Human salivary adenoid cystic carcinoma cell lines with high metastatic potential (SACC-LM) and low metastatic potential (SACC-83) were provided by the Laboratory of Oral and Maxillofacial Surgery, Ninth People's Hospital, School of Medicine, Shanghai Jiao Tong University. Cells were cultured in RPMI-1640 (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) culture medium containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified incubator with 5% CO₂. Upon 70–80% confluence, using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) as transfection medium, miR-21 inhibitor/siRNA (Shanghai GenePharma Co., Ltd., Shanghai, China) or negative control siRNA (Shanghai GenePharma Co., Ltd.) were transfected into SACC-LM cells, according to the manufacturer's protocol. The sequences of miR-21 siRNA and negative control siRNA were 5′-UCAACAUCAGUCUGAUAAGCUA-3′ and 5′-CAGUACUUUUGUGUAGUACAA-3′, respectively. Non-transfected cells were used as a blank control. The final concentration of siRNA was 60 nM. At 5 h after transfection, the culture medium was replaced with fresh RPMI-1640 containing 10% FBS and transfection efficiency was determined using fluorescent images.

miRanda (http://34.236.212.39/microrna/home.do), miRBase (http://www.mirbase.org/) and TargetScan (release 7.1) were searched to predicted the target genes of miR-21. At 48 h after transfection, total RNA and miRNA were extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was obtained using HiScript Reverse Transcriptase (Vazyme Biotech Co., Ltd., Nanjing, China) including 3.575 µg RNA, 2 µl Oligo (dT) 15 or 2 µl hsa-miR-21-5p loop, 4 µl dNTP, 4 µl Hiscript Buffer, 1 µl Reverse Transcriptase and 0.5 µl RNase inhibitor, according to the following protocol: 25°C for 5 min, 50°C for 15 sec, 85°C for 5 min and 4°C for 10 min. mRNA levels of miR-21, PDCD4, PTEN and B-cell lymphoma (Bcl)-2 were detected by qPCR using a SYBR Premix Ex Taq II kit (Takara Bio, Inc., Otsu, Japan) on a 7900HT Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Based on the sequence of miR-21, a stem-loop primer was designed by Primer Premier 5.0 (Premier Biosoft International, Palo Alto, CA, USA) as follows: 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAACATC-3′, 5′-TGCGCTAGCTTATCAGACTGA-3′ (forward), 5′-CCAGTGCAGGGTCCGAGGTATT-3′ (reverse). U6 small nuclear RNA was used as a reference gene, with the primer 5′-CGCTTCGGCAGCACATATAC-3′ (forward); 5′-AAATATGGAACGCTTCACGA-3′ (reverse). The following other primer sequences were used: PDCD4, 5′-AACCAGTCC-AAAGGGAAGG-3′ (forward) and 5′-ACATCCACCTCCTCCACATC-3′ (reverse); PTEN, 5′-CACGACGGGAAGACAAGTTC-3′ (forward) and 5′-TCTGCACGCTCTATACTGCA-3′ (reverse); and Bcl-2, 5′-AGCCTGAGAGCAACCCAAT-3′ (forward) and 5′-AGCGACGAGAGAAGTCATCC-3′ (reverse). GAPDH was used as an internal reference, the sequences were 5′-TCAAGAAGGTGGTGAAGCAGG-3′ (forward); 5′-TCAAAGGTGGAGGAGTGGGT-3′ (reverse). The PCR reaction was performed as follows: 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec and 60°C for 30 sec. All RT-qPCR experiments were performed in triplicate and repeated three times. Each gene expression was quantified using the 2^−ΔΔCq method (18), and their expression was compared between cells transfected with negative control and miR-21 siRNA.

A Cell Counting Kit (CCK)-8 assay was performed to measure cell proliferation and viability. SACC-LM cells were seeded at a density of 8×10³ cells/well in 96-well plates. When the cells reached 70–80% confluence, they were transfected with miR-21 inhibitor or negative control and incubated for 24, 48, 72 or 96 h. Subsequently, 10 µl CCK-8 reagent (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added into each well of the plate and cells were incubated at 37°C for 30 min. Absorbance at 460 nm was measured using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).

A total of 5×10⁵ SACC-LM cells per well were seeded into 6-well plates and cultured until 80% confluence. At 48 h after transfection, a wound was created in the center of the layer of confluent cells adhering to the bottom of the well using a 100 µl pipette tip. Plates were gently washed three times with PBS to remove the cell debris, and serum-free RPMI-1640 medium was added. The scratch width was measured by photo recordings at 0 and 24 h after the wound was created using an inverted optical microscope at ×100 magnification (Olympus Corporation, Tokyo, Japan).

Cell invasion was detected by a Matrigel invasion assay. Diluted Matrigel (100 µl; BD Biosciences, Franklin Lakes, NJ, USA) was added to the upper chamber of each well of a 24-well Transwell plate (Corning Incorporated, Corning, NY, USA). At 48 h after transfection, 1×10⁵ SACC-LM cells in 200 µl serum-free RPMI-1640 medium were seeded in the upper chamber, while 500 µl RPMI-1640 containing 10% FBS was added to the lower chamber. After 24 h incubation at 37°C, residual cells in the upper chamber were carefully removed with a cotton swab, while cells that had invaded to the lower chamber were fixed using 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet for 15 min at room temperature. Cells on the bottom surface were quantified by counting five random fields per well under the light microscope at a magnification of ×100 using Image-Pro Plus version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA), and the mean number of cells passing through the chamber calculated.

At 48 h after transfection, transfected and non-transfected SACC-LM cells were harvested by trypsinization and washed twice in cold PBS. Subsequently, 1.0×10⁵ cells were suspended in 500 µl binding buffer containing 5 µl annexin V-FITC (Nanjing KeyGen Biotech Co., Ltd.) and 5 µl propidium iodide (PI; all from Nanjing KeyGen Biotech Co., Ltd., Nanjing, China), gently mixed with a pipette and incubated at room temperature for 15 min. The apoptotic rate was obtained from the percentage of cells that were double-stained with annexin V-FITC and PI, as detected by a flow cytometer (Facs Canto II; BD Biosciences) using BD FACSDiva version 8.0.1 software.

Then, 48 h after transfection, non-transfected and transfected SACC-LM cells were trypsinized, collected and centrifuged at 671 × g for 5 min at room temperature. Total protein was extracted using protein lysis solution containing RIPA buffer and 1% phenyl methane sulfonyl fluoride (PMSF; Beyotime Institute of Biotechnology, Jiangsu, China). Protein concentrations were measured using a BCA Protein assay kit (Beyotime Institute of Biotechnology). Denatured protein samples (40 µg) were separated by 10% SDS-PAGE and subsequently transferred to polyvinylidene fluoride membranes. After blocking in 5% nonfat dry milk for 2 h at room temperature, the corresponding membranes were incubated with antibodies against PDCD4 (ab80590; 1:7,000; Abcam, Cambridge, UK), Bcl-2 (ab32124; 1:1,000; Abcam), PTEN (ab32199; 1:1,000; Abcam), Snail family transcriptional repressor 1 (Snail1; BZ06198; 1:500; Bioworld Technology, Inc., St. Louis Park, MN, USA) and E-cadherin (BS1098; 1:500; Bioworld Technology, Inc.) at 4°C overnight. GAPDH (BZ00672; 1:7,000; Bioworld Technology, Inc.) was used as an internal control. Subsequently, the membranes were incubated with goat anti-rabbit polyclonal IgG secondary antibodies (ZB-2010; 1:500; OriGene Technologies, Inc., Beijing, China) for 2 h at room temperature. Membranes were washed in washing buffer three times and PDCD4, PTEN, Bcl-2, Snail1, E-cadherin and GAPDH protein bands were visualized using a BCIP/NBT Alkaline Phosphatase Color Development kit (P0321; Beyotime Institute of Biotechnology). Western blot results were normalized to the expression of GAPDH and analyzed using ImageJ version 1.49 software (National Institutes of Health, Bethesda, MD, USA).

Data were analyzed using SPSS statistical software version 16.0 (SPSS, Inc., Chicago, IL, USA). Results are presented as the mean ± standard deviation. Comparisons between two groups were performed using Student's t-test, while comparisons among multiple groups were performed using one-way analysis of variance followed by LSD test. P<0.05 was considered to indicate a statistically significant difference.

Previous reports have indicated that miR-21 expression in SACC tumor tissue samples was significantly higher compared with normal tissue samples (13). In order to investigate the potential role of miR-21 in SACC, the present study measured miR-21 expression in two SACC cell lines (SACC-83 and SACC-LM). RT-qPCR results demonstrated that miR-21 expression was significantly higher in SACC-LM cells, which have a high metastatic potential, compared with SACC-83 cells, which have a low metastatic potential (Fig. 1A), indicating a potential role of miR-21 in SACC cells metastatic ability.

To investigate the effect of miR-21 on cell growth, cells were transfected with miR-21 siRNA or negative control siRNA. RT-qPCR analysis revealed that miR-21 expression was reduced in SACC-LM cells transfected with miR-21 siRNA, compared with cells transfected with negative control siRNA (Fig. 1B). Flow cytometry and CCK-8 assays were performed to determine the effect of miR-21 silencing on cell apoptosis and proliferation in SACC-LM cells, respectively. CCK-8 detection was performed at five time points (0, 24, 48, 72 and 96 h). The apoptotic rate in the miR-21 siRNA, negative control siRNA and blank control groups was 14.5±0.6, 8.43±0.25 and 8.5±1.249%, respectively. In addition, miR-21 siRNA-transfected cells exhibited a significant increase in annexin V staining compared with the two other groups (P<0.01; Fig. 2A and B). As demonstrated in Fig. 2C, miR-21 silencing triggered a decreased cell viability (P<0.05) compared with cells transfected with negative control siRNA, and viability was decreased in a time-dependent manner.

Wound healing migration and Matrigel invasion assays were performed to investigate the effects of miR-21 on cell migration and invasion, respectively. Based on the wound healing assay results, transfection with miR-21 siRNA significantly reduced the migratory ability of SACC-LM cells, compared with cells transfected with negative control siRNA (Fig. 3A and B). In the Matrigel invasion assay, miR-21 silencing in SACC-LM cells reduced the number of cells crossing the membrane, compared with the negative control siRNA group, with means of 675 and 269 cells per random microscopic field, respectively (Fig. 3C and D). Furthermore, the protein expression of metastasis-associated markers was determined by western blot analysis, and the results demonstrated that Snail1 protein expression was decreased, while E-cadherin protein expression was increased, in the miR-21 siRNA group compared with blank control and negative control siRNA groups (Fig. 3E and F). These results indicate that miR-21 may promote cell invasion and migration in SACC-LM cells, which may be associated with the high potential for metastasis of this cell line.

To identify potential target genes of miR-21, several databases were employed, including miRanda, miRBase and TargetScan. The search results indicated that PDCD4, PTEN and Bcl-2 are predicted miR-21 target genes. Therefore, RT-qPCR was performed to further verify the accuracy of the above result. The results demonstrated that PDCD4 and PTEN mRNA levels were upregulated, while Bcl-2 mRNA levels were downregulated, following transfection of SACC-LM cells with miR-21 siRNA, compared with cells transfected with negative control siRNA (Fig. 4A). Therefore, these results demonstrated a potential role for miR-21 in the regulation of the expression of PDCD4, PTEN and Bcl-2.

As PDCD4, PTEN and Bcl-2 are involved in migration, invasion and apoptosis and they are miR-21 target genes, western blot analysis was performed to further determine whether miR-21 regulates the protein expression of these genes in SACC-LM cells. The results demonstrated that transfection of SACC-LM cells with miR-21 siRNA led to a significant increase in PDCD4 and PTEN protein expression, and markedly reduced Bcl-2 protein expression, compared with blank control and negative control siRNA groups (Fig. 4B and C). These results indicate that miR-21 levels were negatively associated with PDCD4 and PTEN expression, and positively associated with Bcl-2 expression. Therefore, miR-21 may reduce apoptosis, and increase cell proliferation and metastasis, by regulating the expression of PDCD4, PTEN and Bcl-2 in SACC.