The human CRC cell lines HCT116 and SW480 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA), and all human CRC cell lines were cultured according to the described ATCC protocol. The cell lines were maintained in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA).

A total of 20 paired CRC tissues with matched adjacent normal tissues from patients were obtained during surgery at the Sixth Affiliated Hospital of Sun Yat-Sen University (Guangzhou, China) between January 2010 and December 2013 (Table I). Patients were diagnosed based on clinical and pathological evidence, and the specimens were immediately snap-frozen and stored in liquid nitrogen tanks. For the use of the clinical materials for research purposes, prior patient consent was obtained and approval from the Institutional Research Ethics Committee was procured.

Total RNA from tissues or cells was extracted using TRIzol (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. Messenger RNA (mRNA) was polyadenylated using a poly(A) polymerase-based first-strand synthesis kit and reverse transcription (RT) of total mRNA was performed using a PrimeScript RT Reagent kit (both from Takara, Dalian, China) according to the manufacturer's protocol. miRNA was reverse transcribed of total mRNA using the RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. Complementary DNA (cDNA) was amplified and quantified using the ABI 7500HT system using SYBR-Green I (both from Applied Biosystems, Foster City, CA, USA). Table II lists the primers used in the reactions. Real-time PCR was performed according to a standard method, as previously described (26). Primers for U6 and miR-874-3p were synthesized and purified by RiboBio (Guangzhou, China). U6 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control for miRNA or mRNA, respectively. Relative fold of expression was calculated using the comparative threshold cycle (2^−ΔΔCt) method.

The miR-874-3p expression plasmid was generated by cloning the genomic pre-miR-874-3p gene into retroviral transfer plasmid pMSCV-puro (Clontech Laboratories, Inc., Tokyo, Japan) to generate plasmid pMSCV-miR-874-3p. pMSCV-miR-874-3p was co-transfected with the pIK packaging plasmid into 293FT cells using the standard calcium phosphate transfection method, as previously described (27). Thirty-six hours after the co-transfection, supernatants were collected and incubated with the cells to be infected for 24 h in the presence of Polybrene (2.5 µg/ml). After infection, puromycin (1.5 µg/ml) was used to select stably transduced cells over a 10-day period. The luciferase reporter system of pTEAD-luc (#83467; Addgene Inc., Cambridge, MA, USA) was used to examine the transcriptional activity of TEAD. The 3′ untranslated regions (3′UTR) of human YAP and TAZ were PCR-amplified from genomic DNA and cloned into the pmirGLO luciferase reporter vector (Promega, Madison, WI, USA). The list of primers used in the clone reactions is presented in Table III. The miArrest plasmids for anti-miR-874-3p and negative control plasmids were constructed and cloned into phU6 plasmids by GeneChem (Shanghai, China). The sequence of anti-miR-874-3p was TCGGTCCCTCGGGCCAGGGCAG. Small interfering RNA (siRNA) for YAP and TAZ knockdown (50 nmol/l) were obtained from RiboBio. Transfection of siRNAs and plasmids was performed using Lipofectamine 3000 (Life Technologies) according to the manufacturer's instructions.

Nuclear/cytoplasmic fractionation was separated using Cell Fractionation kit according to the manufacturer's instructions, and the whole cell lysates were extracted using RIPA buffer (both from Cell Signaling Technology, Beverly, MA, USA). Western blotting was performed according to a standard method, as previously described (28). Antibodies against YAP, TAZ, Bcl-2 and Bcl-xL were purchased from Abcam (Cambridge, MA, USA). The membranes were stripped and reprobed with an anti-α-tubulin antibody (Sigma-Aldrich, St. Louis, MO, USA) as the loading control.

Flow cytometric analysis of apoptosis was carried out using the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, Bedford, MA, USA), and was performed and presented according to the described protocol. Briefly, cells were dissociated with trypsin and resuspended at 1×10⁶ cells/ml in binding buffer with 50 µl/ml FITC Annexin V and 50 µl/ml propidium iodide (PI). The cells were subsequently incubated for 15 min at room temperature, and then were analyzed using a Gallios flow cytometer (Beckman Coulter, Inc. Brea, CA, USA). The cell inner mitochondrial membrane potential (Δψm) was detected by flow cytometry using MitoScreen JC-1 staining kit (BD Biosciences), and was carried out and presented according to the described protocol. Briefly, cells were dissociated with trypsin and resuspended at 1×10⁶ cells/ml in assay buffer, and then incubated at 37°C for 15 min with 10 µl/ml JC-1. Before being analyzed by flow cytometry, the cells were washed twice using assay buffer. Flow cytometric data were analyzed using FlowJo 7.6 software (TreeStar, Inc., Ashland, OR, USA).

Activity of caspase-9 or −3 was analysis by spectrophotometry using caspase-9 colorimetric assay kit or caspase-3 colorimetric assay kit (KeyGen, Nanjing, China), and was carried out and presented according to the described protocol. Briefly, 5×10⁶ cells or 100 mg fresh tumor tissues were washed with cold phosphate-buffered saline (PBS) and resuspended in lysis buffer and incubated on ice for 30 min. Then, 50 µl of the cell suspension, 50 µl of reaction buffer, and 5 µl of caspase-3/−9 substrate were mixed, and then incubated at 37°C for 4 h. The absorbance was measured at 405 nm, and BCA protein quantitative analysis was used as the reference to normal each experiment groups.

Four-week-old BALB/c-nu female mice weighing 15–20 g were maintained in a standard pathogen-free environment where the animals were housed in sterile cages under laminar flow hoods in a 20–26°C temperature controlled room with a 12-h light/12-h dark cycle and fed autoclaved chow and water. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Guangdong Medical College. The 4–6 week-old BALB/c-nu mice were randomly divided into four groups (n=5/group) and the indicated cells (2×10⁶) were subcutaneously inoculated into the inguinal folds of the nude mice. Ten days following cell inoculation, the mice were intraperitoneally injected (50 mg/kg/day) with 5-FU for 4 weeks (29). For the experimental testing to ascertain whether miR-874-3p mimics enhance the sensitivity of CRC cells to 5-FU, animals were injected with 100 µl miR-874-3p (1 mmol/l) or negative control through the lateral tail vein every 4 days for 4 weeks. Tumor volume was determined using an external caliper and calculated using the equation (L × W²)/2. On day 38, tumors were imaged using an IVIS imaging system (Caliper, Alameda, CA, USA). Annimals were euthanized, tumors were excised, weighed and stored in liquid nitrogen tanks.

Cells (4×10⁴) were seeded in triplicate in 24-well plates and cultured for 24 h. Cells were transfected with 100 ng HOP-flash reporter luciferase plasmid, HIP-flash (HOP-flash mutant), pmirGLO-YAP1-3′UTR or -TAZ-3′UTR, luciferase plasmid, plus 5 ng pRL-TK Renilla plasmid (Promega) using Lipofectamine 3000 (Invitrogen) according to the manufacturer's recommendation. Luciferase and Renilla signals were measured 36 h after transfection using a Dual-Luciferase Reporter Assay kit (Promega) according to the manufacturer's protocol.

Cells were co-transfected with HA-Ago2, followed by HA-Ago2 immunoprecipitation using HA-antibody. Real-time PCR analysis of the IP material was used to test the association of the mRNA of YAP1 and TAZ with the RISC complex. The specific processes were performed as previously described (30).

All values are presented as means ± standard deviation (SD). Significant differences were determined using GraphPad 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). One-way ANOVA was used to determine statistical differences between multiple testing. Student's t-test was used to determine statistical differences between two groups. P<0.05 was considered significant. All the experiments were repeated three times.

By analyzing The Cancer Genome Atlas (TCGA) CRC miRNA sequencing datasets, we found that miR-874-3p expression was downregulated in CRC tissues compared with that in adjacent normal colorectal tissues, as well as in the 11 paired primary CRC tissue samples compared with their matched adjacent normal tissue samples (Fig. 1A and B). We further confirmed miR-874-3p expression in our own 20 paired CRC tissues. Consistent with TCGA analysis, we found that the expression level of miR-874-3p was significantly differentially downregulated in 19/20 primary CRC tissue samples compared with their matched adjacent normal tissue samples (Fig. 1C). Thus, these results indicate that downregulation of miR-874-3p may be involved in the progression of CRC.

The transcriptional co-activators YAP and TAZ are major components of the Hippo signaling pathway and play a critical role in the development and progression of multiple cancer types, including CRC (31–33). Using the publicly available algorithms TargetScan and miRanda, we found that transcriptional factors of Hippo signaling, including TAP and TAZ, may be potential targets of miR-874-3p (Fig. 2A). We further constructed stably expressing miR-874-3p CRC cells via exogenously overexpressing miR-874-3p or endogeneously silencing miR-874-3p via virus transduction (Fig. 2B). Western blot analysis revealed that miR-874-3p overexpression reduced the expression levels of TAP and TAZ. In contrast, silencing of miR-874-3p increased the expression of TAP and TAZ, suggesting that miR-874-3p negatively regulated these proteins in CRC cells (Fig. 2C). Furthermore, luciferase assay showed that miR-874-3p overexpression attenuated, while silencing of miR-874-3p elevated the reporter activity driven by the 3′UTRs of TAP and TAZ transcripts, but not by the mutant 3′UTRs of these transcripts within miR-874-3p-binding seed regions in HCT116 and SW480 cells (Fig. 2D and E). Moreover, microribonucleoprotein (miRNP) immunoprecipitation (IP) assay revealed an association of miR-874-3p with TAP and TAZ transcripts (Fig. 2F and G), further indicating the direct repressive effects of miR-874-3p on TAP and TAZ. Collectively, our results suggest that miR-874-3p directly targets TAP and TAZ, leading to activation of Hippo signaling.

YAP and TAZ inactivate the Hippo signaling pathway through nuclear translocation and interaction with TEAD transcription factors (34,35). Thus, we further examined the effects of miR-874-3p on Hippo signaling activity. As shown in Fig. 3A, we found that miR-874-3p overexpression significantly decreased, while silencing of miR-874-3p enhanced TEAD-dependent luciferase activity. Moreover, cellular fractionation and western blot analysis revealed that overexpression of miR-874-3p decreased nuclear accumulation of YAP and TAZ, while silencing of miR-874-3p increased their nuclear expression (Fig. 3B). Real-time PCR analysis revealed that miR-874-3p overexpression significantly decreased, while silencing of miR-874-3p increased expression levels of multiple downstream genes including CTGF, BCL2L1 and cyclin A in the CRC cells (Fig. 3C and D). Thus, these results demonstrated that downregulation of miR-874-3p inactivates the Hippo signaling pathway in CRC cells.

The role of miR-874-3p in the chemoresistance of CRC cells was further investigated following treatment of cells with first-line anti-CRC drug 5-FU (10 µM, 24 h) in vitro (36). As shown in Fig. 4A, we found that miR-874-3p overexpression increased, while silencing of miR-874-3p decreased the apoptosis rate of HCT116 and SW480 cells treated by 5-FU. In addition, miR-874-3p overexpression decreased, while silencing of miR-874-3p increased the mitochondrial potential of HCT116 and SW480 cells under treatment of 5-FU (Fig. 4B). Moreover, we further examined the effect of miR-874-3p on the expression levels of the anti-apoptotic proteins Bcl-2 and Bcl-xL, and the activity of caspase-9 and −3. We found that overexpression of miR-874-3p decreased Bcl-2 and Bcl-xL expression, but enhanced the activity of caspase-9 and −3. Conversely, silencing of miR-874-3p exhibited an opposite effect (Fig. 4C-E). Therefore, these results demonstrated that upregulation of miR-874-3p restores the sensitivity of CRC cells to 5-FU in vitro.

We further examined the effect of miR-874-3p on the chemoresistance of CRC cells in vivo. Mice were randomly divided into four groups (n=5/group) and subcutaneously inoculated (3×10⁶ HCT116 cells/mouse) in the left dorsal flank. Two weeks later, each group of mice was intratumorally injected with 150 µg mimic control, miR-874-3p, anti-miR-874-3p control or the miR-874-3p inhibitor (2 mg/ml) three times each week for 4 weeks, combined with intraperitoneal injection of 5-FU (50 mg/kg/day) (Fig. 5A). As shown in Fig. 5B and C, the tumor volumes and weight were decreased in the miR-874-3p-overexpressing plus 5-FU group, but were markedly increased in the anti-miR-874-3p plus 5-FU group, compared to the respective controls. These findings indicate that upregulation of miR-874-3p sensitizes CRC cells to 5-FU in vivo.

We then explored the functional significance of YAP and TAZ in the chemoresistance of CRC cells. As shown in Fig. 6A and B, individual inhibition of YAP or TAZ in the miR-874-3p-silenced cells abrogated the effects of miR-874-3p downregulation on the apoptotic ratio and mitochondrial potential. Furthermore, the inhibitory effects of miR-874-3p downregulation on caspase-3 and −9 were attenuated by separate silencing of YAP and TAZ in CRC cells (Fig. 6C and D). Taken together, our results indicate that YAP and TAZ are functionally relevant effects of miR-874-3p in the chemotherapeutic response of CRC cells.