RNA-binding protein RNPC1: acting as a tumor suppressor in breast cancer

RNA extraction, reverse transcription and quantitative RT-PCR (qRT-PCR)

Total RNA was extracted from cells and tissues using Trizol reagent (TaKaRa, A-79061), and cDNA was synthesized using Primescript RT Reagent (TaKaRa) following manufacturer's instructions. The following PCR primers were used:

RNPC1 forward, 5′-ACGCCTCGCTCAGGAAGTA3-′

RNPC1 reverse, 5′-GTCTTTGCAAGCCCTCTCAG3-′

β-actin forward, 5′-GCTGTGCT ATCCCTGTACGC3-′

β-actin reverse, 5′-TGCCTCAGGGCAGCGGAACC3-′

qRT-PCR for β-actin and other genes was performed for every cDNA sample. All PCR reactions were performed using the fluorescent SYBR Green I methodology. Real-time quantitative PCR was performed on StepOne Plus Real-Time PCR system (Applied Biosystems, USA) using FastStart Universal SYBR Green Master (Roche, Switzerland) according to the manufacturer's instructions. As a result, the relative gene expression was normalized, with β-actin serving as the internal control. Noticeable, this study showed clearly RNPC1instead of RNPC1a.

Tissue samples

121 pairs of snap-frozen breast tumor and matched normal tissues from adjacent regions were provided by the First Affiliated Hospital with Nanjing Medical University from February 2006 to August 2009, from patients treated surgically for clinical stage I–III breast cancer (aged 34–82 years). All the patients did not receive chemotherapy, radiotherapy or hormone therapy before surgery. Tumor and normal tissue samples had been verified as tumor or non-tumor by histopathological examination of hematoxylin stained paraffin sections. Histologic types were classified according to the World Health Organization (2003). TNM staging was defined according to the American Joint Committee on Cancer (AJCC) (the 6th version, 2002). All the cases were individually categorized by independent pathologists. All the samples’ collection was according to the ethical guidelines of the Declaration of Helsinki and approved by the ethics and research committee of the First Affiliated Hospital of Nanjing Medical University. Before surgery patients are informed that their surgical specimens would possibly be used for research purposes. All the participants provided their written informed consent for inclusion in the data analysis and manuscript publication. Data were analyzed anonymously.

Cell culture

The human breast cancer cell lines (MCF-7, MDA-MB-231, BT474 and ZR-75) and non-malignant breast epithelial cells (MCF-10A) were obtained from the American Type Culture Collection (ATCC, VA, USA) and culture in complete medium of High glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin - streptomycin solution at 5% CO₂ and 37°C incubator. Cell line SUM1315 was provided by Stephen Ethier (University of Michigan). The 184A1 immortalized breast epithelial cell line was provided by Ceshi Chen (Kunming Institute of Zoology).

Plasmid construction and lentivirus packaging

Lentivirus packaging cells were transfected with PGLV3-h1-GFP-puro vector (GenePharma, Shanghai, China) or pGLV5-h1-GFP-puro vector (GenePharma, Shanghai, China) containing either the RNPC1a knockdown (shRNPC1a) or RNPC1a overexpression (RNPC1a), and a scrambled sequence (SCR) or a negative control sequence (NC), respectively, following the manufacturer’s instructions. Three shRNA plasmids (sh1, sh2, sh3) were constructed against different RNPC1a targets, including a scrambled sequence as a negative control (Additional file 1: Table S1). All plasmids were verified by sequencing (GenePharma, Shanghai, China). Cells were plated in 6 wells dishes at 30% confluence and infected with the retroviruses. Meanwhile, polybrene (5 μg/ml) was added with the retroviruses to enhance the target cells infection efficiency. Stable pooled populations of breast cancer cells were generated by selection using puromycin (2 μg/ml) for 2 weeks. For knockdown, one construct (sh3), with ≥85% knockdown efficiency, was used for further studies (Additional file 1: Figure S1).

Colony formation assay

Cell used for colony formation analysis were seeded into 6-well plates (500 cells/wells) and cultured normally for 15–20 days. The colonies were fixed in paraform and stained with Giemsa after washed by phosphate-buffered saline (PBS) twice, then dried at room temperature. The colonies in each well were counted, and all cell colonies contained 50 or more cells.

Cell counting kit (CCK-8) assay

Cell proliferation was assessed using CCK-8 kit (Dojindo, Japan) according to the manufacturer’s instructions/protocol. Cells diluted serum-free medium, 2,000 cells/wells were seeded in a 96-well cell culture plate, grown at 37°C on the day of measuring the growth rate of cells, 100 μl of spent medium was replaced with an equal volume of fresh medium containing 10% CCK8, then cells continued to be incubated at 37°C for 3 h, and the absorbance was finally determined at 450 nm using a micro plate reader (5082 Grodig, Tecan, Austria).

Wound healing assay

Breast cancer cells were seeded into 6-well plates, and allowed to grow until 100% confluens. Then the cell layer was gently scratched through the central axis using a sterile plastic tip and loose cells were washed away. Quantification of cell motility by measuring the distance between the invading fronts of cells in three random selected microscopic fields (200×) for each condition and time point (0, 18 h).

Cell migration and invasion assays

In vitro cell migration and invasion assays were performed as described previously [31]. Images of three random fields (200×) were captured from each membrane, and the number of migratory or invasive cells was counted.

Tumorigenesis in nude mice

BALB/C female nude mice (4-6-weeks old, 18–22 g) were randomly divided into two groups (each containing 7 mices). Stable RNPC1a-expression MDA-MB-231 cells or control cells (1 × 10⁶ cells in 0.1 ml PBS) was subcutaneously orthotopically injected into mammary fat pads of the mice and the growth of tumors was followed up for 6 weeks. Tumor volume was measured weekly using a caliper, calculated as (tumor length × width²)/2. After 6 weeks, mice were sacrificed and checked for final tumor size. Mouse studies were conducted according to the Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of Nanjing Medical University. All the samples’ collection was according to the ethical guidelines of the Declaration of Helsinki and approved by the ethics and research committee of the First Affiliated Hospital of Nanjing Medical University.

Western blotting analysis

Western blot analysis was performed as described previously [32]. The primary antibodies used were anti-rabbit RBM38 (Santa Cruz), p21 (Santa Cruz), p53 (Santa Cruz), p53 (Millipore), Vimentin (Abcam), anti-mouse E-cadherin (Abcam). The secondary antibodies were purchased from Cell Signaling technology. The intensity of the bands was determined using densitometric analysis. GAPDH (Santa Cruz) was used to as loading control.

DNA histogram analysis

Cell cycle was assessed by flow cytometry (Becton Dickinson, San Jose, CA, USA). For cell cycle analysis, cells were collected, washed with PBS and fixed in ethanol at −20°C for 8 h before being collected by centrifugation. Then cells were washed with PBS, and resuspended in 500 μl of PBS with 0.2% Triton X-100, 10 mM EDTA, 100 μg/ml RNase A, and 50 μg/ml propidium iodide (PI) at room temperature for 30 min.

Statistical analysis

The data were analyzed using the SPSS 12.0 software (SPSS, Chicago, IL, USA). All experiments in this study were repeated in triplicate, unless otherwise specified. Student t-test was used to analyze the statistical significance of the differences between groups. χ² test and Fisher Exact test were used to assess the correlation between RNPC1 and clinicopathologic parameters. For all the tests p values < 0.05 was considered statistically significant.

RNPC1 was lower expressed in human breast cancer cells

RNPC1 expression in five breast cancer cell lines and two breast epithelial cell lines were quantified by qRT-PCR and Western blot (Figure 1A, p < 0.05). Among the seven cell lines analyzed, RNPC1 was found lower expression in breast cancer cells compared to normal mammary breast epithelial MCF-10A and 184A1 cells. Among breast cancer cells, MCF-7, BT474, ZR-75 cells expressed relatively higher levels of RNPC1, and low expression or barely detectable levels were found in MDA-MB-231, SUM1315. Noticeable, this study showed that clearly MB-231 could instead of MDA-MB-231.

RNPC1 protein and mRNA expression were down-regulated in human breast cancer tissue

RNPC1a inhibited proliferation and growth in human breast cancer cells in vitro

To further address the functions of RNPC1 in breast cancer cells, we infected MCF-7 cells and MDA-MB-231 cells and selected stably infected cells. The over-expressed cell lines were named as MCF-7-RNPC1a or MB-231-RNPC1a, while the matched control cell lines were named as MCF-7-NC or MB-231-NC, respectively. The silenced cell line was named as MCF-7-shRNPC1a or MB-231-shRNPC1a, while the matched control cell lines were named as MCF-7-SCR or MB-231-SCR, respectively. We confirmed the expression levels using both Western blot (Figure 2A and E) and qRT-PCR (Figure 2B and F, both p < 0.001).

The growth of the stable cell lines over 6 days was determined using Cell counting kit (CCK-8) assay. As shown in Figure 2C and Figure 2D, RNPC1a overexpression led to significantly decreased cell proliferation (p < 0.05), while RNPC1a knockdown led to significantly increased cell proliferation (Figure 2G and H, both p < 0.05). To further study the mechanism by which RNPC1a overexpression or knockdown affected proliferation, cell cycle progression was analyzed using flow cytometry. MCF-7-RNPC1a cells showed a delayed G1 phase compared to MCF-7-NC cells (65.28 ± 1.495 vs 54.28 ± 1.121) (Figure 3B, p < 0.05), while MB-231-RNPC1a cells also showed a delayed G1 phase compared to MB-231-NC cells (37.74 ± 2.559 vs 28.44 ± 1.033) (Figure 3B, p < 0.05). RNPC1a overexpression inhibited the proliferation of breast cancer cells via a delay in cell cycle progression. We obtained the similar results in RNPC1a knockdown MCF-7 (Additional file 2: Figure S2).

Since anchorage-independent growth is strongly correlated with tumorigenicity [33]. The ability of MCF-7 or MB-231 cell lines to form colonies was much fewer when RNPC1a was over-expressed (Figure. 3C, p < 0.05). The ability of MCF-7 or MB-231 cell lines to form colonies was much more when RNPC1a was knockdown (Figure. 3D, p < 0.05).

RNPC1a suppressed migratory and invasive potential

As shown in Figure 4A and C, determined by their migration in the wound gap after 18 h, distance migrated of RNPC1a overexpression decreased by 69 μm (Figure 4A, p < 0.01), while RNPC1a knockdown increased by 110 μm (Figure 4C, p < 0.01) compared to the control cells, respectively. We conducted three-dimensional cell migration assay using transwell chambers and invasion assay with Matrigel-precoated transwell chambers. We found that RNPC1a overexpression exhibited significantly decrease ability of migration and invasion (Figure 4B, both p < 0.01). RNPC1a knockdown exhibited significantly increase ability of migration and invasion (Figure 4D, both p < 0.05). Besides, we obtained the similar results of MCF-7 cells (Additional files 3: Figure S3).

RNPC1a down-regulate mutp53 and up-regulate p21 protein expression in breast cancer cells

Previous study affirmed that translational of wild-type p53 (wtp53) was repressed by RNPC1a [17]. However, our study found wtp53 protein was no significantly altered in RNPC1a over-expressed or silent MCF-7 cells (Figure 5A). Level of p21 protein was increased in RNPC1a over-expressed MCF-7 and MDA-MB-231 cells (Figure 5A and B). Mutp53 protein was decreased in RNPC1a over-expressed MDA-MB-231 cells. When RNPC1a was silenced, p21 protein was decreased in MCF-7 and MDA-MB-231 cells, while mutp53 was increased (Figure 5B).

RNPC1a up-regulate E-cadherin and down-regulate vimentin protein expression in breast cancer cells

We observed that the RNPC1a knockdown in MCF-7 cells led to a spindle-shaped fibroblastic morphology. This morphological change might suggest the phenotypic change of EMT. In addition, RNPC1a over-expressed MDA-MB-231 cells lost their fibroblast-like morphology, which was accompanied by a cobblestone-like epithelial morphology (data not shown). As shown in Figure 5C and Figure 5D the levels of E-cadherin expression was increased in the RNPC1a over-expressed cells, while decreased in the RNPC1a knockdown cells. The levels of Vimentin expression was increased in the RNPC1a knockdown MDA-MB-231 cells, while decreased in the RNPC1a over-expressed cells in MDA-MB-231. But MCF-7 cells were not obviously changed in the protein level of the mesenchymal markers such as Vimentin.

RNPC1a suppressed tumorigenesis in nude mice

To evaluate the tumor-suppressive functions of RNPC1a in vivo, tumorigenicity of MDA-MB-231 cells expressing RNPC1a was evaluated in nude mice. Over-expressed RNPC1a and control cells were injected into mammary fat pads of the mice. Control cells were discovered tumors after 2 weeks, while tumors derived from over-expressed RNPC1a cells were discovered after 4 weeks (Figure 6A, p < 0.05). RNPC1a over-expressed cells formed smaller tumor volume and weight compared to the control cells (Figure 6B, p < 0.01).