Bladder cancer and adjacent non-cancerous tissue (ANT) specimens were collected from 46 patients who underwent surgical resection of tumor lesions at the Shanghai Tenth People's Hospital of Tongji University (Shanghai, China) between January 2012 and December 2015. The clinical characteristics of these patients are shown in Table I. None of the patients had received any chemotherapy or radiotherapy prior to surgery. Following surgery, tissue specimens were immediately placed into liquid nitrogen or were snap-frozen in liquid nitrogen, and stored at -80°C until further use. All patients were diagnosed with invasive bladder cancer according to the 2002 version of the American Joint Committee on Cancer/Union for International Cancer Control tumor, lymph node and metastasis (TNM) staging system (21). This study was approved by the Ethics Committee of Shanghai Tenth People's Hospital of Tongji University, and informed consent was obtained from all patients or their relatives.

To predict the target genes of miR-612 we conducted a bioinformatics analysis using a panel of different algorithms including DIANAmT (http://www.microrna.gr/microT) miRanda (http://www.microrna.org/microrna/home.do) miRDB (http://www.mirdb.org/) PICTAR5 (http://www.pictar.org/) RNA22 (http://cbcsrv.watson.ibm.com/rna22.html) TargetScan (http://www.targetscan.org/) and miRWalk (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/).

miRWalk2.0 is an updated version of the database miRWalk that can used to validated miRNAs binding sites from the human, mouse and rat on their target genes (http://zmf.umm.uniheidelberg.de/apps/zmf/mirwalk2/) (22,23). MiRWalk2.0 not only recorded the gene sequence of micrornas binding sites, but also combines this information with a comparison of binding sites resulting from 12 existing miRNA-target prediction programs (DIANA-microTv4.0, DIANA-microT-CDS, miRanda-rel2010, mirBridge, miRDB4.0, miRmap, miRNAMap, doRiNA i.e., PicTar2, PITA, RNA22v2, RNAhybrid 2.1 and Targetscan 6.2), we predicted 21 possible target genes in 7 databases (DIANAmT, miRanda, miRDB, miRWalk PICTAR5, RNA22 and Targetscan). Subsequently, based on the combined score of mirna-612 on Targetscan, 9 possible target genes above 95 score were selected (LRRC41, DUSP14, ZNF318, GABRA5, SMAD6, YTHDF1, HOXA13, TSG101 and ME1).

To further narrow the screening range, we retrieved the 9 genes on PubMed by keywords '(Tumor) AND LRRC41', '(Tumor) AND DUSP14', '(Tumor) AND ZNF318', '(Tumor) AND GABRA5', '(Tumor) AND SMAD6', '(Tumor) AND YTHDF1', '(Tumor) AND HOXA13', '(Tumor) AND TSG101', '(Tumor) AND ME1', excluding the genes that have been studied in bladder cancer (HOXA13{Hu, 2017 #120} (24) and no high expression in other tumors [LRRC41 (25), DUSP14 (26), ZNF318 (27), GABRA5 (28)].

Human bladder cancer T24, UMUC3, 5637 and J82 cell lines, and the immortalized human normal bladder epithelial cell line SV-HUC-1 were obtained from type culture collection of the Chinese Academy of Sciences (Shanghai, China). SV-HUC-1 cells were maintained in F12K medium (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), while T24, UMUC3 and 5637 cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and J82 cells were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.). Cells were cultured at 37°C in a humidified incubator with 5% CO₂. All cell culture media were supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA).

Human miR-612 mimic (miR-612) and the corresponding negative control mimic (miR-NC), as well as malic enzyme 1 siRNA (si-ME1) and the corresponding negative control siRNA (si-NC), were purchased from GenePharma Co., Ltd. (Shanghai, China). A plasmid carrying malic enzyme 1 (ME1) cDNA was purchased from RiboBio Co., Ltd. (Guangzhou, China). For transient gene transfection, bladder cancer T24 cells were grown and transiently transfected with these reagents using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol for 6 h at 37°C. The cells were then subjected to different assays.

Total RNA was isolated from frozen tissues or cultured cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The concentration and purity of the RNA samples were measured by a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). For detection of miR-612, 1 µg total RNA was reverse transcribed into cDNA using a One Step Prime script miRNA cDNA Synthesis kit (Qiagen, Inc., Valencia, CA, USA), and qPCR was performed using the KAPA SYBR FAST qPCR kit (Kapa Biosystems, Inc., Boston, MA, USA), with U6 mRNA used to normalize the miR-612 expression level. The primers used were as follows: miR-612 forward, 5-GCAGGGCTTCTGAGCTCCTTAA-3, U6 forward, 5-CAAATTCGTGAAGCGTTCCATAT-3; the common reverse primer was purchased from RiboBio Co., Ltd. The amplification procedure was as follows: 5 min at 95°C, followed by 40 cycles at 95°C for 30 sec and 65°C for 45 sec.

For detection of the ME1 mRNA level, cDNA was synthesized using the PrimeScript RT Reagent kit (Takara Bio, Inc., Tokyo, Japan), according to the manufacturer's protocol. qPCR for mRNA detection was also performed using the KAPA SYBR FAST qPCR kit (Kapa Biosystems, Inc.), The primers used were as follows: ME1 forward, 5′-GCAGGTCTCCTTGCAGCTCT-3′ and reverse, 5′-TCCAAGGCCATCACA ATCAG-3′; GAPDH forward, 5′-ATGTCGTGGAGTCTACTGGC-3′ and reverse, 5′-TGACCTTGCCCACAGCCTTG-3′. The PCR parameters for relative quantification were as follows: 2 min at 95°C, followed by 40 cycles of 45 sec at 57°C and 45 sec at 72°C. The ME1 mRNA level was normalized to that of GAPDH. The relative expression levels of miR-612 and ME1 were analyzed by the 2^−∆∆Cq method (29).

The cell proliferation rate was measured using the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Briefly, the transfected cells were seeded into 96-well plates at a density of 1×10³ cells/well and grown for up to 5 days. Subsequently, 10 µl CCK-8 reagent was added into each well, and the cells were further incubated at 37°C for 2 h. Next, the optical density value was measured at 450 nm using a microplate spectrophotometer obtained from BioTek Instruments, Inc. (Winooski, VT, USA).

For the tumor cell colony formation assay, 1,000 transfected cells/well were seeded into 6-well plates and cultured for 14 days. Subsequent to culturing, the cell colonies were washed three times with phosphate-buffered saline (PBS), then fixed with 75% ethanol and stained with 0.1% crystal violet solution. Cell colonies with ≥50 cells were counted under a light microscope (Olympus Corporation, Tokyo, Japan). The experiments were performed in triplicate and repeated at least once.

Transwell chambers (Corning, Inc., Lowell, MA, USA) containing 6.5-mm-diameter polycarbonate filters with an 8-µm pore size were used to measure the cell migration capacity. Briefly, 5×10⁴ transfected cells in RPMI-1640 medium (200 µl) without FBS were seeded into each insert (24-well plates), and 600 µl cell culture medium containing 10% FBS was added to the bottom chamber. The cells were allowed to migrate for 16 h at 37°C and 5% CO₂. For the tumor cell invasion assay, the filter of the Transwell chamber was precoated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA), and the cells were allowed to invade for 24 h. The remaining experimental procedures were the same as those of the tumor cell migration assay. At the end of the experiments, the cells remaining in the upper inserts were carefully removed using a cotton swab, whereas cells that had migrated or invaded into the reverse side of the filters were fixed with 70% ethanol for 30 min and stained with 0.1% crystal violet for 10 min. Images of five randomly selected microscopic fields were captured under a microscope (Olympus Corporation), and then the number of cells was counted. The experiments were performed in triplicate and repeated at least once.

The wild-type 3′-UTR segment and a mutant 3′-UTR segment of ME1 cDNA were amplified using PCR, and then inserted into the luciferase gene using the psiCHECK-2 vector (Promega Corporation, Madison, WI, USA), according to the manufacturer's instructions. Luciferase assay was then performed in T24 cells, during which tumor cells (1×10⁵) were seeded into 24-well plates and grown overnight. On the following day, 100 ng luciferase reporter vectors and 100 nM miR-612 or miR-NC were transfected into the T24 cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 48 h. Thereafter, the cell protein was extracted and the luciferase activity was measured using a luciferase reporter assay system (Promega Corporation), according to the manufacturer's protocol.

Total cellular protein was extracted from the transfected cells using a radioimmunoprecipitation assay buffer (Sigma-Aldrich) containing protease inhibitor, and the protein concentration of these samples was measured using a bicinchoninic acid assay kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Protein samples of 20 µg each were separated with 10% sodium dodecyl sulfate-polyacrylamide gels by electrophoresis and then blotted onto nitrocellulose membranes (Sigma-Aldrich; Merck KGaA). For western blotting, the membranes were blocked with 5% non-fat milk for 1 h at 37°C and immunoblotted at 4°C overnight with primary antibodies against: ME1 (sc-365891, 1:1000, secondary antibodies: Mouse), E-cadherin (sc-52327, 1:1000 secondary antibodies: Mouse), N-cadherin (sc-53488, 1:1000, secondary antibodies: Mouse), vimentin (sc-373717, 1:1000, secondary antibodies: Mouse), matrix metalloproteinase-9 (MMP-9) (sc-12759, 1:1,000, secondary antibodies: Mouse), and anti-β-actin (sc-47778, 1:1000, secondary antibodies: Mouse) (all purchased from Santa Cruz Biotechnology, Dallas, TX, USA). The membranes were subsequently incubated with the fluoresence-conjugated secondary antibodies (926-68072 or 926-32210, LI-COR Biosciences, Shanghai, China) for 1 h at room temperature, and the protein bands were visualized and quantified by using the Odyssey two-color infrared laser imaging system (LI-COR Biosciences, Lincoln, NE, USA).

The animal experiments conducted in the present study were approved by the Animal Care and Use Committee of Tongji University. Briefly, 6 male nude BALB/c mice (age, 6 weeks old; weight, 18-22 g) were purchased from Shanghai SIPPR-Bk Lab Animal Co., Ltd. (Shanghai, China) and housed in specific-pathogen-free conditions with a 12-h light/dark cycle (temperature, 18–22°C; humidity, 50–60; irradiated feed and double-distilled water). Bladder cancer T24 cells stably expressing miR-612 or miR-NC were harvested, washed twice with PBS and resuspended in PBS. A total of 5×10⁶ cells were then subcutaneously injected into the left flank regions of each nude mouse. Tumor cell xenograft formation was measured every 5 days using a Vernier caliper, and the tumor volume was calculated using the following formula: Volume (mm³) = 0.5 × width² × length. At 5 weeks after tumor cell inoculation, the mice were sacrificed by cervical dislocation. Tumor xenografts were resected, weighed and then stored at −80°C until further use.

SPSS software (version 16.0; SPSS, Inc., Chicago, IL, USA) was used for all statistical analyses. The experimental data were statistically analyzed using the Student's t-test or one-way analysis of variance, and are expressed as the mean ± standard deviation from three independent experiments. The χ² test or Fisher's exact test was performed to examine the correlation of miR-612 expression with the clinicopathological data of bladder cancer patients, whereas the association of miR-612 with ME1 expression was statistically analyzed using Spearman's correlation test. P<0.05 was regarded to indicate a statistically significant difference.

In the present study, the levels of miR-612 expression in 46 pairs of bladder cancer and ANT specimens were initially assessed using RT-qPCR. It was observed that the miR-612 expression level was significantly lower in bladder cancer tissues compared with that in ANTs (P<0.01; Fig. 1A). Next, the association of miR-612 expression with the clinicopathological features of the patients was examined, and loss of miR-612 expression was found to be significantly associated with advanced TNM stages (Fig. 1B) and tumor distant metastasis (Fig. 1D). Furthermore, the receiver operating characteristic curve analysis demonstrated that tissue miR-612 levels may be a potential biomarker for distinguishing bladder cancer patients from the controls (area under the curve, 0.958; Fig. 1C). Consistently, miR-612 expression was also significantly lower in T24, UMUC3, 5637 and J82 bladder cancer cells as compared with that in the normal bladder epithelial cell line SV-HUC-1. T24 cells exhibited the lowest miR-612 levels among these four bladder cancer cell lines (Fig. 1E).

To determine the biological effects of miR-612 on bladder cancer cell growth, miR-612 mimic or negative control was transfected into T24 cells, since this cell line had the lowest level of miR-612 expression (Fig. 1E). The data revealed that the miR-612 mimic was successfully transfected into the cells and increased miR-612 expression; miR-NC transfection had no significant effect on the T24 cells (Fig. 2A). The CCK-8 and colony formation assays demonstrated that miR-612 overexpression in T24 cells significantly reduced tumor cell growth (Fig. 2B) and colony formation (Fig. 2C). Furthermore, the nude mouse experiments indicated that T24 cells with miR-612 overexpression resulted in slower growth of the tumor xenografts as compared with that for miR-NC-transfected T24 cells (Fig. 2E). Consistently, the weight (Fig. 2F) and tumor xenograft size (Fig. 2D) of the miR-612-transfected group were significantly reduced in comparison with those of the miR-NC-transfected group.

Next, the effects of miR-612 on the metastatic capacity of bladder cancer cells were assessed in vitro, and it was observed that restoration of miR-612 expression in T24 cells using the miR-612 mimic significantly reduced the tumor cell migration and invasion abilities in vitro (Fig. 3A and B). Furthermore, the expression of several accepted EMT-associated markers was also affected by the miR-612 mimic. Compared with the miR-NC group, the expression levels of N-cadherin, vimentin and MMP-9 were significantly decreased by miR-612 mimic transfection, whereas the expression level of E-cadherin was significantly upregulated in the miR-612 group (Fig. 3C). During EMT, N-cadherin, vimentin and MMP-9 levels are upregulated, and E-cadherin levels are downregulated. Thus, our findings indicated that miR-612 overexpression inhibited EMT.

Since miRNAs alter the expression of their target genes to exert their biological functions in cells, bioinformatic analysis was performed using several software (DIANAmT, miRanda, miRDB, miRWalk, PICTAR5, RNA22 and Targetscan) to predict the possible targets of miR-612. Subsequently, relevant publications on these genes were retrieved from Pubmed, and genes that have been widely studied in bladder cancer and were not highly expressed in other tumors were excluded. Finally, it was detected that miR-612 was able to bind to the 3′-UTR of ME1 cDNA (Fig. 4A). This binding ability of miR-612 to the ME1 3′-UTR was then confirmed using a luciferase reporter assay. The results revealed that miR-612 was able to precisely bind to the wild-type 3′-UTR of ME1, which resulted in significantly inhibited luciferase activity (Fig. 4B). Furthermore, it was demonstrated that miR-612 overexpression in T24 cells significantly downregulated the mRNA (Fig. 4C) and protein (Fig. 4D) levels of ME1. The ex vivo data further supported this observation, since the level of ME1 mRNA was markedly higher in bladder cancer tissues as compared with that in ANTs (Fig. 4E). In addition, the level of ME1 mRNA was inversely associated with the level of miR-612 in bladder cancer tissues (Fig. 4F; r=−0.403; P<0.0001). These results indicated that ME1 is indeed the direct target of miR-612 in bladder cancer.

To further confirm the effect and biological functions of ME1 in bladder cancer cells, ME1 expression was knocked down in T24 cells using si-ME1 and compared against the si-NC group. The data demonstrated that si-ME1 significantly reduced the ME1 mRNA and protein expression levels in T24 cells, as compared with the miR-NC-transfected T24 cells (Fig. 5A and B). In addition, functional assays revealed that ME1 knockdown significantly inhibited the tumor cell growth (Fig. 5C), colony formation (Fig. 5D), migration (Fig. 5E) and invasion capacity (Fig. 5F), and affected the tumor cell EMT marker levels (Fig. 5G) in T24 cells, mimicking the effects of miR-612 overexpression on T24 cells (Figs. 2 and 3).

Furthermore, to better verify that ME1 was a direct target of miR-612, the T24 cells were simultaneously co-transfected with the miR-612 mimic or miR-NC and ME1 overexpression plasmid. We found that ME1 overexpression using ME1 cDNA transfection in miR-612-expressing T24 cells was able to partially reverse miR-612 overexpression-induced reduction of ME1 expression in T24 cells (Fig. 6A and B). The ME1 overexpression also functionally rescued the inhibitory effect of miR-612 overexpression on tumor cell growth, colony formation, migration and invasion (Fig. 6C–F). Taken together, the results indicated that miR-612 inhibited tumor cell growth, colony formation, migration, invasion and EMT by targeting ME1 in vitro (Fig. 7).