Cycloheximide (CHX) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-PTEN and anti-USP13 were purchased from Millipore (Bedford, MA, USA). Anti-p-AKT (Ser 473), anti-AKT and anti-β-actin were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). The real-time PCR Master mix kit was purchased from Takara (Dalian, China).

HCT-116 CRC cell lines were purchased from the American Type Culture Collection (ATCC, Dallas, TX, USA). Caco2, CO-115 and 293T cell lines were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). HCT-116, Caco2, CO-115 and 293T cells were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), penicillin (100 U/ml) and streptomycin (100 μg/ml) in a humidified atmosphere of 5% CO₂ at 37°C. The cells were examined for mycoplasma contamination periodically and were always found to be negative. Cell transfections were performed using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions.

Total RNA was extracted from cell lines using Trizol (Invitrogen). RT-qPCR of miR-135b was performed using TaqMan miRNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) and TaqMan human miRNA assay (Assay ID: RNU6B, 001093; miR-135b, 002261). For detection of the mRNAs of PTEN, total RNA was extracted by TRIzol (Invitrogen) and subjected to reverse transcription using the PrimeScript II first-strand cDNA synthesis kit (Takara) according to the manufacturer’s instructions. The primers for human PTEN were: forward, 5′-CAAGATGATGTTTGAAACTATTCCAATG-3′ and reverse, 5′-CCTTTAGCTGGCAGACCACAA-3′. RT-qPCR was performed with the 7500 Fast real-time PCR system (Applied Biosystems). miR-135b expression was normalized to that of RNU6B. PTEN expression was normalized to GAPDH. Reverse-transcriptase (RT) reactions, including no-template controls and RT minus controls, were run in a GeneAmp PCR 9700 Thermocycler (Applied Biosystems). After the Ct values of the target genes were subtracted by that of the internal control RNU6B/GAPDH, the relative expression level of each target gene was obtained using the 2^−ΔΔCt method.

Glucose consumption was measured in the cell lysates with a Glucose uptake colorimetric assay kit (BioVision, San Francisco, CA, USA). The extracellular lactate was measured in the medium using a lactate assay kit (BioVision) according to the manufacturer’s instructions.

For luciferase reporter experiments, the 3′-UTR segment of the USP13 gene was amplified by PCR using human cDNA as templates and inserted into the pGL3-control vector (Promega, Madison, WI, USA). A mutant construct was generated from the perfect complementary site using the Quik change site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA).

MiRNASelect™ pEP- miR-Null control lentiviral vector and miRNASelect™ pEP-hsa-mir-135b lentiviral expression vector were obtained from Cell Biolabs (San Diego, CA, USA). Human miRNA inhibitors against human miR-135b in the lentiviral vector were obtained from OriGene (Rockville, MD, USA). Expression lentiviral vector for shRNA against USP13 was constructed into a pLKO.1 vector. The target sequences were: 5′-CGATTTAAATAGCGACGATTA-3′ (sh-13-1) and 5′-GCC AGTATCTAAATATGCCAA-3′ (sh-13-2). Negative control vector containing scrambled shRNA was obtained from Addgene (Cambridge, MA, USA). pLenti-C-Myc-DDK tagged USP13 or PTEN lentiviral expression vector was purchased from OriGene. The plasmids were prepared with plasmid maxi kit and transfected in 293T cells (Invitrogen) with the Lipofectamine™ 2000 to produce lentiviral particles. The cells were infected with lentiviral particles for 2 h. The cells were then selected with 2.5 mg/ml puromycin for 72 h.

Cells were lysed in a RIPA buffer containing complete protease/phosphatase inhibitor cocktail (Cell Signaling Technology, Inc.). The lysate was centrifuged at 12,000 rpm for 15 min at 4°C and the supernatant was collected. Protein concentration was determined by the Bradford protein assay. The proteins were separated by SDS-PAGE gel electrophoresis and then transferred onto PVDF membranes (Millipore). The membranes were blocked with 10% BSA in TBST for 1 h and incubated with antibodies overnight at 4°C. After washing with TBST three times, the membranes were probed with HRP-conjugated secondary antibodies in TBST for 1 h at room temperature, then washed with PBST three times. The immunobinding signals were detected by a chemiluminescence method (ECL, Millipore).

To directly evaluate the effect of miR-135b on USP13, the luciferase assay was used. Stable CRC cell lines that constitutively express human miR-135b were generated. The cells were transfected with firefly luciferase reporter vector and the control vector pRL-CMV (coding for Renilla luciferase, Promega) or plus miR-135b inhibitor lentiviral particles. Forty-eight hours after transfection, firefly and Renilla luciferase activities were measured using the Dual-Luciferase reporter assay system (Promega), and the relative reporter activity was normalized to Renilla luciferase activity. Each assay was repeated in six independent experiments.

Cell proliferation was examined by using a Cell Counting kit-8 (Dojindo, Kumamoto, Japan). The cells were plated in 96-well plates at 2×10³ cells/well and cultured in growth medium for 24 h. After 24 h, CCK-8 (10 μl) was added to each well containing 100 μl of DMEM medium. The plate was incubated for 2 h at 37°C. Absorbance was measured at 450 nm using a microplate reader (Tecan, Switzerland).

Data were analysed using the Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.

miR-135b was reported to be upregulated in CRCs and its overexpression was due to loss of APC and PTEN. APC was proven to be a target of miR-135b and formed a feedback loop with miR-135b (36). We determined whether miR-135b affected the levels of PTEN. To examine this potential effect, we generated stable Caco2 cell lines that constitutively express human miR-135b (Fig. 1A). Overexpression of miR-135b decreased the levels of PTEN protein. We then examined the expression of PTEN mRNA by RT-qPCR. In RT-qPCR analysis, no statistically significant change in PTEN mRNA expression was observed between the control and miR-135b overexpressing groups (Fig. 1B).

The role of ubiquitination in modulating PTEN protein has been identified (37,38). The regulation of PTEN levels by miR-135b may be the results of protein stability regulation. We determined the PTEN stability in Caco2 cells with treatment of cycloheximide (CHX). As shown in Fig. 1C, upregulation of miR-135b decreased the stability of PTEN. These results suggested that miR-135b may regulate the stability of PTEN in post-translational levels.

miR-135b is predicted not to target PTEN mRNA. To determine the function of miR-135b in the regulation of PTEN stability we used the computational algorithms of TargetScan (ver. 5.2) for prediction analysis to identify target genes of miR-135b. USP13 is a deubiquitinase that mediates deubiquitination of the target proteins. USP13 was reported to stabilize the PTEN protein by direct binding and deubiquitination of PTEN (39). Along with these observations, reassessment of computational prediction suggested that miR-135b potentially targets USP13 gene (Fig. 2A). Overexpression of miR-135b suppressed USP13 protein expression in Caco2 cells (Fig. 2B). We also found that over-expression of miR-135b significantly reduced the luciferase activity of plasmids containing the 3′-UTR of USP13 (Fig. 2C). The putative mutated target sites of USP13 3′-UTR were introduced to further investigate the direct regulatory effect. The reporter assay showed that the effects of miR-135b repression were eliminated when the target sites of USP13 3′-UTR were mutated (Fig. 2D). Moreover, the suppressive effects of miR-135b on the USP13 3′-UTR-carrying luciferase were significantly reduced by the expression of miRNA inhibitor against human miR-135b (Fig. 2E). These results indicated that miR-135b regulated the expression of USP13 through a direct seed sequence interaction.

The Warburg effect is defined by an increased glucose uptake via glycolysis as a cellular resource, and is a common phenotype of cancer cells (40). AKT was reported to play a critical role in regulating the Warburg effect (41). USP13 can regulate AKT phosphorylation by stabilizing PTEN protein. Thus, USP13 can suppress glycolysis and cell proliferation (39). We hypothesized that miR-135b might regulate glycolysis through the downregulation of USP13/PTEN. Overexpression of miR-135b increased AKT phosphorylation (Fig. 3A). Additionally, glucose uptake and glycolysis of CRC cells were promoted by miR-135b (Fig. 3B).

To determine whether miR-135b-mediated upregulation of glycolysis depends on USP13, we restored the expression of USP13 in miR-135b stably expressing CRC cells. Glucose uptake and glycolysis in miR-135b-overexpressing CRC cells were decreased with the restoration of USP13 (Fig. 4A). Similarly, overexpression of PTEN decreased glucose uptake and glycolysis (Fig. 4B). Moreover, miR-135b promoted the proliferation of HCT-116 CRC cells but not with the restoration of USP13 or overexpression of PTEN (Fig. 4C). CO-115 CRC cells have mutations leading to the premature stop codon of PTEN. Thus, we overexpressed miR-135b in CO-115 cells. We found that miR-135b did not affect glucose uptake and glycolysis in CO-115 cells (Fig. 4D). Therefore, miR-135b promotes cell proliferation and glycolysis via the downregulation of USP13 or PTEN.

PTEN is a negative regulator of the PI3K pathway and overexpression of miR-135b partly depends on PI3K pathway. We determined whether the levels of USP13 affect the expression of endogenous miR-135b. Knockdown of USP13 in Caco2 cells decreased the levels of PTEN (Fig. 5A). By contrast, the levels of endogenous miR-135b were significantly increased in USP13-knockdwon Caco2 cells (Fig. 5A). We knocked down the expression of USP13 in CO-115 cells. However, knockdown of USP13 did not affect the levels of endogenous miR-135b in CO-115 cells (Fig. 5B). These results suggested that loss of USP13 upregulates the levels of endogenous miR-135b by reducing the stability of PTEN.