Hep-2 cells and U2OS cells were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Science (Shanghai, China). The cells were maintained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (Sigma-Aldrich) at 37°C in 5% CO₂ at a humidity of 95%. Transfection of Hep-2 cells was performed with Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA) following the manufacturer’s instructions. Transfections were performed in triplicate for each experiment.

To predict the miRNAs that target hTERT and the conserved sites bound by the seed region of these miRNAs, different target prediction programs were used, including TargetScan (http://www.targetscan.org/), miRanda (http://www.microrna.org/microrna/home.do) and starBase(http://starbase.sysu.edu.cn/).

Total RNA was extracted from Hep-2 cells or U2OS cells using TRIzol reagent (Invitrogen Life Technologies), strictly following the instructions. The synthesis of cDNA was carried out using the RevertAid First strand cDNA synthesis kit (Fermentas; Thermo Fisher Scientific, Waltham, MA, USA). qPCR was performed using TaqMan miRNA assay kits (Applied Biosystems, Foster City, CA, USA) on an Applied Biosystems 7500 thermal cycler. The expression of miRNA was normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primers for hTERT were 5′-CTTCCACTCCCCACATAGGA-3′ (hTERT-F) and 5′-GTA CAGGGCACACCTTTGGT-3′ (hTERT-R). The primers for GAPDH were 5′-AGAAGGCTGGGGCTCATTTG-3′ (GAPDH-F) and 5′-AGGGGCCATCCACAGTCTTC-3′ (GAPDH-R). PCR amplification was conducted in a volume of 20 μl containing 3 μl RT product, 10 μl 1X SYBR Select master mix and 7 μl all-in-one miRNA qPCR primer. The reaction conditions were as follows: 50°C for 2 min and 95°C for 20 sec, followed by 40 cycles of 95°C for 10 sec, 60°C for 34 sec and 70°C for 10 sec. Melting analysis was carried out at the end of the amplification cycle to verify non-specific amplification. The expression level was quantified using the 2^−ΔΔCt method.

The 3′-UTR segments of hTERT containing the miR-299-3p binding sites were amplified by PCR from Hela cell genomic DNA. The primers were 5′-CCGCTCGAGTGGCCACCCGCCCACAG-3′ (hTERT-F) and 5′-GTTTAGCGGCCGCTTTTACTCCCACAGCACCTCC-3′ (hTERT-R). Next, the PCR product was cloned into the XhoI/NotI sites downstream of the Renilla luciferase (Rluc) reporter gene pmiR-RB-Report™ vector (RiboBio, Guangzhou, China), which was named pmiR-hTERT-3′-UTR-wt. Mutations in the predicted miR-299-3p binding sites were established using pmiR-hTERT-3′-UTR-wt as a template, and was named pmiR-hTERT-3′-UTR-mut. The pmiR-RB-Report vector containing a synthetic Renilla luciferase (hRluc) gene and a synthetic firefly luciferase (hluc) gene encoding firefly luciferase was used as the internal control. The recombinant plasmid, designated as pmiR-RB-hTERT-3′-UTR, was confirmed by restriction enzyme digestion and DNA sequencing. Hela cells were co-transfected in 96-well plates with miR-299-3p mimic or negative control (RiboBio), pmiR-hTERT-3′-UTR-wt or pmiR-hTERT-3′-UTR-mut by Lipofectamine 2000. Forty-eight hours after transfection, luciferase activity was measured using the dual luciferase assay system (Promega, Heidelberg, Germany). The firefly luciferase activity of each sample was normalized to Renilla luciferase activity.

Western blot analysis was performed as routinely described. Briefly, the protein was homogenized in modified RIPA buffer containing 0.5% sodium dodecyl sulfate (SDS) in the presence of proteinase inhibitor cocktail (Complete mini; Roche Diagnostics, Basel, Switzerland). Proteins (40 μg) were separated by electrophoresis on a 10% SDS-PAGE gel and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). Subsequently, the membrane was blocked in 5% non-fat milk and incubated with rabbit anti-hTERT antibody (1:1000 dilution; Epitomics, Burlingame, CA, USA) at 4°C overnight, followed by incubation with a secondary antibody (1:5000; LI-COR, Lincoln, NE, USA) for 4 h at room temperature. The same membrane was probed for GAPDH (1:1000 dilution; Epitomics) as the loading control. The relative density of hTERT to GAPDH was analyzed with an Odyssey^® infrared imaging system (LI-COR).

Hep-2 Cells were seeded in a 96-well plate at a concentration of 10³ cells per well. Following transfection, the cells were maintained at 37°C for 24, 48 and 72 h, respectively. The cells were treated with CCK-8 (Dojindo Inc., Kunamoto, Japan) for 4 h at 37°C. Subsequently, the medium was removed and the pellet was dissolved in 100 μl DMSO. Finally, the number of cells was determined using a microplate reader at a wavelength of 450 nm.

SPSS 11.5 software (SPSS Inc., Chicago, IL, USA) was used for the data analysis. Student’s t-test was performed to analyze the differences between the data obtained from three independent experiments. P<0.05 was considered to indicate a statistically significant difference.

Fig. 1 shows the correlation between miR-299-3p expression and hTERT protein in Hep-2 and U2OS cells. Western blot analysis revealed that hTERT was positively expressed in the Hep-2 cells, while it was negatively expressed in U2OS cells (Fig. 1B). In addition, qPCR was used to measure the expression of miR-299-3p in the two cell lines. As shown in Fig. 1C, higher levels of miR-299-3p were observed in U2OS cell lines compared with those in Hep-2 cells. An inverse correlation was observed between the expression of miR-299-3p and hTERT protein in these two cell lines. These results indicated that the high expression of hTERT in Hep-2 cell lines was inversely correlated with the endogenous expression of miR-299-3p.

To investigate the association between miR-299-3p and hTERT, the expression of hTERT was measured following knockdown of miR-299-3p in vitro (Fig. 2A). RT-qPCR analysis demonstrated that transfection of Hep-2 cells with miR-299-3p inhibitor contributed to the expression of hTERT mRNA compared with the control group transfected with a scrambled inhibitor negative control (Fig. 2B). In addition, the expression of hTERT protein was increased in the presence of miR-299-3p downregulation (Fig. 2C). The effect of overexpression of miR-299-3p induced by miR-299-3p mimic on the expression of hTERT protein was investigated by RT-qPCR and western blot analysis (Fig. 2D–F). It was observed that upregulation of miR-299-3p reduced the expression of hTERT mRNA and protein. These results indicated that miR-299-3p negatively regulated hTERT expression.

In Hela cells co-transfected with the reporter plasmids and miR-299-3p mimic or negative control duplex, the activity of the luciferase reporter containing wild-type 3′-UTR was significantly suppressed by miR-299-3p mimic. To be precise, the luciferase activity declined by 36% in comparison with the negative control (P<0.05). However, the luciferase activity of the mutant reporter recovered by 24% compared with the wild type, indicating that miR-299-3p may suppress the expression of hTERT by binding with the 3′-UTR of hTERT mRNA. Together, the results revealed that miR-299-3p regulated the expression of endogenous human hTERT by directly targeting the 3′-UTR of hTERT mRNA (as shown in Fig. 3).

To investigate the effects of miR-299-3p overexpression on the growth of Hep-2 cancer cells, CCK-8 assay was performed on Hep-2 cells 24, 48 and 72 h after treatment with miR-299-3p mimic, inhibitor and negative control, respectively. As demonstrated in Fig. 4A, the growth of Hep-2 cancer cells notably decreased 72 h after transfection of miR-299-3p mimic compared with the mock and negative control. However, downregulation of miR-299-3p contributed to a significant increase in cell growth compared with the other two groups 72 h after transfection with miR-299-3p inhibitor. All of these results demonstrated that miR-299-3p affected the growth of Hep-2 cancer cells.