Twenty-five gastric cancer tissues and adjacent nontumor tissues were obtained from the Department of General Surgery, Changhai Hospital (Shanghai, China) for RT-PCR analysis. All of the tissue samples were obtained from surgical removal and immediately snap frozen and stored in liquid nitrogen until use. The study protocol was approved by the Shanghai Changhai Hospital Ethics Committee. Informed consent was obtained from all patients.

Cell lines BGC-823, SGC-7901 and HEK 293T used in this study were purchased from the American Type Culture Collection (ATCC). BGC-823 and SGC-7901 were cultured and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). HEK 293T was cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS. Cells were maintained at 37°C in a humidified atmosphere with 5% CO₂.

Total RNA was isolated from the gastric cancer tissues, adjacent nontumor tissues and gastric cancer cell lines using Trizol according to the manufacturer’s instructions. Purified mRNA and miRNAs were detected by qRT-PCR assay using All-in-One miRNA qRT-PCR detection kit (GeneCopoeia, Rockville, MD, USA). All primers are listed in Table I. U6 small RNA was used as an internal control for normalization and quantification of miR-17/20a expression. β-actin was used as an internal control for normalization and quantification of UBE2C expression.

The luciferase reporter construct was constructed by cloning the human UBE2C mRNA sequence into the pMIR-Report construct (Ambion Inc., Austin, TX, USA). The wild-type or mutant UBE2C mRNA fragment was amplified and cloned into the luciferase reporter via SpeI and HindIII sites. All the primers are listed in Table I. Luciferase reporter assays were performed as follows. HEK 293T cells plated in a 96-well plate were co-transfected with 100 nM single-stranded miRNA inhibitors or negative control oligonucleotides, 10 ng of firefly luciferase reporter and 5 ng of pRL-TK (Promega, Madison, WI, USA) using the INTERFERin^® reagent (Polyplus-Transfection SA, Brant, France). Cells were collected 36 h after the last transfection and analyzed using the Dual-Luciferase Reporter assay system (Promega).

RNA oligos were chemically synthesized and purified by Genepharma Co. Ltd. (Shanghai, China). The sequence of the human miR-17 inhibitor was 5′-CUACCUGCACUGUAAGCACUUUG-3′ and the human miR-20a inhibitor was 5′-CUACCUGCACUAUAAGCACUUUA-3′. Negative control oligonucleotides for the miRNA inhibitor were 5′-CAGUACUUUUGUGUAGUACAA-3′. The sequences of the UBE2C siRNAs were 5′-GAAGUACCUGCAAGAAACCUACUCATT-3′ (sense) and 5′-UGAGUAGGUUUCUUGCAGGUACUUCTT-3′ (antisense) and for the control siRNAs were 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) and 5′-ACGUGACACGUUCGUAGAATT-3′ (antisense). The transfections were performed with INTERFERin reagent. The final concentration of miRNA inhibitors was 100 nM. The final concentration of siRNAs was 20 nM.

The in vitro growth of gastric cancer cells was measured using the MTT assay. Cells (5,000) were seeded into each well of 96-well plates and transfected with miR-17/20a inhibitors or UBE2C siRNAs at a final concentration of 100 or 20 nM, respectively. On the day of harvest, 100 μl of spent medium was replaced with an equal volume of fresh medium containing MTT (0.5 mg/ml). The plates were incubated at 37°C for 4 h, and then the medium was replaced by 100 μl of DMSO (Sigma, St. Louis, MO, USA) and plates were shaken at room temperature for 10 min. The absorbance was measured at 570 nm.

Total cellular extracts (20 μg) were separated by a 4–20% Tris-glycine gel and then transferred to a PVDF membrane (Immobilon-P transfer membranes; Millipore Corp., Bedford, MA, USA). Following the transfer, the blots were blocked with 5% non-fat dry milk in PBS + 0.1% Tween-20 for 2 h and washed three times with PBS + 0.1% Tween-20 at 4°C. The blots were then probed with a 1:200 dilution of the primary antibody against UBE2C (AB3935; Abcam, Cambridge, MA, USA), cyclin A (H-432; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or β-actin (c-11; Santa Cruz Biotechnology). The blots were then probed with a 1:750 dilution of the secondary antibodies for 1 h at 4°C, followed by washes in PBS + 0.1% Tween-20 and detection was carried out using an Odyssey Scanning system.

All statistical analyses were carried out using the SPSS 16.0 statistical software package. All data are presented as the mean ± standard deviation. Student’s t-test was used to analyze the difference between two experimental groups and a two-tailed P<0.05 was taken to indicate statistical significance.

The miR-17/20 cluster, which is located on chromosome 13q31, have a similar sequence and are hypothesized to have the same target genes. In order to investigate the biological significance and its underlying mechanisms of the dysregulation of miR-17/20a in gastric cancer, the potential target genes of miR-17/20a that function in gastric cancer pathogenesis were further analyzed. Newly published CLASH data in HEK 293 cells provided us with the direct evidence for miRNA:mRNA pairing (19). The CLASH data showed that both miR-17 and miR-20a targeted the coding DNA sequence (CDS) of UBE2C which is involved in many cellular processes including cell proliferation, migration, survival and other cellular functions (20). All genes targeted by miR-17 and miR-20a in the CLASH data are listed in Table II and Table III. After retrieving miR-17/20a potential binding sequences in the human, mouse, rat and horse, the alignments with miR-17 and miR-20a are illustrated in Fig. 1. Alignment results indicated that miR-17/20a may target the coding sequence of UBE2C using a 3′ end mode.

To determine whether miR-17/20a are capable of targeting and regulating UBE2C expression in gastric cancer cells, we performed the following experiments. Previous research has reported that miR-17/20a are upregulated in gastric cancer (9); thus, we performed a loss-of-function study. We created the luciferase reporter plasmid with the wild type or the mutant targeting sequence of UBE2C mRNA (Fig. 2A). The inhibitors of miR-17 and/or miR-20 were transfected into HEK 293T cells. Luciferase assay was used to test the regulation of UBE2C by the miRNAs. The results showed that inhibition of miR-17 or miR-20a decreased the luciferase activity, and inhibition of both miR-17 and miR-20a caused a more significant decrease in luciferase activity, whereas the reporter plasmid with the mutant sequence of UBE2C produced no change in luciferase activity (Fig. 2A). These results revealed that miR-17/20a positively regulated UBE2C expression. Then, we explored whether the endogenous UBE2C in gastric cancer cells was regulated similarly. Two gastric cancer cells (BGC-823 and SGC-7901) were transfected with miR-17 and/or miR-20a inhibitors, and UBE2C mRNA and protein levels were examined by qRT-PCR and western blot analysis, respectively. The levels of UBE2C mRNA (Fig. 2B) and protein (Fig. 2C) were consistently and substantially downregulated by inhibition of miR-17 and miR-20a in the gastric cancer cells. A more significant decrease in UBE2C expression was also found in the cells which were transfected with both the miR-17 and miR-20a inhibitors. Our results indicated that miR-17/20a directly targeted UBE2C mRNA and positively regulated expression of UBE2C at both the mRNA and protein levels.

Previous research reported that miR-17/20a are upregulated in many cancers including gastric cancer (9) and overexpression of UBE2C is associated with worse patient outcomes in a number of cancers (21,22). Our results demonstrated that UBE2C was positively regulated by miR-17/20a in gastric cancer cells. Next, we explored the correlation between miR-17/20a expression levels and the mRNA level of UBE2C in gastric cancer tissues. To determine the levels of miR-17/20a in gastric cancer samples, total RNAs were extracted from gastric cancer tissues, and the expression levels of miR-17/20a were analyzed using qRT-PCR and normalized to an endogenous control (U6 RNA). As shown in Fig. 3A, miR-17 and miR-20a were significantly increased in gastric cancer tissues vs. adjacent nontumor tissues (Fig. 3A), which agreed with a previous study (9). qRT-PCR assay was performed to detect the mRNA expression of UBE2C in the same gastric cancer samples and was normalized to β-actin. As shown in Fig. 3B, UBE2C mRNA levels were significantly increased in the gastric cancer tissues vs. the adjacent nontumor tissues, which was similar to previous findings in other cancers (21). After normalization to the expression value in normal tissues, RNA levels of miR-17/20a and mRNA levels of UBE2C in gastric cancer tissues were analyzed by Pearson’s correlation coefficient analysis. Markedly, UBE2C mRNA levels were positively correlated with miR-17 (r=0.469, P<0.001) and miR-20a (r=0.507, P<0.001) expression levels in gastric cancer tissues (Fig. 3C). Collectively, these results suggest that high endogenous miR-17/20a may target and upregulate the expression of UBE2C in gastric cancer.

We then investigated the biological significance and its underlying mechanisms of the upregulation of miR-17/20a in gastric cancer. Cell proliferation is a key characteristic during tumorigenesis. However, the association of miR-17/20a with gastric cancer cell proliferation is unknown. Since overexpression of UBE2C increased the proliferation of cancer cells by modulating degradation of target genes such as cyclin A (23), and given that UBE2C is a direct target of miR-17/20a, we hypothesized that miR-17/20a may affect gastric cancer cell viability through UBE2C. In order to investigate this hypothesis, we performed the following experiments. Both miR-17 and miR-20a inhibitors or UBE2C siRNAs were transiently transfected into BGC-823 and SGC-7901 cells. In the cell proliferation assay, downregulation of miR-17 and miR-20a in BGC-823 and SGC-7901 cells resulted in significant suppression of cell proliferation which was similar to UBE2C siRNAs (Fig. 4A). Western blot analysis results determined that transfection of miR-17/20a inhibitors or UBE2C siRNAs decreased the expression of UBE2C and its downstream gene cyclin A in BGC-823 and SGC-7901 cells (Fig. 4B). Our results demonstrated that downregulation of UBE2C expression by miR-17/20a contributes, at least in part, to the suppression of the growth of gastric cancer cells.