂O₂ for 30 min to block the endogenous peroxidase (POD) activity. Following antigen retrieval (AR) by microwave (heating), 5% bovine serum albumin (BSA) was applied to block non-specific binding. The sections were then incubated with the primary antibodies at 4°C overnight. Anti-Ki67 (ab15580) and anti-GAPDH (ab181602) were obtained from Abcam and used at the following dilutions: anti-Ki67 (1:1,000) and anti-GAPDH (1:3,000). After being rinsed with PBS 3 times for 5 min each, the sections were treated with biotinylated secondary antibody (1:200; ab6728; Abcam) for 1 h, followed by incubation with streptavidin-horseradish peroxidase (HRP) at 37°C for 20 min. Diaminobenzidine (DAB) substrate was used as a color developing agent for the visualization of Ki67-positive cells.

The tissue slides were pre-hybridized in a hybridization solution (Boster Bioengineering Co., Ltd., Wuhan, China) at 37°C for 2 h. Subsequently, 10 picomoles of digoxigenin-labeled detection probes (Boster Bioengineering Co., Ltd.) complementary to lncRNA CASC9 were added and hybridized overnight at 37°C. Following stringent washes, an immunological reaction was carried out using a mouse monoclonal antibody to digoxigenin (1:200; ab6212; Abcam;), followed by the addition of alkaline phosphatase-conjugated streptavidin dilution (BD Biosciences) to detect the streptavidin dilution probes. The slides were mounted with aqueous mounting medium (Maixin Biotechnology, Fuzhou, China).

Apoptosis assay was performed using an In Situ Cell Death Detection kit (Roche Diagnostics, Basel, Switzerland). Briefly, the sections were blocked by incubation in 3% H₂O₂ in methanol for 5 min at 25°C. Subsequently, the sections were labeled with TdT labeling reaction mix at 37°C for 1 h. Nuclei exhibiting DNA fragmentation were visualized by incubation in 3,3′-diaminobenzidine (DAB) for 15 min at 25°C. The sections were observed under a light microscope (BX51; Olympus, Tokyo, Japan) and photographed.

Data are presented as the means ± standard deviation (SD). Statistical analysis was performed using SPSS 16.0 software (SPSS, Chicago, IL, USA). Two-tailed Student's t-test was applied to compare the differences between 2 groups and one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison was employed to compare the differences among 3 independent groups. The correlation between lncRNA CASC9 expression and miR-195, miR-497 or CHK1 mRNA expression in the BC tissues was identified using Pearson's correlation analysis. A value of P<0.05 was considered to indicate a statistically significant difference.

Although CASC9 has been reported to play a role in the carcinogenesis and progression of multiple types of human malignancies, its biological roles in BC remain poorly understood. In this study, initially, we carried out RT-qPCR analysis to detect the expression of lncRNA CASC9 in 17 pairs of BC tissues and corresponding para-cancerous tissues. As presented in Fig. 1A, lncRNA CASC9 expression was significantly upregulated in the BC tissues compared with the matched adjacent normal tissues (P<0.01). To further investigate the differences in lncRNA CASC9 expression between the BC tissues and their matched non-cancerous tissues, we performed ISH analysis to visualize the expression of lncRNA CASC9. As shown by ISH analysis, the BC tissues exhibited higher expression levels of lncRNA CASC9 than the matched non-cancerous tissues (Fig. 1B). Consistently, lncRNA CASC9 expression was markedly upregulated in the BC cell lines (MDA-MB-231, MDA-MB-468, MCF7 and MDA-MB-415) compared with the normal human mammary epithelial cell line, MCF-10A (P<0.01, Fig. 1C). The MDA-MB-231 cells (lowest endogenous lncRNA CASC9 expression) were selected for overexpression experiments. The MDA-MB-415 cells (highest endogenous lncRNA CASC9 expression) were selected for knockdown experiments. Taken together, these findings indicated that lncRNA CASC9 expression was significantly upregulated in the BC tissues and cell lines.

To explore the biological role of CASC9 in BC, overexpression experiments were conducted using the MDA-MB-231 cells. In addition, knockdown experiments were performed using the MDA-MB-415 cells. The transfection efficiency was determined by RT-qPCR analysis (Fig. 2A). As shown by the results of MTT assays, MDA-MB-231 cell proliferation was markedly accelerated by transfection with CASC9 mimics compared with the negative control group, whereas CASC9 knockdown markedly attenuated MDA-MB-415 cell proliferation (P<0.01; Fig. 2B). As shown in Fig. 2C, transfection with CASC9 mimics promoted MDA-MB-231 cell cycle progression, while cell cycle arrest was observed in the MDA-MB-415 cells transfected with si-CASC9 (P<0.01). Additionally, CASC9 overexpression was found to markedly suppress MDA-MB-231 cell apoptosis compared with the negative control group, whereas MDA-MB-415 cell apoptosis was significantly promoted by CASC9 knockdown (P<0.01; Fig. 3).

In addition, we examined the effects of lncRNA CASC9 on the expression of cell proliferation-associated proteins and apoptosis-associated proteins in the BC cells. Lower expression levels of CCND1, CDK4 and Bcl2, and a higher expression level of caspase-3 were observed in the MDA-MB-231 cells transfected with CASC9 mimics compared with the negative control group (P<0.01; Fig. 4A), whereas CASC9 knockdown was found to elevate the expression levels of CCND1, CDK4 and Bcl2, and to decrease the expression of caspase-3 in comparison with the negative control group (P<0.01; Fig. 4B). On the whole, these results suggest that CASC9 facilitates BC cell proliferation, accelerates cell cycle progression and suppresses cell apoptosis.

Recent studies have suggested that lncRNAs function as miRNA sponges to participate in the occurrence and development of multiple types of human neoplasms (29,30). To elucidate the underlying mechanisms through which lncRNA CASC9 facilitates BC cell proliferation and suppresses cell apoptosis, we applied miRanda online software to predict the potential targets of lncRNA CASC9. The miR-195/497 cluster, frequently reported to be involved in diverse types of human tumors, was selected as a candidate target of lncRNA CASC9 (Fig. 5A). Moreover, we constructed luciferase reporter vectors carrying the predicted miR-195/497 binding site (wild-type lncRNA CASC9) or its corresponding mutant fragment (mutant lncRNA CASC9). As illustrated in Fig. 5B, the co-transfection of wide-type lncRNA CASC9 and miR-195 or miR-497 markedly reduced the luciferase activity, while the co-transfection of mutant lncRNA CASC9 and miR-195 or miR-497 failed to affect the luciferase activity (P<0.01). To validate the interaction between lncRNA CASC9 and the miR-195/497 cluster, anti-Ago2 RIP assays were carried out to pull down the endogenous miRNAs interacting with lncRNA CASC9 in the MDA-MB-231 cells. miR-195 and miR-497 were found to be notably enriched by lncRNA CASC9 mimics in comparison with the negative control group (P<0.01; Fig. 5C). These data indicate that lncRNA CASC9 interacts with the miR-195/497 cluster in BC cells.

As illustrated in Fig. 6A, miR-195 and miR-497 expression levels were found to be markedly downregulated by transfection with CASC9 mimics in the MDA-MB-231 cells compared with the negative control group, whereas CASC9 knockdown notably upregulated the miR-195 and miR-497 expression levels in the MDA-MB-415 cells (P<0.01). To further elucidate the potential mechanisms through which lncRNA CASC9 promotes BC cell proliferation and survival, the potential targets of the miR-195/497 cluster were predicted using the miRanda algorithm (Fig. 6B). Among all the putative targets of the miR-195/497 cluster, CHK1 attracted our attention for its crucial role in the tumorigenesis and progression of various types of human malignancies. A previous study identified CHK1 as a target of miR-497 in hepatocellular carcinoma (31).

To investigate whether lncRNA CASC9 regulates CHK1 expression, we transfected the BC cells with CASC9 mimics or si-CASC9. The results of western blot analysis demonstrated that CHK1 protein expression was significantly enhanced by transfection of the MDA-MB-231 cells with CASC9 mimics compared with the negative control, whereas CASC9 knockdown was discovered to suppress CHK1 expression in the MDA-MB-415 cells (P<0.01; Fig. 6C). Notably, Pearson's correlation analysis revealed that lncRNA CASC9 expression negatively correlated with miR-195 and miR-497 expression in the BC tissues; on the contrary, lncRNA CASC9 expression was found to positively correlate with CHK1 mRNA expression in the BC tissues (P<0.01; Fig. 6D). These results suggest that lncRNA positively regulates the expression of CHK1 through sponging the miR-195/497 cluster in BC cells.

To determine whether the effects of CASC9 on BC cell proliferation and apoptosis are mediated by the miR-195/497 cluster, we downregulated the expression of miR-195 or miR-497 in the MDA-MB-415 cells transfected with si-CASC9. As shown in Fig. 7A, the inhibitory effects of CASC9 knockdown on BC cell proliferation were partially reversed by the downregulation of miR-195 or miR-497. Furthermore, miR-195 or miR-497 downregulation was found to attenuate MDA-MB-415 cell apoptosis induced by CASC9 knockdown (P<0.01; Fig. 7B). The results of western bot analysis also revealed that the use of the miR-195 or miR-497 inhibitor significantly upregulated the expression of CHK1 in the MDA-MB-415 cells transfected with si-CASC9 (P<0.01; Fig. 8A). As shown in Fig. 8B, the use of the miR-195 or miR-497 inhibitor partially reversed the inhibitory effects of si-CASC9 on CCND1, CDK4 and Bcl2 expression, and attenuated the promoting effects of si-CASC9 on caspase-3 expression in the MDA-MB-415 cells (P<0.01). These data indicate that the promoting effects of CASC9 on BC cell proliferation and survival are mediated by the miR-195/497 cluster.

With the aim of investigating the effects of CASC9 on breast tumor growth in vivo, we performed tumor xenograft model assays. As presented in Fig. 9A, CASC9 knockdown markedly inhibited breast tumor growth in vivo (P<0.01). Immunohistochemistry revealed that Ki67 expression in the tumors removed from the nude mice was significantly suppressed in the mice injected with cells transfected with si-CASC9 compared with the mice injected with cells from the negative control group (Fig. 9B). Furthermore, TUNEL staining demonstrated that CASC9 knockdown markedly facilitated cell apoptosis in the tumors separated from the nude mice (Fig. 9C). Additionally, CASC9 knockdown was found to suppress CHK1 mRNA and protein expression in the separated tumors (P<0.01; Fig. 9D and E). Taken together, these data suggest that the knockdown of CASC9 inhibits breast tumor growth in vivo.