miR‑124 inhibits cell growth through targeting IQGAP1 in colorectal cancer
- Authors:
- Published online on: September 27, 2018 https://doi.org/10.3892/mmr.2018.9518
- Pages: 5270-5278
Abstract
Introduction
MicroRNAs (miRNA/miR) represent a cluster of small non-coding RNA molecules involved in controlling gene expression, translation and cellular biological behaviors. In recent years, growing evidence suggests that miRNAs serve a crucial role in tumor development by functioning as oncogenes or tumor suppressors, depending on their target genes and downstream signaling pathways (1). Of these, miR-124, first identified to be highly expressed in the brain, has been disclosed to be aberrantly downregulated or silenced in multiple tumors including liver, breast, cervical, nasopharyngeal and lung cancer (2–6), which arises at least partly from DNA methylation (7–9). Furthermore, miR-124 has also been demonstrated to suppress cell growth, invasion, epithelial-mesenchymal transition, and even metastasis through targeting a series of oncogenes, including rho-associated protein kinase, EZH2, signal transducer and activator of transcription 3 (STAT3), Slug and forkhead box protein Q1 (FOXQ1) (3,5,10,11). Given that miR-124 regulates various malignant phenotypes in tumor development by repressing different target genes, it is helpful to understand the role of miR-124 regulatory networks in tumors in order to identify novel miR-124-targeting genes.
Colorectal cancer (CRC) represents one of the leading causes of cancer-associated mortality worldwide (12). The expression of miR-124 is negatively correlated with grade of CRC differentiation, but positively associated with poor prognosis, therefore represents an independent prognostic factor for CRC (13,14). Previous studies have demonstrated the tumor-suppressive properties of miR-124 in the regulation of tumor growth and metastasis (15–17). In addition, miR-124 has been demonstrated to sensitize the response of tumor cells to radiotherapy in CRC, through inhibiting paired mesoderm homeobox protein 1 (18). Notably, an integrating analysis demonstrated that miR-124 mediates crosstalk within the Toll-like receptor signaling pathway in Crohn's disease, ulcerative colitis and CRC (19), which indicates that the involvement of miR-124 in CRC progression begins at a very early stage of pathology.
IQ motif containing GTPase activating protein 1 (IQGAP1) is one of the largest known scaffold proteins, participating in protein-protein interactions and integrating diverse signaling pathways. Evidence implicates IQGAP1 as an essential regulator of the mitogen activated protein kinase 1 (MAPK) (20–22) and Wnt/β-catenin signaling pathways (23–25) that serve crucial roles in the progression of multiple tumors. IQGAP1 is demonstrated to be overexpressed in CRC tissues compared with the normal counterparts (26) and tends to be expressed more at the invasive front than at the upper portions within the carcinoma tissues, which is associated with higher rates of distant metastasis (26,27). Notably, in silico predictions indicate that miR-124 has a conserved binding site within the 3′untranslated region (UTR) of IQGAP1 mRNA. Therefore, it was hypothesized that overexpression of IQGAP1 in CRC is, at least partly, due to silencing of miR-124.
Materials and methods
Tissue samples and cell lines
A panel of 30 pairs of primary CRCs and their matched adjacent normal mucosa, were obtained from patients (16 males and 14 females; aged 31–84 years) who underwent surgical resections, between June 2016 to December 2016 at the First People's Hospital of Yunnan province (Kunming, China). Tissues were snap-frozen in liquid nitrogen immediately after resection and then stored at −80°C for further use. The clinical features of all 30 CRC patients, including clinical stage, tumor location and treatment prior to surgical resection are presented in Table I. All patients whose tissue samples were collected for the study signed the informed consent. This project was approved by the Ethics Committee of the First People's Hospital of Yunnan Province.
Human CRC cell lines, including HT29, SW480, SW620, DLD1, LoVo and HCT116, and 293T cells, were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). CRC cells were maintained routinely in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin solution. The normal colonic epithelial cell line FHC was kindly donated by Dr. Liang Peng from Guangzhou Medical University (Guangzhou, China). FHC cells were maintained in Dulbecco's modified Eagle's medium (DMEM): F12 medium (Gibco; Thermo Fisher Scientific, Inc.) with supplements following ATCC protocol. 293T cells were maintained in DMEM medium containing high glucose supplemented with 10% FBS. All the cells mentioned were cultured in a 37°C humidified atmosphere containing 5% CO2.
Bioinformatics analyses
The analysis of the microarray dataset (accession no. GSE20916) (28) was performed using bioinformatics tool R2 (http://r2.amc.nl) following the manufacturer's protocol. The 2 subgroups of normal colon tissues differed in collection method. One consisted of 24 samples is collected by surgery, the other of 10 samples is collected by colonoscopy. The colon tumor subgroup consisting of 30 samples were also from colonoscopy. Targetscan version 7.1 (http://targetscan.org), Pictar (http://pictar.mdc-berlin.de) and miRecords (http://c1.accurascience.com/miRecords/) were used for prediction of miR-124 target genes.
Oligonucleotide and cell transfection
Hsa-miR-124 mimics and its scrambled controls were synthesized by Shanghai Genepharma Co., Ltd. (Shanghai, China). The sequences of the hsa-miR-124 mimics were as follows: Sense 5′-UAAGGCACGCGGUGAAUGCC-3′ and antisense 5′-CAUUCACCGCGUGCCUUAUU-3′. The sequences of the scramble controls were as follows: Sense 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense 5′-ACGUGACACGUUCGGAGAATT-3′. For transfection, cells were seeded into 6-well clusters at a density of 3×106 cells/well and transfected using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol, with 30 nM miR-124 mimics or scramble controls for 48 h at 37°C before further experimentation in assays or RNA/protein extraction.
Lentivirus packaging and stable cell line establishment
IQGAP1-knockdown and negative control lentivirus particles were packaged by co-transfecting 2.5 µg IQGAP1-short hairpin (sh)RNA plasmids or shRNA scramble control plasmids (GeneCopoeia, Inc., Rockville, MD, USA) using psi-LVRU6GP vector as backbone (GeneCopoeia, Inc.) with lentiviral packaging plasmids into 293T cells, using Lenti-Pac™ HIV Expression Packaging Systems (GeneCopoeia, Inc.) according to the manufacturer's protocol. The target sequence for IQGAP1-shRNA #1 and #2 are as follows: 5′-GGTTATCACCCTCATTCGTTC-3′ and 5′-GGCTTATGAGTACCTTTGTCA-3′. For lentivirus infection, cells were incubated with viral supernatant in the presence of 8 µg/ml polybrene for 24 h, followed by Puromycin (Invitrogen; Thermo Fisher Scientific, Inc.) selection until drug-resistant colonies became visible.
RNA extraction and quantitative polymerase chain reaction (qPCR)
Total RNA was isolated from CRC tissues and cells using the TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Mature miR-124 expression in cells was determined using a Hairpin-it™ miRNAs qPCR kit (Shanghai Genepharma, Co., Ltd.). RNU6B was used as an endogenous control. IQGAP1 mRNA expression was determined by using SYBR green qPCR assay (Takara Bio, Inc., Otsu, Japan). The thermocycling conditions were as follows: Denaturation at 95°C for 3 min, followed by 40 cycles of amplification at 95°C for 12 sec and extension at 62°C for 40 sec. GAPDH was used as the endogenous control. The sequence of primers for IQGAP1 was as follows: Forward: 5′-GGGACCAACCAAAGTGTGTCAAC-3′, reverse: 5′-CTGCTCATTATTGCCTGTCTTGGA-3′. Primer sequence for GAPDH: Forward: 5′-TGACTTCAACAGCGACACCCA-3′, reverse: 5′-CACCCTGTTGCTGTAGCCAAA-3′. Data was analyzed using the 2−ΔΔCq method (29). This experiment was repeated 3 separate times.
Cell growth assay and cell colony formation assay
Cell growth was detected by using a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer's protocol. For colony formation assay, cells were trypsinized and plated on 6-well plates at a density of 3×102 cells/well and cultured at 37°C for 10 days. The colonies were stained with 0.1% crystal violet solution containing 80% methanol for 5 min at room temperature. The number of colonies defined as >50 cells/colony were counted at ×40 magnification by using a light microscope. The assays were performed in triplicate of wells in 3 separate experiments.
Western blot analysis
Cultured cells were lysed in radioimmunoprecipitation assay buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) with 1% PMSF, for 15 min on ice Subsequently, cell lysates were centrifuged at 14,000 × g for 30 min and the supernatant was harvested. Protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). A total of 30 or 50 µg protein/lane was loaded onto a 10% SDS-PAGE minigel and transferred onto a polyvinylidene difluoride membrane. Membranes were blocked using Tris-buffered saline containing 20% Tween-20 and 5% non-fat milk at room temperature for 2 h. Following probing with 1:1,000 diluted anti-IQGAP1 (cat. no. 29016; Cell Signaling Technology, Inc.), anti-phosphorylated (p)-extracellular signal regulated kinase (ERK)1/2 (cat. no. 4370; Cell Signaling Technology, Inc.), anti-ERK1/2 (cat. no. 4695; Cell Signaling Technology, Inc.) and anti-β-catenin (cat. no. 9562; Cell Signaling Technology, Inc.) respectively at 4°C overnight, the blots were subsequently incubated with anti-rabbit horseradish peroxidase-conjugated anti-immunoglobulin (Ig)G (cat. no. 7074; Cell Signaling Technology, Inc.) for 1 h at room temperature. Signals were visualized using ECL Substrates (Pierce; Thermo Fisher Scientific, Inc.). β-actin (1:3,000; cat. no. sc-47778; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was used as an endogenous protein for normalization. This experiment was repeated 3 separate times.
Luciferase reporter assay
A fragment of wild type 3′UTR of IQGAP1 containing the putative miR-124 binding site was amplified by PCR. The PCR product was sub-cloned into a psiCHECK-2 vector (Promega Corporation, Madison, WI, USA) immediately downstream of the luciferase gene sequence. A corresponding psiCHECK-2 construct containing the 3′UTR of IQGAP1 with a mutant seed sequence of miR-124 was also synthesized.
293 cells were plated in triplicate wells in 24-well clusters at a density of 1×105 cells/well, then co-transfected with 0.1 µg psiCHECK-2 construct and 30 nM miR-124 mimic or scramble control using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Following incubation for 48 h at 37°C, luciferase activity was detected using a dual-luciferase reporter assay system (Promega Corporation) and normalized by Renilla activity. The experiment was repeated 3 separate times.
Statistical analysis
Statistical analyses were performed using the SPSS software, version 15.0 (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism software version 6.01 (Graphpad Software, Inc., La Jolla, CA, USA). Comparisons of miR-124 and IQGAP1 expression between CRC tissues and paired adjacent colonic epithelial tissues were performed using a Wilcoxon's paired test. All data are presented as the mean ± standard deviation. Comparisons among multiple groups were performed using one-way analysis of variance followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.
Results
IQGAP1 expression is aberrantly upregulated in CRC
IQGAP1 has been reported to be overexpressed in multiple tumors, including CRC. Therefore gene expression profiles of human CRC cohorts were first analyzed to observe the status of IQGAP1 expression. As demonstrated in Fig. 1A, IQGAP1 was significantly upregulated in the cohort of CRC tissues compared with the other 2 subgroups of normal colon tissues collected by surgery and colonoscopy respectively (P<0.001). qPCR was performed to detect IQGAP1 mRNA level in clinical specimens of CRC. In the cohort of 30 cases of paired CRC tissues and adjacent colon epithelia, it was demonstrated that IQGAP1 was significantly upregulated in tumors compared with the matched normal epithelia (P=0.0062; Fig. 1B). IQGAP1 protein levels in CRC cell lines: HCT116, HT29, SW480, SW620, LoVo and DLD1, are presented in Fig. 1C. Since HCT116 and SW480 cells expressed a high level of IQGAP1, they were selected for establishing stable IQGAP1-knockdown cells.
Knockdown of IQGAP1 inhibits CRC cell growth and colony formation ability by suppressing phosphorylation of ERK and antagonizing β-catenin activity
Next, the role of IQGAP1 in CRC growth and the potential underlying mechanism was investigated. Stable IQGAP1 knockdown of CRC cells was generated by introducing IQGAP1 shRNA-packaged lentiviral particles into HCT116 and SW480 cells which express relatively higher levels of IQGAP1 compared with the other cell lines investigated in the present study. In the loss-of-function assays, downregulation of IQGAP1 significantly reduced cell growth (P<0.05) and colony formation ability (Fig. 2A-C). Immunoblotting demonstrated that in the IQGAP1-knockdown cells, phosphorylation of ERK1/2 was impaired as well as β-catenin activity, (Fig. 2D), demonstrating the augmenting effect of IQGAP1 on MAPK and β-catenin signaling.
miR-124 is aberrantly downregulated in CRC tissues
To demonstrate the expression pattern of miR-124 in CRC, qPCR was performed to detect the miR-124 level in the clinical specimens and cell lines. In the mentioned cohort of 30 cases of paired CRC tissues and adjacent colon epithelia, it was demonstrated that miR-124 was significantly downregulated in CRCs compared with the adjacent normal tissues (P=0.005; Fig. 3A). The miR-124 expression level was further examined in CRC cell lines and miR-124 expression in all 6 CRC cell lines mentioned was significantly decreased compared with the normal colonic epithelial cell line FHC (P<0.0001; Fig. 3B). Notably, HCT116 and SW480 cells that expressed relatively high IQGAP1 expression levels (Fig. 1C), were demonstrated to express relatively low levels of miR-124 out of these CRC cell lines, therefore were chosen for restoration of miR-124 in further assays (Fig. 3B).
IQGAP1 is a direct target gene of miR-124 in CRC
By utilizing bioinformatics algorithms (Targetscan/Pictar/miRecords), it was identified that IQGAP1 was a potential target gene of miR-124. The predicted binding site of miR-124 within the 3′UTR of IQGAP1 mRNA is presented in Fig. 4A. It was hypothesized that miR-124 may target IQGAP1 in CRC. To investigate this further, an miR-124 mimic was introduced into HCT116 and SW480 cells. With the restoration of miR-124 expression, IQGAP1 mRNA expression was demonstrated to be significantly decreased by qPCR (P<0.05). In addition, its protein level was demonstrated to be decreased by immunoblotting (Fig. 4B and C), while suppression of ERK1/2 phosphorylation and β-catenin was also simultaneously observed (Fig. 4C). To further investigate if the predicted binding site of miR-124 to 3′UTR of IQGAP1 mRNA is responsible for the downregulation of IQGAP1, the 3′UTR of IQGAP1 was cloned into a luciferase reporter vector wild-type (wt)-IQGAP1 and a corresponding mutant version (mut-IQGAP1) was also constructed. wt-IQGAP1 and miR-124 mimic or scrambled control were co-transfected into 293T and HCT116 cells to perform a luciferase reporter assay. In 293T and HCT116 cells, the luciferase activity of miR-124 transfected cells was significantly reduced compared to the scrambled control cells (P<0.05; Fig. 4D and E). Furthermore, miR-124-mediated repression of luciferase activity was abolished by the mutant putative binding site (Fig. 4D and E).
Restoration of miR-124 expression suppresses cell growth and colony formation ability of CRC
To investigate the effects of miR-124 on cell growth of CRC, the cell growth ability of miR-124-transfected HCT116 and SW480 cells was measured. First of all, significant restoration of miR-124 expression was performed by mimic transfection in the two cell lines, HCT116 and SW480 (P<0.05; Fig. 5A). CCK-8 assays demonstrated that restoration of miR-124 expression significantly repressed cell growth ability in HCT116 and SW480 cells after 72 h (P<0.05; Fig. 5B and C). Furthermore, significantly decreased colony formation activity was also observed in the two aforementioned cell lines (P<0.05; Fig. 5D). These results suggested that miR-124 serves a suppressive role in tumor growth in CRC and its absence may confer malignant potentials to CRC cells.
Discussion
IQGAP1, the best characterized member of the IQGAP family, functions as a scaffold protein in the cytoplasm to curb, compartmentalize and coordinate multiple signaling pathways in a variety of cell types, and participates in cell-cell interaction, cell adherence, and movement via actin/tubulin-based cytoskeletal reorganization (30). In recent years, studies demonstrated that IQGAP1 is frequently aberrantly overexpressed in multiple tumors and integrates and mediates several pro-oncogenic signaling pathways to promote tumorigenesis and metastasis (31–34). IQGAP1 may bind directly to B-Raf, dual specificity mitogen-activated protein kinase kinase 1 and ERK, facilitating epidermal growth factor-induced activation of the MAPK cascade. IQGAP1 binding to β-catenin enhances β-catenin nuclear translocation, initiating transcription of cyclin D1 (35). Notably, a previous study by Jameson et al (36) verified that blockage of IQGAP1-ERK1/2 interactions by introducing a specific IQGAP1 WW domain peptide, inhibits RAS- and RAF-driven tumorigenesis and bypasses acquired resistance to the BRAF inhibitor, which validates IQGAP1 inhibition as a promising therapeutic strategy for tumors.
Dysregulation of miRNAs have been demonstrated to account for malignant phenotypes in tumors. Growing evidence proves that miR-124 is a miRNA, which exerts tumor suppressive effects but is frequently absent in tumors (2–6). Despite this miR-124 has been validated to target oncogenic genes including STAT3, FOXQ1 and Slug (5,10,11) and the present study aimed to identify the mechanistic link of miR-124 to other potential target genes contributing to CRC development. Since IQGAP1 is predicted as a potential target gene of miR-124 by in silico prediction, it was hypothesized that there is a regulative link between miR-124 and IQGAP1. In the present study, IQGAP1 was demonstrated to be overexpressed in CRC tissue. Knockdown of IQGAP1 in CRC cells could impede cell growth and colony formation ability, which is considered to arise from repression of phosphorylated (p)-ERK1/2 and β-catenin expression. Similarly, restoration of miR-124 induced the inhibition of cell growth and colony formation ability, and repressed p-ERK1/2 and β-catenin. Given that miR-124 bound to the 3′UTR of IQGAP1 mRNA and led to a decrease of IQGAP1 expression, it was demonstrated that the absence of miR-124 was responsible, at least in part, for the aberrant upregulation of IQGAP1 and subsequent enhanced activation of MAPK and β-catenin signaling in CRC.
From the perspective of tumor treatment, it is urgent to investigate promising therapeutic targets and invent efficient approaches for tumor targeting. For example, the results of the present study indicated that IQGAP1 is a potential target for CRC treatment and further highlighted that miR-124 is in theory a potential way to antagonize IQGAP1 similar to the WW domain peptide of IQGAP1 mentioned. Recently, nanoparticles have emerged as a popular concept for targeted delivery of therapeutic agents to tumors (37–39). A type of combined polymeric nanoparticle (PNP) entrapping phosphatidyl inositol 3 kinase inhibitors or irinotecan suppresses or even eliminates lung metastasis of CRC in a BALB/C mice model, but negligible accumulation of PNPs is detected in the organs including the spleen, liver and kidneys indicating good tissue selectivity of PNPs and organ safety (40). Notably, delivery of nanoparticles conjugated with miR-20a or anti-miR-155 relieves aberrant expression of their protein targets and eventually reduces tumor growth and liver metastasis in murine colon cancer models (41,42). In addition, considering miR-124 suppresses tumor progression through targeting a list of tumor-promoting proteins, miR-124-entrapped nanoparticles are anticipated to be a novel therapeutic option for treating tumors with absence of miR-124.
In conclusion, the present study provided evidence that accounted for aberrant upregulation of IQGAP1 in CRC and newly described a potential mechanism underlying dysregulated miR-124 indulging CRC progression. Therefore, it would be worthwhile to estimate the implication of miR-124-targeted therapy for clinical management of CRC.
Acknowledgements
The authors would like to thank Prof Liang Peng from Guangzhou Medical University (Guangzhou, China) for his technical assistance.
Funding
The present study was supported by grants from the National Natural Science Foundation of China (grant no. 81502128) and Joint Foundation of Kunming Medical University and Yunnan Provincial Science and Technology Department (grant no. 2014FB085).
Availability of data and materials
The dataset GSE20916 analyzed during the present study is publically available in GEO Datasets resource of NCBI [https://www.ncbi.nlm.nih.gov/gds/?term=GSE20916].
Authors' contributions
QG and YZ designed the study. JF, WZ and YW performed the experiments. PW analyzed the data and performed statistical analysis. YZ wrote the paper. All authors read and approved the manuscript.
Ethics approval and consent to participate
All patients whose tissue samples were collected for the study signed the informed consent. This project was approved by the Ethics Committee of the First People's Hospital of Yunnan Province.
Patient consent for publication
All patients whose tissue samples were collected for the study signed an informed consent.
Competing interests
The authors declare that they have no competing interests.
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