After obtaining Our University Ethics Committee approval and informed consent from all study participants, tissue samples and blood samples were drawn at the Department of Otorhinolaryngology, Shengjing Hospital, China Medical University in 2010 and 2011. Up to 8 ml of whole blood were collected from each participant in an ethylene diamine tetracetic acid tube. Blood samples were centrifuged at 1,200 × g for 10 min at 4°C to separate the blood cells, and the supernatant was transferred into microcentrifuge tubes and then centrifuged a second time at 12,000 × g for 10 min at 4°C to completely remove the cellular components. Plasma was aliquoted and stored at −80°C until use. Blood samples were processed and plasma was frozen within 4 h of collection.

Total RNA (2 μg) was reverse-transcribed using Transcript First-strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) according to the manufacturer’s protocol. In short, the 50 μl reactions were incubated for 60 min at 42°C, 10 min at 70°C, and then stored at 4°C. qRT-PCR analyses were performed using the Bulge-Loop™ miRNA qRT-PCR Detection kit (Ribobio Co., Guangzhou, China) and TransStart™ Green qPCR SuperMix (TransGen Biotech) according to with the manufacturer’s protocol with the Rotor-Gene 6000 system (Qiagen, Hilden, Germany). Briefly, the reactions were incubated at 95°C for 30 sec, followed by 40 cycles of 95°C for 30 sec, 60°C for 20 sec, 70°C for 1 sec. The relative expression level for miRNA-126 was computed using the comparative CT method. It is important to note that to control for possible diversity in the amount of starting RNA, miRNA expression was normalized to small nucleolar RNA U6.

The CellSearch system (Veridex, Warren, NJ, USA) is the only test sanctioned by the United States Food and Drug Administration for enumeration of CTCs in clinical practice. Blood samples (5 ml) from patients were drawn into CellSave tubes, which were maintained at room temperature and processed within 72 h of collection. CTCs were defined as nucleated epithelial cell adhesion molecule (EpCAM)-positive cells, lacking cluster of differentiation (CD)45 but expressing cytoplasmic cytokeratins 8, 18 and 19. All CTC evaluations were performed by qualified and trained personnel.

Specimens were lysed using lysis buffer [50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM Na₃VO₄, 1 mM NaF, protease inhibitor cocktail]. The extracts were incubated on ice for 20 min, centrifuged at 12,000 × g for 20 min at 4°C and supernatants collected. Protein concentrations were determined using Bradford assay (Bio-Rad, Hercules, CA, USA), and proteins were resolved by 10% Bis-Tris gel electrophoresis, transferred to a nitrocellulose membrane and western blot analysis performed. Anti-Camsap1 and anti-β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

The human laryngeal cancer cell line Hep-2 was purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The cells were cultured in PRMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS) in a humidified cell incubator with an atmosphere of 5% CO₂ at 37°C. Exponentially growing cells were used for experiments.

Hep-2 cells were transfected with Precursor Molecules mimicking miR-126 (Pre-miR-126) (Applied Biosystems, Foster, CA, USA) or Camsap1 siRNA (sc-92757, Santa Cruz) by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

All experiments with animals were performed according to the guidelines of the China Medical University Ethics Committee. NU/NU Nude mice (Crl: NU-Foxn1nu) aged 6-8-weeks were purchased from Charles River (Wilmington, MA, USA). Hep-2 cells (3×10⁷ in 200 μl) were subcutaneously injected into the axilla of each mouse. After the tumor diameters reached 3–5 mm, the mice were divided randomly into three groups (untreated, miR-126 mimic, Camsap1 knockdown) and received a 100 μl intratumoral injection of PBS, miR-126 mimic, Camsap1 siRNA. Three injections were administered at 9 a.m., 3 p.m. and 9 p.m. every three days. Tumor growth was then monitored for 30 days. Every five days until the end of the experiment, one mouse from each group was randomly selected to be anesthetized, photographed and sacrificed. For each tumor, measurements were made using calipers, and tumor volumes were calculated as follows: length × width² × 0.52. Tumors were subsequently fixed in 4% paraformaldehyde for 24 h, then embedded in paraffin.

Additional mice (n=60) were used to establish xenografts to obtain survival curves. Mice with xenografted tumors (as described above) that reached 3–5 mm in diameter were divided into three treatment groups (n=20 for each). Survival was monitored until the experiments were terminated due to heavy tumor burden.

For immunohistochemical staining of CD31, endogenous peroxidase activity was blocked in 4-μm tumor sections with 3% hydrogen peroxide for 30 min. Antigen retrieval was performed in citrate buffer (10 mM, pH 6.0) for 30 min at 95°C in a pressure cooker. CD31 antibodies (Sigma-Aldrich, Carlsbad, CA, USA) were incubated with sections at 1:500 overnight at 4°C. Sections were then incubated with a biotinylated secondary antibody for 1 h at RT, followed by incubation with a streptavidin horse-radish peroxidase (HRP) complex (Beyotime, Beijing, China) for 60 min at room temperature. Bound antibody was visualized with 3,3’-diaminobenzidine tetrahydrochloride (DAB, Beyotime). Sections were also counterstained with hematoxylin (Beyotime). Microvessel density was detected using the method of Ivkovic-Kapic et al (15). Regions of highest vessel density were located at low magnification (×40), then the number of vessels present were counted at ×200 magnification. Three high magnification fields were counted for each tumor section and the mean microvessel density value was recorded for each. Any individual endothelial cells, or endothelial cell cluster, that was clearly separated from adjacent microvessels was counted as a single microvessel.

The miRNA targets predicted by TargetScan (http://www.targetscan.org/) are based on the presence of conserved 8 mer, 7 mer and 6 mer sites that match the seed region of each miRNA (16).

Tubulin was absorbed for 1 min onto glow-discharged formvar- and carbon-coated grids. The samples were stained in 1.5% uranyl acetate for 25 sec. Images were recorded on a FEI Morgagni 268D transmission electron microscope. Tubulin oligomers were imaged at ×120,000 magnification resulting in a pixel size of 0.53 nm.

Transfected cells were washed with PBS, fixed in 4% paraformaldehyde, permeabilized in 1% Triton X-100 for 5 min, and blocked with 5% bovine serum albumin in PBS containing 0.5% Triton X-100 for 1 h. Camsap1 expression and tubulin expression were detected using anti-Camsap1 (Santa Cruz) antibody and anti-tubulin (Santa Cruz) antibody, respectively. Cells were washed with PBS and incubated with appropriate secondary fluorophore-conjugated antibody for 1 h at room temperature. Secondary antibody used for detection of Camsap1 was Alexa Fluor^® 488 donkey anti-goat IgG (H+L) (Invitrogen) and tubulin was Alexa Fluor^® 594 rat anti-mouse IgG (H+L) (Invitrogen).

Thirty-four separate protein sequences of Camsap1 from a wide range of organisms were extracted from NCBI. An alignment of these sequences was made using Mega 5.0. To determine the phylogenetic relationships of these sequences, we used maximum likelihood (ML), neighbor-joining (NJ), and Bayesian Markov chain Monte Carlo (MCMC) approaches to infer three individual trees.

All statistical analyses were carried out using SPSS version 17.0 (Statistical Package for the Social Sciences). The experiments were conducted in triplicates. All numerical data are expressed as the means ± SD. Differences among the mean values were evaluated using Student’s t-test. P-values <0.05 were considered statistically significant.

Using qRT-PCR assays, we measured the circulating levels of miR-126 in the patients. Based on this result, we analyzed the potential relationship between the circulating levels of miR-126 and the clinicopathological characteristics of the sampled patients. Results are summarized in Table I. No correlation was found with sex, age, lymph node metastasis, T classification and clinical stage (P>0.05). However, miR-126 expression was significantly associated with differentiation of LSCC (P<0.05). Western blot analysis and real-time PCR analysis were performed in order to determine the levels of Camsap1 protein and mRNA in LSCC specimens. Both Camsap1 protein and mRNA in cancer tissue was significantly higher than that in matched normal tissue (P<0.05, Fig. 1).

In the present study, we isolated and enumerated the CTCs in patients using the CellSearch^® system (Fig. 2A). The patients with <10 CTCs showed a higher survival rate when compared with the patients with >10 CTCs (P<0.05, Fig. 2B). miR-126 expression was closely correlated with the favorable prognosis of the patients with LSCC, whereas Camsap1 expression was correlated with a poor prognosis (P<0.05, Fig. 2B). The plasma level of miR-126 and the number of CTCs were significantly negatively correlated (r=−0.848, P<0.01; Fig. 2C). The plasma level of miR-126 was also negatively related with Camsap1 expression (r=−0.937, P<0.05; Fig. 2C). However, Camsap1 expression was positively related with the number of CTCs (r=−0.776, P<0.01; Fig. 2C).

The antitumor properties of miR-126 and Camsap1 were further evaluated using LSCC mouse models. As shown in Fig. 3A, compared to untreated mice, both miR-126 mimics and Camsap1 knockdown mice had a significant lower tumor volume (P<0.05). Correspondingly, the tumor weights of miR-126 mimics or Camsap1 knockdown mice also were lower than that of untreated ones (P<0.05, Fig. 3B). In addition, the survival rate of mice with miR-126 mimics and Camsap1 knockdown was significantly improved (P<0.05; Fig. 3C). Furthermore, the anti-angiogenic effects were shown in vivo by anti-CD31 immunohistochemistry following injection of miR-126 mimics or Camsap1 knockdown into tumors (P<0.05; Fig. 3D).

To clarify the mechanism of miR-126 or Camsap1 in LSCC cells, TargetScan were used to determine whether Camsap1 is a target gene of miR-126 or not. The prediction results are shown in Fig. 4A. Furthermore, microtubules formed bundles and aggregated together in untreated cells by using negative stain electron microscopy (Fig. 4B). After knockdown treatment of miR-126 mimics or Camsap1, microtubules did not form such aggregates in LSCC cells. These results indicated Camsap1 induced the formation of bundles by directly interacting with microtubules (Fig. 4B). Interestingly, we demonstrated that Camsap1 and tubulin colocalized in the cytoplasm by using immunofluorescence (IF) (Fig. 4C).

To further study the functions of CAMSAP1 protein, we made use of it in a phylogenetic analysis of the family. Fig. 5 shows a tree generated by maximum likelihood analysis of a codon-based alignment. It appears that a CAMSAP1 gene arose in the ancestors of simple animals. During the evolution of the vertebrates, this gene was multiplied so that extant vertebrate genomes encode three classes of CAMSAP1 genes. These results indicated that we could get more information on CAMSAP1 gene from adjacent species, such as Gorilla.