Long non-coding RNA MALAT-1 modulates metastatic potential of tongue squamous cell carcinomas partially through the regulation of small proline rich proteins

This study was approved by Ethics Committee of Peking University Health Science Center (IRB00001053-08043). TSCC samples were obtained from 127 patients of the Department of Oral & Maxillofacial Surgery, Shenzhen Hospital, Peking University. A summary of cohort characteristics was listed in Table 1. A detailed description of tumor characteristics was listed in Additional file 1: Table S1. Adjacent normal mucosa tissues located at least 1.5 cm far from the macroscopically unaffected margins of the tumor were defined as normal controls. All the TSCC samples were graded in 4 groups according to common criteria of SCC staging: Stage1 (less than 2 centimeters in size and has not spread to lymph nodes in the area; n = 23), Stage2 (more than 2 cm in size, but less than 4 cm, and has not spread to lymph nodes in the area; n = 55), Stage3 (more than 4 cm in size/ has spread to only one lymph node on the same side of the neck as the cancer; n = 38), Stage4 (has spread to tissues around the lip and oral cavity/ has spread to more than one lymph node on the same side of the neck as the cancer, to lymph nodes on one or both sides of the neck, or to any lymph node that measures more than 6 cm/ has spread to other parts of the body, n = 11). The TSCC tissues were collected from patients undergoing surgical excision. Matched samples of TSCC (n = 127) and normal oral squamous cell mucosa (n = 127) were subjected to real-time PCR analysis. All patients were informed about the aims of specimen collection and gave signed written consent in accordance with the ethical guidelines of Peking University.

Total RNA was isolated from tissues by using a AxyPrepTM Blood Total RNA MiniPrep Kit (Axygen, US) according to the manufacturer’s instruction. First strand cDNA was synthesized with a RevertAidTM First Stand cDNA Synthesis Kit (Fermentas, US) using random hexamar primer. Quantitative PCR was performed through BioRad Chromo4 real-time PCR system. The primer sets for amplifying MALAT-1 and other related genes were listed in Table 2. Since “housekeeping” gene may have differential expression in the tissue types being evaluated [17], we compared the expression of 16 reference genes in 30 paired TSCC, ANT and LNM samples (Additional file 2: Figure S1). The sequences of the selected reference genes were listed in the Additional file 1: Table S2. We selected ACTB as the reference gene in analyzing the results. At the end point of PCR cycles, melt curves were made to check product purity. The level of MALAT-1 was expressed as a ratio relative to the β-actin mRNA in each sample. Exploratory data analysis using box plot was applied to visually identify the expression level of target mRNA.

Human tongue squamous cell carcinoma cell line CAL 27 and SCC-25 (CRL-2095™ & CRL-1628™) was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) where they were characterized by mycoplasma detection, DNA -Fingerprinting, isozyme detection and cell vitality detection. These cell lines were purchased in August 2012 and immediately expanded and frozen so that they could be restarted every 3 to 4 months from a frozen vial of the same batch of cells. CAL 27 and SCC-25 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO, US) supplemented with 10 % fetal bovine serum (PAA) and 1 % penicillin/ streptomycin (Life Technologies Inc., US).

To generate lentiviruses expressing MALAT-1 shRNA and control shRNAs, HEK293T cells grown on 10 cm dish were transfected with 6 μg of MALAT-1 shRNAs (cloned in PLKO.1) or control vector, 6 μg of pREV, 6 μg of pGag/Pol, and 2 μg of pVSVg. 12 h after transfection, cells were cultured with DMEM medium containing 20 % FBS for an additional 36 h. The culture medium containing lentivirus particles was centrifuged at 10000 × g for 2 min and then used for infection. 24 h after infection, cells were cultured with fresh medium for another 24 h, followed with further experiment. The knockdown efficiency was evaluated by real-time PCR analysis. The shRNA sequences targeting MALAT-1 are “ATG GAG GTA TGA CAT ATA AT” and “GGG AGT TAC TTG CCA ACT TG” [18].

Cell proliferation was measured by Cell Proliferation Reagent WST-1 (Roche, USA) as introduced previously [19]. Cells were counted and plated in 96-well culture plates (1 × 10³ per well); WST-1 assay measuring the activity of mitochondrial dehydrogenases was performed following the manufacturer’s instructions at 0-, 1-, 2-, 3-, and 4-day time points.

Migration assays were performed using 24-well Trans-well units with 8 mm pore size polycarbonate inserts (BD Biosciences, US). Trans-wells were coated overnight with 10 mg/ml of fibronectin in PBS at 48 °C, followed by incubation with 1 % BSA for 1 h at 37 °C. The SCC-25 and CAL27 cells transfected with shRNA (MALAT-1 shRNA) or plasmids (SPRR expression vectors and mock vectors) were detached with trypsin/EDTA, washed once with DMEM containing 10 % FBS, and re-suspended in DMEM containing 1 % FBS at 2 × 10⁵ cells/ml. Aliquots (100 microliters) of cell suspensions were directly added to the upper side of each chamber. Following incubation for 12 h, the cells on the upper side of the membrane were removed, whereas the cells that migrated to the underside were fixed with 3 % formaldehyde and stained with 0.3 % crystal violet for 10 min. The number of cells on the underside of the membrane was counted in five different fields with a light microscope at 100×, and the mean and SD was calculated from three independent experiments.

After washing the cells with 50 mM potassium phosphate buffer (pH 7.4), the total RNA of each sample was extracted by RNeasy Mini Kit (Qiagen, US). The procedure for the extraction of the total RNA was according to the manufacturer’s instruction. The quality of the extracted RNA was confirmed with Bioanalyzer 2100 (Agilent Technologies, US). GeneChip(R) arrays (Affymetrix) were used as the DNA microarrays. DNA microarray analysis was performed with Bio Matrix Research. Statistical analysis after data acquisition and normalization of expression data was performed using GeneSpring (Agilent Technologies, US). For the pathway- or function-based category classification, the Munich Information Center for Protein Sequence (MIPS) was used.

Cells were washed with PBS and lysed in a buffer containing 50 mM Tris-HCl (pH 6.8), 2 % SDS, 10 % glycerol, phosphatase inhibitors (100 mM Na₃VO₄, 10 mM NaF) and protease inhibitor (1 mM PMSF). Equal amounts of protein were loaded on a SDS-PAGE and transferred to PVDF membrane. After blocking with 5 % non-fat milk in TBS-T (containing 0.1 % Tween-20), the membranes were incubated with specific primary antibodies, followed by HRP-conjugated secondary antibodies. Proteins were visualized by fluorography using an enhanced chemiluminescence system. Antibodies for SPRR1B, 2A (Abcam, US), 2E (Abnova, US) and β-actin (Sangon,Shanghai, China) were purchased as the primary antibodies for the approach.

The animal experiments were approved by the Ethics Committee of Peking University Health Science Center (IRB00001053-09028). Six-week-old male nude mice (Zi Guang Laboratory Animal Technology Co. Ltd., Guangdong, China) were placed under general anesthesia with 1 % pentobarbital sodium (Sigma). SCC-25/CAL 27 cells (5 × 10⁶) were injected subcutaneously (15 mice each group, and additional 15 mice for CAL27-Mock and CAL27-MALAT1KD cells). Metastasis was assayed by gross examination at autopsy and by PCR for Alu sequences in various organs. Control cells including SCC-25 and CAL27 cells caused grossly evident metastasis within the first 8 weeks and all animals were sacrificed at this time point. On the contrary, mice receiving MALAT-1 shRNA-transfectants were healthy at 8 weeks, but several were sacrificed for comparison, while the remaining mice were followed for an additional 4 weeks to determine if metastatic tumors developed. The volume of xenograft was calculated as v = 3/4πab² (a = length, b = width). The average volume of the xenografts at sacrifice were listed in the Additional file 1: Table S3. Grossly obvious tumors and metastases were dissected and fixed immediately with 4 % paraformaldehyde for pathological analysis (Some of the animal models as well as metastases were shown in the Additional file 3: Figure S2).

The cloned SPRR1B & 2A cDNA fragment were inserted into pcDNA3.1 expression vector to construct the expression vectors. To produce stable transfectants, pcDNA-SPRR1B & 2A as well as mock plasmids were stably transfected into the CAL27/SCC25 line using Lipofectamine 2000 reagent (LF2000, Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations. Selection was performed via the addition of 1 mg/ml G418. The transfectants from the backbone vector and pcDNA3-SPRR1B/2A were designated as mock-CAL27/SCC25 and SPRR1B/2A-CAL27/SCC25, respectively.

GraphPad Prism software (Version 5.0) was used to analyze the obtained data. Results of the MALAT-1 lncRNA expression for paired TSCC and ANT samples or paired TSCC and local lymph-node metastasis were compared using paired t-test. Results of the MALAT-1 lncRNA expression for different TSCC groups were compared using non-parametric Mann-Whitney test. Data of in-vitro experiments were analyzed using the chi-square test or Fisher exact test. Differences of the metastasis between different groups of mouse models were analyzed using Chi-square test. P-values less than 0.05 were considered statistically significant.