Twenty laryngeal carcinoma tissues used in this study were obtained from Taizhou People's Hospital in China. Specimens were snap-frozen in liquid nitrogen, incuding 10 laryngeal carcinomas with stage I and II, and 10 laryngeal carcinomas with stage III and IV. The collection and use of the patient samples were reviewed and approved by Institutional Ethics Committees, and written informed consent from all patients was appropriately obtained.

Hep-2 and TU212 cells were purchased from Chinese Academy of Sciences Cell Bank. Cells were maintained in DMEM medium supplemented with 10% fetal bovine serum. Cells were transfected using Lipofectamine 2000 (Invitrogen, USA) at the time of 50-60% confluent. 48 h after transfection, cells were harvested for further studies.

For expression plasmid construct, wild-type RB cDNA sequence without 3'UTR was selected and cloned into Pgenesil-1 vector. 2'-O-methyl (OMe)-oligonucleotides were chemically synthesized and purified by GenePharma Co., Ltd. (Shanghai, China). The amount of oligonucleotides transfected was 50 nmol/L. Sequences as follows: miR-106b, 5'- UAAAGUGCUGACAGUGCAGAU-3'; anti-miR-106b (As-miR-106b), 5'-AUCUGCACUGUCAGCACUUUA-3'; scrambled miRNA (negative control), 5'-UUGUACUACACAAAAGUACUG-3'.

Trizol reagent was used to isolate total RNA from cells 48 h after transfection. The RT-real-time PCR was carried out with the miRNA detection kit (Ambion, USA). Amplification reaction protocol was performed for 40 cycles consisting 95°C for 3 min, 95°C for 15 sec, 60°C for 30 sec. Both RT and PCR primer were purchased from Ambion. 5S RNA was used for normalization. Relative quantification was conducted using amplification efficiencies derived from cDNA standard curves and obtained relative gene expression. Relative gene expression was calculated via a 2^ΔΔCt method.

Cells were plated at 10⁴ cells per well in 96-well plates with six replicate wells. After transfection as described previously, 20 μl of MTT (5 g/L, Sigma, USA) was added into each well at each day of consecutive 4 days after treatment and the cells were incubated for additional 4 h, the supernatant was then discarded. 200 μl of DMSO was added to each well to dissolve the precipitate. Optical density (OD) was measured at wave length of 550 nm. The data are presented as the mean ± SD, which are derived from triplicate samples of at least three independent experiments.

Cells were washed with PBS, fixed with 70% ethanol for at least 1 h. After extensive washing, the cells were suspended in HBSS (Hank's Balanced Salt Solution) containing 50 μg/mL PI and 50 μg/ml RNase A and incubated for 1 h at room temperature, and analyzed by FACScan (Becton Dickinson, USA). Cell cycle analysis was analyzed by ModFit software. Experiments were performed in triplicate. Results were presented as % of cell in a particular phase.

Equal amounts of protein per lane were separated by 8% SDS-polyacrylamide gel and transferred to PVDF membrane. The membrane was blocked in 5% skim milk for 1 h and then incubated with a specific antibody for 2 h. The antibodies used in this study were: antibodies to RB (Santa Cruz, USA). The antibody against β-actin (Santa Cruz, USA) was used as control. The specific protein was detected by using a SuperSignal protein detection kit (Pierce, USA). The band density of specific proteins was quantified after normalization with the density of β-actin.

The human RB 3'UTR (bases 813-959) were amplified and cloned into the XbaI site of the pGL3-control vector (Promega, USA), downstream of the luciferase gene, to generate the plasmids pGL3-WT-RB-3'UTR. pGL3-MUT-RB-3'UTR plasmids were generated from pGL3-WT-RB-3'UTR by deleting the binding site (bases 883-889) for miR-106b "GCACUUU". For the luciferase reporter assay, cells were cultured in 96-well plates, transfected with the plasmids and As-miR-106b using Lipofectamine 2000. 48 h after transfection, luciferase activity was measured using the Dual Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to renilla luciferase activity for each transfected well.

Statistics was determined by ANOVA, or t test using SPSS11.0. Statistical significance is determined as P < 0.05.

To explore miR-106b expression in laryngeal carcinomas, we examined 20 human laryngeal carcinoma specimens with different clinical stages using Real time PCR. As shown in Figure 1, the levels of miR-106b increased markedly in laryngeal carcinomas with stage III and IV in comparison to those with stage I and II (P < 0.01). And we also found high miR-106b expression in Hep-2 and TU212 laryngeal carcinoma cells (Figure 1).

As-miR-106b and miR-106b mimic oligonucleotides were employed to change miR-106b expression in Hep-2 and TU212 cells to evaluate the significance of miR-106b in laryngeal carcinoma. In both two cells, miR-106b expression significantly decreased in As-miR-106b group and increased in miR-106b group 48 h after transfection (Figure 2A). MTT assay data showed that a statistically significant cell proliferation inhibition was found in As-miR-106b group of Hep-2 cells, compared with control groups respectively. Similar trend was observed in TU212 cells (Figure 2B). There was no difference between blank control group and negative control group in the whole experiment. Next we analyzed the cell cycle distribution by FACS. As-miR-106b treated cells represented significant ascends in G0/G1 phase in comparison to untreated Hep-2 and TU212 cells (Figure 2C). However, we did not observe a significant difference in the rate of growth inhibition between miR-106b group and blank control group; although a slightly increasing trend of cell survival rate and G0/G1 phase was seen in Hep-2 and TU212 cells. These results raise the possibility that there exists a threshold value for miR-106b up-regulation. Taken together, reduction of miR-106b can induce cells arrest at G0/G1 phases, thereby inhibiting cell proliferation in laryngeal carcinoma cells.

To further explore the molecular mechanism of As-miR-106b induced cell cycle in laryngeal carcinoma cells, bioinformatics analysis of miR-106b potential target genes was performed through the databases TargetScan http://www.targetscan.org and PicTar http://www.pictar.bio.nyu.edu, We found that tumor suppressor RB associated with cell cycle contained the highly conserved putative miR-106b binding sites (Figure 3A). To determine whether RB is directly regulated by miR-106b, Western blot analysis and Luciferase reporter assay were employed. Western blot analysis showed that a notable induction of RB expression was detected after knockdown of miR-106b in Hep-2 and TU212 cells (Figure 3B). Further, we created pGL3-WT-RB-3'UTR, and pGL3-MUT-RB-3'UTR plasmids. Reporter assay revealed that inhibition of miR-106b triggered a marked increase of luciferase activity of pGL3-WT-RB-3'UTR plasmid both in Hep-2 and TU212 cells, without change in luciferase activity of pGL3-MUT-RB-3'UTR (Figure 3C). These data indicate that RB is a direct target of miR-106b in laryngeal carcinoma.

Having demonstrated RB as a direct target of miR-106b, we next examined the importance of RB in miR-106b-mediated cell proliferation. The cell cycle distribution analysis showed that upregulation of miR-106b significantly reduced cell cycle G0/G1 phase arrest induced by serum starvation (Figure 4A). Then we transfected Rb without 3'UTR into Hep-2 cells. Western blot assay showed that transfection with RB without 3'UTR overrided RB expression targeted by miR-106b (Figure 4B). As shown in Figure 4C, the cells transfected RB significantly induced G0/G1 phase arrest. However, when we transfected with RB without 3'UTR and miR-106b, expression of RB largely abrogated the effect of miR-106b on cell cycle distribution. These findings suggest that RB is a major target of miR-106b involved in laryngeal carcinoma cell proliferation.

We further explored the correlation of between miR-106b and RB expression in laryngeal carcinomas. We tested RB expression in these 20 human laryngeal carcinoma specimens and found RB expression was down-regulated in laryngeal carcinomas with stage III and IV in comparison to those with stage I and II (Figure 5A). Further, Pearson correlation showed that a significant negative correlation existed between miR-106b and RB expression in laryngeal carcinoma tissues (R = 0.673, P < 0.005) (Figure 5B).