miR-152 inhibits the proliferation and invasion of chordoma cells by targeting HOXC8.

Introduction

Chordoma is an uncommon type of cancer characterized by a slow growth rate, high recurrence, and local invasion.[1,2] The overall survival of patients with chordoma is poor, even after surgical resection followed by radiation therapy.[3] Although increasing numbers of studies have investigated the molecular mechanisms underlying chordoma progression,[4,5] there are still no reliable biomarkers for chordoma prediction or treatment.

MicroRNAs (miRNAs) are endogenous RNAs ranging from 20 to 25 nucleotides in length.[6] miRNAs have been widely recognized to cause mRNA degradation or translation repression by binding to the 3′-untranslated region (UTR) of their target genes.[6,7] Emerging evidence suggests that miRNAs are abnormally expressed in human cancers,[8,9] and can function as either tumor suppressors or oncogenes, depending on the target genes regulated.[8,9] miRNAs were also reported to modulate many essential cell behaviors including cell proliferation, invasion, and apoptosis.[8,9] miR-152 is a member of the miR-148/152 family that shows reduced expression patterns in various types of cancer.[10] miR-152 functions as a tumor suppressor by targeting genes in cancers including nasopharyngeal carcinoma, endometrial cancer, and ovarian cancer.[11-13] miR-152 inhibited cell migration and invasion by interacting with DNA (cytosine-5)-methyltransferase 1 in nasopharyngeal carcinoma.[11] Furthermore, miR-152 inhibited endometrial cancer cell proliferation and arrested the cell cycle at G2/M phase by targeting cell division cycle 25B (CDC25B).[12] A previous study by Li et al.[13] showed that miR-152 participated in the proliferation and metastasis of ovarian cancer through targeting ERBB3. However, the function of miR-152 in chordoma requires further investigation.

Homeobox (HOX) genes encode transcription factors that can in turn modulate various cell processes.[14] Numerous studies have investigated the role of homeobox C8 (HOXC8) in human cancers, and found that its expression was dysregulated in cervical cancer, non-small cell lung cancer, breast cancer, and osteosarcoma.[15-17] For example, HOXC8 expression was upregulated in cervical cancer and its overexpression promoted cell proliferation.[15] Moreover, HOXC8 was revealed as a trigger for non-small cell lung cancer cell proliferation and migration activation through inducing transforming growth factor-β1 expression.[16] However, the molecules regulating HOXC8 expression in cancer development are not fully understood. HOXC8 was found to be regulated by miR-196s, resulting in abnormal breast cancer cell migration and metastasis.[17]

To the best of our knowledge, the relationship between HOXC8 and miR-152 in chordoma progression has not been determined. The current study therefore focused on investigating the effects of miR-152 and HOXC8 on chordoma cell behavior, and examining the association between miR-152 and HOXC8 to provide more evidence for the mechanisms underlying chordoma progression.

Materials and methods

Cell culture

Human chordoma cell lines (U-CH1 and U-CH2) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and incubated in Iscove’s modified Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) and RPMI-1640 medium (Invitrogen) at a ratio of 1:4, supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillin/streptomycin (Beyotime, Haimen, Jiangsu, China). Human embryonic kidney 293T (HEK-293T) cells were also obtained from ATCC and cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin/streptomycin. The incubator was maintained at 37°C and 5% CO2.

Cell transfection

Cells were transfected with miR-152 mimic (5′-UCAGUGCAUGACAGAACUUGG-3′), miR-152 inhibitor (5′-CCAAGUUCUGUCAUGCACUGA-3′), negative control miRNA (NC-miRNA, 5′-UUCUCCGAACGUGUCACGUTT-3′), and pcDNA3.1-HOXC8 using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were collected after 48 hours of transfection for further analyses. miRNAs were purchased from GenePharma (Shanghai, China), and the pcDNA3.1-HOXC8 expression vector and pcDNA3.1 vector were purchased from GenScript (Nanjing, China).

RNA isolation and reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was isolated using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. cDNA was synthesized using a PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). RT-qPCR was conducted using SYBR® Premix Ex Taq™ II (Takara) with an ABI 7500 Real-time PCR system (Applied Biosystems, Foster City, CA, USA). The following primers were used: miR-152 forward, 5′-GATCATTGGCCTTGCCAGTA-3′; reverse, 5′-GTGTGTAGAGGTCAGGAAGT-3′, and U6 snRNA forward, 5′-GGTAGCCGCGGTTGAAATGG-3′; reverse, 5′-CAGTAAGCAGTAAAGTCGA-3′. miR-152 levels were calculated using the 2−ΔΔCt method with U6 snRNA as an internal control. The RT-qPCR procedure was carried out as follows: 95°C for 2 minutes, 95°C for 10 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, for 40 cycles.

Protein isolation and western blot

Total protein was extracted using cell lysis buffer (Beyotime). Protein concentrations were determined using a bicinchoninic acid protein assay kit (Beyotime), and 50-µg samples were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Beyotime). The membranes were blocked in 5% bovine serum albumin, followed by incubation overnight with primary antibodies (anti-HOXC8: ab86236; anti-GAPDH: ab181602; Abcam, Cambridge, MA, USA) at 4°C. The membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (ab6721, Abcam) for 2 hours at room temperature and the protein signals were visualized using a BeyoECL Plus kit (Beyotime) and observed using a ChemiDoc MP system (Bio-Rad, Hercules, CA, USA). HOXC8 levels were analyzed using Image J 1.44 software (NIH, Bethesda, MA, USA) with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control.

Cell proliferation assay

Cells were plated into 96-well plates and allowed to attach for 24 hours, followed by the addition of 10 μL MTT (10 mg/mL, Beyotime) to each well. After further incubation for 4 hours, the medium was removed and 200 μL dimethylsulfoxide was added. Finally, the absorbance was measured at 490 nm using a microplate reader. All samples were analyzed in triplicate.

Colony-formation assay

Cells were seeded in six-well plates (1,000 cells/well) and grown in the corresponding medium. After incubation for 14 days, the cells were washed with phosphate-buffered saline, fixed with 4% paraformaldehyde for 30 minutes, and stained with 0.5% crystal violet for 30 minutes. The numbers of colonies were counted under a microscope.

Transwell invasion assay

Cell invasion was assayed using Transwell insert chambers (Corning, Inc., Corning, NY, USA). Cells (1 × 105) were seeded in the upper chamber in the corresponding medium without FBS, and the lower chamber was filled with medium supplemented with FBS. After incubation for 24 hours, invading cells were fixed with 4% paraformaldehyde for 30 minutes and stained with 0.5% crystal violet for 5 minutes. Cell numbers were counted in five independent fields using an inverted microscope (Olympus, Tokyo, Japan), and the invasion ability was presented as fold-change compared with the control group.

Luciferase reporter assay

The target gene of miR-152 was determined using TargetScan (http://www.targetscan.org/vert_72/). The HOXC8 3′-UTR containing the wild-type (wt) or mutant (mut) miR-152-binding site was cloned into a psiCHECK2 vector (Promega, Madison, WI, USA). Cells were co-transfected with 100 ng wt or mut reporter plasmid and 100 nM miR-152 mimic/miR-NC. Luciferase activity was then measured after 48 hours of transfection using a Luciferase System Kit (Promega).

Statistical analysis

Differences between groups were assessed by Student’s t-test, one-way analysis of variance (ANOVA), and Tukey’s post-hoc test. Data were presented as mean ± standard deviation and analyzed using SPSS 17.0 software (SPSS, Chicago, IL, USA). A P value <0.05 was considered significant.

Results

Downregulation of miR-152 promotes chordoma cell proliferation and invasion

We measured the expression of miR-152 in chordoma cell lines (U-CH1 and U-CH2) and normal HEK-293T cells. miR-152 expression levels were decreased in U-CH1 (0.43 ± 0.06) and U-CH2 (0.39 ± 0.07) cells compared with HEK-293T (1.24 ± 0.07) cells (P < 0.001) (Figure 1a). U-CH1 and U-CH2 cell lines were then transfected with synthetic miRNAs. miR-152 expression was dramatically increased by miR-152 mimic but decreased by miR-152 inhibitor compared with miR-NC in both cell lines, as determined by RT-qPCR (P < 0.001) (Figure 1b). MTT assay revealed that proliferation of U-CH1 and U-CH2 cells were decreased by miR-152 mimic but enhanced by miR-152 inhibitor (P < 0.001) (Figure 1c). Colony-formation assay showed that the number of colonies was significantly increased in cells transfected with miR-152 inhibitor compared with miR-NC (P < 0.01) (Figure 1d). Transwell migration assay also demonstrated that cell migration was significantly decreased by ectopic expression of miR-152 compared with cells transfected with miR-NC (P < 0.01) (Figure 1e).

Figure 1.

miR-152 overexpression inhibits chordoma cell proliferation, colony formation, and cell migration. (a) miR-152 expression in human chordoma cell lines (U-CH1 and U-CH2) and human embryonic kidney 293T (HEK-293T) cells measured by RT-qPCR. (b) miR-152 expression, (c) cell proliferation, (d) colony formation, and (e) cell invasion in human chordoma cell lines (U-CH1 and U-CH2) transfected with synthetic mRNAs (stained with crystal violet). **P<0.01; ***P<0.001. miR-152, microRNA-152; RT-qPCR, reverse transcription-quantitative PCR; miR-NC, negative control miRNA.

HOXC8 was a direct target of miR-152

Identification of the miR-152 target gene is crucial for exploring the mechanisms of miR-152 in chordoma. The online software TargetScan identified HOXC8 as a direct target of miR-152 (Figure 2a). Luciferase reporter assay was used to validate the relationship between miR-152 and HOXC8. Luciferase activity was significantly decreased by transfection with miR-152 mimic in cells transfected with HOXC8-wt (P < 0.001) but not in cells transfected with HOXC8-mut (Figure 2b). To confirm the miR-152 regulation of HOXC8 expression, we detected HOXC8 expression in cells transfected with miR-152 mimic, miR-152 inhibitor, or miR-NC. miR-152 overexpression significantly decreased HOXC8 expression while transfection with miR-152 inhibitor increased the expression of HOXC8 (Figure 2c). These results suggested that HOXC8 was a direct target gene of miR-152.

Figure 2.

HOXC8 was a direct target of miR-152. (a) Putative binding site between miR-152 and HOXC8 3′-UTR. (b) Luciferase activity in cells transfected with HOXC8-wt was repressed by miR-152 mimic. (c) HOXC8 expression in human chordoma cell lines (U-CH1 and U-CH2) transfected with synthetic mRNAs. ***P<0.001. ns, not significant; miR-152, microRNA-152; miR-NC, negative control miRNA; HOXC8, homeobox C8; wt, wild-type: mut, mutant; UTR, untranslated region; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

miR-152 regulates chordoma cell behavior through regulating HOXC8

To confirm that miR-152 regulated chordoma cell behavior by targeting HOXC8, we co-transfected the miR-152 mimic and HOXC8 construct into U-CH1 and U-CH2 cells and examined HOXC8 levels by western blot. HOXC8 levels were significantly enhanced by the HOXC8 construct (Figure 3a), and the HOXC8 construct partially reversed the inhibitory effect of miR-152 mimic on HOXC8 expression (Figure 3a). Furthermore, HOXC8 overexpression significantly promoted chordoma cell proliferation (P < 0.001), colony formation (P < 0.01), and cell invasion (P < 0.01) (Figure 3b-3d). Rescue experiments showed that transfection with the HOXC8 construct attenuated the inhibitory effects of miR-152 on chordoma cell behavior (Figure 3b-3d). Collectively, these results confirmed that miR-152 regulated chordoma cell behavior through targeting HOXC8.

Figure 3.

HOXC8 overexpression reversed the inhibitory effects of miR-152 mimic on chordoma cell behavior. (a) HOXC8 expression, (b) cell proliferation, (c) colony formation, and (d) cell invasion in human chordoma cell lines (U-CH1 and U-CH2) transfected with HOXC8 construct or miR-152 mimic. **P<0.01; ***P<0.001. miR-152, microRNA-152; HOXC8, homeobox C8; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Discussion

Over 2,588 mature human miRNAs have been collected in the miRBase to date (http://www.miRBase.org/),[18] many of which have been reported to be associated with the progression of human cancers.[7-9,17] Zhang et al.[19] revealed that overexpression of miR-16-5p suppressed chordoma cell proliferation, invasion, and metastasis by targeting SMAD family member 3 (Smad3) in vitro and in vivo, suggesting a tumor suppressive role of miR-16-5p in chordoma. In contrast, Osaka et al.[20] revealed that high miR-155 expression was correlated with advanced disease stage, the presence of metastasis, and poor outcome in patients with chordoma, indicating an oncogenic role of miR-155. They also found that inhibition of miR-155 expression suppressed the proliferation, migration, and invasion of chordoma cells.[21]

In the current study, miR-152 expression was significantly downregulated in chordoma cell lines (U-CH1 and U-CH2) compared with HEK-293T cells. Functional assays showed that reduced miR-152 expression promoted chordoma cell proliferation, colony formation, and cell invasion in vitro, while transfection with miR-152 mimic significantly decreased the malignant behavior of chordoma cells. These results indicated that miR-152 had a tumor suppressive role in chordoma, consistent with its role in other human cancers, including nasopharyngeal carcinoma, endometrial cancer, and ovarian cancer.[11-13] Information on the downstream targets of miRNAs will help us to understand the roles of miRNAs.[7-9,11-13] Several miR-152 target genes have recently been validated in human cancers.[11-13] HOXC8 is located at chromosome 12 and plays an oncogenic role in human cancers.[14-17,21,22] Moreover, HOXC8 is regulated by miRNAs such as miR-196s in breast cancer.[17] However, the role of HOXC8 in chordoma remains undetermined. We identified HOXC8 as a direct target of miR-152 by luciferase reporter assay and western blot, and functional assays revealed that HOXC8 overexpression could promote cell proliferation and invasion and reverse the effects of miR-152 mimic on chordoma cell behavior.

This study had some limitations. Notably, the study only explored the biological roles of miR-152 and HOXC8 in chordoma cell lines, and further studies are needed to validate their biological roles and connection in chordoma tissues and animal models.

In conclusion, this study determined the roles of miR-152 and HOXC8 in chordoma. The results indicated that miR-152 downregulation and HOXC8 overexpression could both stimulate chordoma cell proliferation, colony formation, and cell invasion. In addition, HOXC8 overexpression could rescue the suppressive effect of miR-152 mimic on chordoma cell behavior, indicating that miR-152 affects chordoma cell behavior via targeting HOXC8. miR-152 might thus be a promising target for the treatment of chordoma, though further studies in chordoma tissues and in vivo are required to confirm the current findings.