miR-4324 functions as a tumor suppressor in colorectal cancer by targeting HOXB2.

OBJECTIVE: MicroRNAs (miRNAs) are reported to have crucial roles in human cancers; however, their role in colorectal cancer (CRC) remains largely unknown.
METHODS: In this study, we analyzed the expression of miR-4324 in CRC cell lines using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). We also examined miR-4324 expression in CRC tumor tissues using a miRNA expression dataset obtained from the Gene Expression Omnibus. We validated the connection between miR-4324 and homeobox B2 (HOXB2) using a luciferase activity reporter assay and western blotting. The effects of miR-4324 and HOXB2 on CRC cell malignant behaviors in vitro were further investigated.
RESULTS: miR-4324 expression was significantly decreased in both CRC tumor tissues and cell lines. Overexpression of miR-4324 suppressed CRC cell proliferation, migration, and invasion. In contrast, overexpression of HOXB2 promoted CRC malignant cell behaviors. Furthermore, we validated HOXB2 as a direct target of miR-4324.
CONCLUSIONS: miR-4324 expression was decreased in CRC. miR-4324 regulates CRC cell proliferation, migration, and invasion by targeting HOXB2.

Introduction

The incidence of colorectal cancer (CRC) is reported to be dramatically increased in Asian countries relative to other parts of the world.[1] Overall survival for patients with localized CRC is about 10% to 15%; however, metastases are highly likely to occur within 5 years.[2,3] To date, multiple biomarkers have been identified that could be used as diagnostic or treatment biomarkers for CRC.[4] However, mechanisms related to CRC progression remain to be elucidated.

MicroRNAs (miRNAs) are a class of non-coding RNAs 18 to 24 nucleotides long and widely expression in mammals.[5] miRNA are reported to have dual roles in human cancers as either tumor suppressor genes or oncogenes.[6] miRNAs regulate gene expression by 3′-untranslated region (3′-UTR) binding, which affects almost all cell behaviors, including cell proliferation, differentiation, and apoptosis.[7]

miR-4324 is a newly identified miRNA that has been shown to have crucial roles in human cancers. Li et al.[8] investigated miRNA expression in nasopharyngeal carcinoma tissues using the miR assay method and found that miR-4324 expression was downregulated in tumor tissues. Moreover, these authors validated the expression of miR-4324 in nasopharyngeal carcinoma using in situ hybridization.[8] Recently, Wang et al.[9] revealed that miR-4324 was downregulated in breast cancer and associated with the loss of phosphate and tensin homolog (PTEN), predicting the poor prognosis of cancer patients. Inamoto et al.[10] revealed that urothelial carcinoma of the bladder patients with deregulation of miR-4324 had a poorer overall survival rate. However, these results did not investigate the biological roles of miR-4324 in cancers. Ge et al.[11] showed that miR-4324 was downregulated in bladder cancer and regulated cancer cell proliferation and metastasis by regulating Rac GTPase activating protein 1/STAT3/estrogen receptor 1. Collectively, these results demonstrate a tumor suppressive role of miR-4324 in these cancer types.

Homeobox B2 (HOXB2), a member of the HOX protein family, has been shown to be expressed at an increased level in cervical cancer and to promote cancer progression.[12] Moreover, HOXB2 was confirmed as a functional target of long non-coding (lnc)RNA HOXB-AS1/miR-885-3p and promoted glioblastoma cell proliferation, migration, and invasion.[13]

In this study, we analyzed the expression of miR-4324 in CRC cell lines and normal cell lines. The relationship between miR-4324 and HOXB2 was analyzed by using bioinformatics analysis, luciferase activity reporter assay, and western blotting. In addition, we analyzed the effects of miR-4324/HOXB2 on CRC cell proliferation, migration, and invasion in vitro.

Materials and methods

This work was an in vitro experiment based on cell lines. Therefore, ethical approval and patient consent were not required.

Cell lines

CRC cell lines (HCT-116, SW480, and SW620) and a normal colon epithelium cell line (FHC) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen/Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen) in a 37°C humidified incubator with 5% CO2.

Microarray analysis

A miRNA expression dataset of CRC tissues, GSE123040, was downloaded from Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/gds/) and used to identify aberrantly expressed miRNAs in CRC.

Cell transfection

miR-4324 mimic and its negative control (NC-mimic) were obtained from GenePharm (Shanghai, China). The pcDNA3.1 containing the open reading frame of HOXB2 (pHOXB2) and the corresponding control were purchased from GenScript (Nanjing, China). Cell transfection was accomplished using Lipofectamine 2000 (Invitrogen) according to the provided protocols.

Reverse transcription-quantitative PCR

The RNA from cultured cells was extracted using Trizol reagent (Invitrogen). Complementary DNA was synthesized from the extracted RNA using Prime-Script miRNA cDNA Synthesis Kit (Takara, Dalian, Liaoning, China). Reverse transcription quantitative-PCR was conducted at Applied Biosystem 7500 system (Foster City, CA, USA) using SYBR Green Mix (Takara) using the following procedure: 95°C for 10 minutes followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. The 2−ΔΔCt method was used to analyze the expression level of miR-4324, using U6 small nuclear (sn)RNA as internal standard.

Western blot

Protein from cultured cells was extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Haimen, Jiangsu, China). An equal amount of protein sample was isolated using 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane. After blocking with 5% fat-free milk, the membranes were incubated with primary antibodies (anti-HOXB2: ab220390, anti-GAPDH: ab181602; Abcam, Cambridge, MA, USA) overnight at 4°C. After incubation with horseradish peroxidase-conjugated secondary antibody (ab6721, Abcam), the band signals were developed using BeyoECL kit (Beyotime). Expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control.

Cell proliferation assay

Cell proliferation rate was analyzed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) method. Ten microliters of MTT solution was added to each well at indicated time points and further incubated for 4 hours. Then, 50 µL of dimethyl sulfoxide was added to each well. Optical density was measured at 570 nm using a microplate reader.

Scratch wound assay

Cells were seeded into a 12-well plate and incubated to approximately 100% confluence. A tip was used to create a scratch on the cell surface. Then, cells were washed with PBS to remove cell debris and photographed at 24 hours.

Transwell invasion assay

Cell invasion ability was analyzed with a Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) pre-coated chamber (Corning, New York, NY, USA). Cells in DMEM without FBS were seeded in the top chamber, and the lower chamber was filled with DMEM containing 10% FBS. After incubation for 48 hours, invasive cells were fixed with 4% paraformaldehyde, stained with crystal violet, and counted under the microscope.

Dual-luciferase reporter assay

The TargetScan algorithm (www.targetscan.org/vert_72) was used to search for putative targets of miR-4324. Wild-type (wt) or mutant (mt) 3′-UTR of HOXB2 was inserted into pMIR-REPORT to generate wt HOXB2 and mt HOXB2, respectively. Cells were co-transfected with synthetic miRNAs or luciferase reporter vectors using Lipofectamine 2000. Following a 48-hour incubation, relative luciferase activity was analyzed with the Dual-Luciferase Assay system (Promega, Madison, WI, USA).

Statistical analysis

Data are presented as mean ± standard deviations following analysis using SPSS V_13.0 software (SPSS Inc., Chicago, IL, USA). Student’s t-test was used to analyze differences between two groups. One-way analysis of variance followed by Tukey’s post hoc test was used to analyze differences among multiple groups. P-values < 0.05 were considered statistically significant.

Results

miR-4324 was downregulated in CRC

To evaluate the function of miR-4324 in CRC, we first examined miR-4324 expression in CRC tissues compared with normal tissues. As presented in Figure 1a, miR-4324 expression was lower in CRC tissues than in normal tissues (P < 0.01). Moreover, miR-4324 expression in CRC cell lines and the normal cell line was analyzed with RT-qPCR. We found that miR-4324 expression was reduced in CRC cell lines compared with the normal cell line (Figure 1b) (SW480 and HCT-116 vs. FHC: P < 0.01, SW620 vs. FHC: P < 0.001). The SW620 and SW480 cell lines were selected for subsequent analyses because they had the lowest miR-4324 expression in the cell lines investigated.

Figure 1.

Expression of miR-4324 was decreased in CRC. (a) Expression of miR-4324 was decreased in CRC cell lines relative to that in a normal epithelial cell line. (b) Expression of miR-4324 was decreased in CRC cell lines (SW480, SW620, HCT-116) relative to that in a normal epithelial cell line. **P < 0.01, ***P < 0.001. miR-4324, microRNA-4324; CRC, colorectal cancer; FHC, normal colon epithelium cell line.

miR-4324 regulates CRC cell proliferation, migration, and invasion

To manipulate the expression of miR-4324, synthetic miRNAs were transfected into CRC cell lines. We found that transfection with miR-4324 mimic increased the levels of miR-4324 in CRC cells (Figure 2a; P < 0.001). A cell proliferation assay revealed that miR-4324 mimic significantly decreased cell proliferation ability in CRC cells (Figure 2b; P < 0.001). The wound-healing assay revealed that miR-4324 mimic significantly decreased cell migration (Figure 2c; P < 0.001). Additionally, the Transwell invasion assay revealed that cell invasion ability was significantly inhibited by miR-4324 mimic (Figure 2d; P < 0.01).

Figure 2.

Overexpression of miR-4324 inhibited CRC cell proliferation, migration, and invasion. Effects of miR-4324 mimic on (a) miR-4324 expression, (b) cell proliferation, (c) cell migration, and (d) cell invasion in CRC cell lines SW480 and SW620. *P < 0.05, **P < 0.01, ***P < 0.001. miR-4324, microRNA-4324; CRC, colorectal cancer; NC-mimic, negative control of miR-4324 mimic, OD, optical density.

HOXB2 was a direct target of miR-4324

TargetScan predicted that HOXB2 contains a putative binding site for miR-4324 (Figure 3a). The luciferase activity reporter assay revealed that luciferase activity in CRC cells transfected with wt HOXB2 was significantly inhibited by miR-4324 mimic (Figure 3b; P < 0.001). Western blot further revealed that HOXB2 expression was reduced by miR-4324 mimic in CRC cells (Figure 3c).

Figure 3.

HOXB2 was a direct target of miR-4324. (a) TargetScan software was used to predict the putative binding site for miR-4324 in the 3′-untranslated region (UTR) of HOXB2. (b) Luciferase activity in CRC cells transfected with luciferase reporter vectors and synthetic miRNAs. (c) Protein expression of HOXB2 in CRC cells transfected with synthetic miRNAs. ***P < 0.001. miR-4324, microRNA-4324; CRC, colorectal cancer; wt, wild-type; mt, mutant; HOXB2, homeobox B2, NC-mimic, negative control of miR-4324 mimic, GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

miR-4324 regulates CRC cell behaviors by targeting HOXB2

To further investigate the role of HOXB2 in CRC, we transfected pHOXB2 into CRC cell lines. We found that HOXB2 expression was significantly increased by pHOXB2 (Figure 4a). Cell proliferation (P < 0.001), migration (P < 0.01), and invasion (P < 0.01) were significantly increased by pHOXB2 (Figure 4b-4d). Moreover, we found that overexpression of HOXB2 partially reversed the effects of miR-4324 on CRC cell proliferation (P < 0.05), migration (P < 0.01), and invasion (P < 0.01) (Figure 4b-4d).

Figure 4.

Overexpression of HOXB2 promotes CRC cell proliferation, migration, and invasion. Effects of pHOXB2 (containing the open reading frame of HOXB2) on (a) HOXB2 expression, (b) cell proliferation, (c) cell migration (magnification: 200×), and (d) cell invasion (magnification: 200×) in CRC cell lines. **P < 0.01, ***P < 0.001. miR-4324, microRNA-4324; CRC, colorectal cancer; wt, wild-type; mt, mutant; HOXB2, homeobox B2; OD, optical density; NC-mimic, negative control of miR-4324 mimic, GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Discussion

The application of bioinformatics methods has identified multiple genes including protein coding genes and non-coding genes associated with cancer progression.[14,15] Numerous miRNAs have been shown to be abnormally expressed in CRC. For instance, expression of miR-483-3p was reported to be downregulated in oxaliplatin-resistant CRC cell lines; it regulates cell migration and apoptosis by targeting FAM171B.[16] Another study revealed that miR-198 was able to inhibit CRC cell proliferation but also promote apoptosis by targeting ADAM metallopeptidase domain 28 and inhibiting the JAK/STAT (Janus kinase/signal transducer and activator of transcription) pathway.[17] miR-10b was shown to inhibit CRC progression in vitro and in vivo by targeting fibroblast growth factor 13.[18] However, numerous miRNAs with altered expression in CRC remain to be explored.

miR-4324 has been demonstrated to be abnormally expressed in several human cancers and to function as a tumor suppressor.[8-11] In this study, we showed that miR-4324 expression was reduced in CRC tissues and cell lines compared with normal tissues and cell line. We also showed that overexpression of miR-4324 inhibited CRC cell growth, migration, and invasion. These results indicated that, consistent with the previous published work, miR-4324 may function as a tumor suppressor in CRC.

It is widely accepted that miRNAs exert their biological functions by targeting downstream target genes; therefore, we were interested in identifying the target of miR-4324 in CRC to fully understand its biological role. Multiple targets of miR-4324 have previously been identified in human cancers.[8-11] Using TargetScan software, we found that HOXB2 was a putative target of miR-4324. The HOX genes are crucial regulators in embryonic development.[19] Thirty-nine HOX genes are classified into four subfamilies: HOXA, HOXB, HOXC, and HOXD, located on chromosomes 7, 17, 2, and 12, respectively.[20] We found that overexpression of HOXB2 could promote CRC cell proliferation, migration, and invasion in vitro, indicating that HOXB2 functions as a tumor promoter in CRC. Using the luciferase activity reporter assay, western blotting, and a functional assay, we confirmed that HOXB2 was a functional target for miR-4324.

In conclusion, we showed that expression of miR-4324 was reduced in CRC. This is the first work to investigate the connection between miR-4324 and HOXB2 in CRC. Altogether, our results provide novel insights into the mechanisms related to CRC progression. Importantly, further efforts are needed to validate the relationship between miR-4324 and HOXB2 in CRC.