Knockdown of long non-coding MIR210HG inhibits cell proliferation, migration, and invasion in hepatoblastoma via the microRNA-608-FOXO6 axis.

OBJECTIVE: Hepatoblastoma is the most common liver tumor. Recent research has found that long non-coding (lnc)RNAs are involved in multiple types of cancers, but the potential mechanism of lncRNA MIR210HG in hepatoblastoma remains unknown. The present study explored the molecular mechanism of MIR210HG in hepatoblastoma progression.
METHODS: The cell counting kit-8 was used to detect cell viability, and Transwell assays assessed cell migration and invasion. Luciferase reporter assays showed the relationship between MIR210HG and microRNA (miR)-608 and between miR-608 and forkhead box O6 (FOXO6). Functional tests were verified in vivo by a tumor xenograft model. The expression of MIR210HG, miR-608, FOXO6, E-cadherin, N-cadherin, and vimentin was determined by quantitative reverse transcription polymerase chain reaction and western blotting.
RESULTS: MIR210HG was shown to be highly expressed in hepatoblastoma tissues and cell lines. Knockdown of MIR210HG reduced proliferation, migration, and invasion in liver cancer cells, and suppressed tumor growth in vivo. MIR210HG competitively combined with miR-608, and miR-608 decreased FOXO6 expression.
CONCLUSION: Our study demonstrated that knockdown of MIR210HG inhibits hepatoblastoma development through binding to miR-608 and downregulating FOXO6. Our results provide novel insights for hepatoblastoma treatment involving the MIR210HG-miR608-FOXO6 axis.

Introduction

Hepatoblastoma is the most common primary liver tumor found in children, accounting for 65% of pediatric liver malignancies and predominantly occurring before the age of 2 years. It usually involves the transformation of immature liver precursor cells, and is not associated with hepatitis virus infection or hepatocirrhosis. Standard treatments for primary hepatoblastoma include complete resection, neoadjuvant chemotherapy, and liver transplantation. Patients whose hepatoblastomas are completely resected have a high survival rate; however, those with unresectable metastatic hepatoblastoma have a poor prognosis and low survival rate. Therefore, there is a need to develop effective diagnostic biomarkers to identify patients with hepatoblastoma early and to explore the mechanism of hepatoblastoma.

Long non-coding (lnc)RNAs are transcribed RNA molecules up to 200 nucleotides in length that do not encode proteins. Recent studies have found that lncRNAs have a range of functions in cancer, including in angiogenesis, tumorigenesis, and tumor metastasis. Moreover, genome-wide analysis showed a difference in the expression of lncRNAs between hepatoblastoma and healthy liver tissue. MIR210HG is encoded by the MIR210 host gene and has been shown to function in colon cancer, invasive breast cancer, and non-small cell lung cancer.[6-8] Additionally, Yan et al reported that MIR210HG is an oncogenic lncRNA in hepatocellular carcinoma (HCC), suggesting it has the potential to be a biomarker for hepatoblastoma in clinical diagnoses. However, the function of MIR210HG in hepatoblastoma is poorly understood. In this study, we explored the mechanism underlying the role of MIR210HG in hepatoblastoma, and show that it affected the proliferation, migration, and invasion of HuH-6 cells via the microRNA (miR)-608/Forkhead box (FOX)O6 axis.

Materials and methods

Samples

Thirty human hepatoblastoma tissue samples and adjacent healthy liver tissue samples were obtained from patients undergoing resection surgery at the Affiliated Hospital of Qingdao University. The present study followed the guidelines of The Declaration of Helsinki, and all patients provided their written informed consent. Tissue samples were immediately kept in liquid nitrogen and then stored at −80°C for further analysis. This study was approved by the ethics committee of the Affiliated Hospital of Qingdao University (approval number SYFY-WALL-25777).

Cell culture

Human hepatoblastoma cell lines SSMC-7721 and HuH-6, and HepG2, and the human normal liver cell line L-02 were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) mixed with 10% fetal bovine serum (FBS, Gibco) and 1% antibiotics (streptomycin and penicillin; Sigma-Aldrich, St Louis, MO, USA) in a humidified incubator with 5% CO2 at 37°C. HuH-6 and HepG2 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and SSMC-7721 and L02 cells were from the Shanghai Institute of Cell Biology (Chinese Academy of Sciences, Shanghai, China).

Short hairpin and small interfering RNA transfection

MIR210HG short hairpin RNA (shRNA) and negative control shRNA were purchased from Genepharma (Shanghai, China). The target sequence was 5′-GAGGAGGGACGCUGCGGCGCGT-3′. Cells were transfected with MIR210HG shRNA or NC shRNA using Lipofectamine RNAiMAX transfection reagent according to the manufacturer’s instructions (Thermo Fisher Scientific).

A negative control siRNA (inh-NC) was also used to control for nonspecific binding. Four groups of HepG2 cells were transfected with sh-NC + inh-NC, sh-MIR210HG + inh-NC, sh-NC + miR-608 inhibitor, and sh-MIR210HG + miR-608 inhibitor using Lipofectamine 3000.

Quantitative reverse transcription polymerase chain reaction (RT-qPCR)

Total RNA was extracted from cells and tissues using TRIzol reagent according to the manufacturer’s instructions (Invitrogen, Thermo Fisher Scientific), and cDNA synthesis was performed using the PrimeScript RT Reagent Kit with gDNA Eraser following the manufacturer’s instructions (Takara Biotechnology, Dalian, China). Briefly, 1 μg RNA was combined with 2 μl 5 × RNA reverse transcriptase in DEPC water to 10 μl, then incubated at 40°C for 15 minutes and 85°C for 5 s. RT-qPCR was carried out using 0.2 μl cDNA, 10 μl SYBR Green Premix Ex Taq kit (Takara Biotechnology), 0.5 μl forward primer, 0.5 μl reverse primer, and DEPC water to 20 μl on an ABI 7500 real-time RT-PCR system (Applied Biosystems, Thermo Fisher Scientific). PCR conditions were 95°C for 30 s, then 40 cycles of 95°C for 5 s and 60°C for 30 s.

Primer sequences were: MIR210HG forward primer 5′-GCTTGGTAGAGTGTCACGCC-3′, reverse primer 5′-CATCTGACCGAGCCAGTTTG-3′; miR-608 forward primer 5′-GGTGTTGGGACAGCTCCGTA-3′, reverse primer 5′-GTATCAACGCAGAGTACTTT-3′; FOXO6 forward primer 5′-GGCCGCGCTCGTGTACC-3′, reverse primer 5′-TACACGAGCGCGGCCG-3′; GAPDH forward primer 5′-AATGGACAACTGGTCGTGGAC-3′, reverse primer 5′-CCCTCCAGGGGATCTGTTTG-3′; and U6 forward primer 5′-GCCAGCTCCTACATCTCAGC-3′, reverse primer 5′-AGCCTGACTTGCTAGTGGATTAT-3′.

Data analysis used the 2−ΔΔCt method and gene expression was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for MIR210HG and FOXO6, and U6 for miR-608.

Cell viability assay

The cell counting kit (CCK)-8 kit (Dojindo, Kumamoto, Japan) was used to determine cell viability. Hepatoblastoma cells were seeded at 1 × 104 cells/well in a 96-well plate. After the required treatments, they were cultured with CCK-8 according to the manufacturer’s instructions. After 2 hours, their optical density at 450 nm was detected using a microplate reader.

Transwell assay

Twelve-well culture plates with 8-μm micropore inserts (Corning Inc., Corning, NY, USA) were used for Transwell cell migration assays. After the required treatments, 3 × 104 hepatoblastoma cells were suspended in 200 μL FBS-free DMEM medium and cultured in the upper compartment of the chamber. A total of 600 μL DMEM medium mixed with 20% FBS was applied to the lower compartment. Cells were cultured for 48 hours, then those that attached to the lower surface of the chamber were fixed with ethanol and stained with 0.5% crystal violet. Cells in five random fields of view were counted under the microscope.

The same 12-well culture plates with 8-μm micropore inserts were used for cell invasion assays, and the top side of the insert was coated with Matrigel. The same procedure as described above for the cell migration assay was followed.

Vector construction

Wild-type or mutant sequences of MIR210HG (Figure 1a) or FOXO6 (Figure 2a) containing miR-608 binding sites were amplified, digested, and ligated into the pGL3 luciferase vector (Promega, Madison, WI, USA) to investigate the binding between MIR210HG or FOXO6 with miR-608.

Figure 1.

Dual luciferase assay showing that miR-608 targets MIR210HG. (a) Bioinformatics analysis predicted that MIR210HG binds with miR-608. (b) RT-qPCR showing that MIR210HG decreased the expression of miR-608 in HuH-6 and HepG2 cells. (c, d) Dual-luciferase reporter assay showing that MIR210HG binds miR-608 in HuH-6 and HepG2 cells. **p < 0.01, ***p < 0.001, ns, not significant.

miR, microRNA; RT-qPCR, quantitative reverse transcription polymerase chain reaction.

Figure 2.

miR-608 targets the 3′-UTR of FOXO6 in hepatoblastoma cells. (a) Bioinformatics analysis predicted that miR-608 binds with FOXO6. (b, c) Dual-luciferase reporter assay showing that miR-608 binds FOXO6 in HuH-6 and HepG2 cells. (d, e) RT-qPCR showing that miR-608 decreased the expression of FOXO6 in HuH-6 and HepG2 cells. (f–i) Western blots showing that miR-608 decreased the expression of FOXO6 in HuH-6 and HepG2 cells. GAPDH was the internal control. *p < 0.05, ***p < 0.001, ns, not significant.

miR, microRNA; UTR, untranslated region; FOXO6, forkhead box O6; miR, microRNA; RT-qPCR, quantitative reverse transcription polymerase chain reaction.

Dual luciferase reporter assay

The indicated luciferase reporter vector and appropriate miRNA (MIR210HG-WT, MIR210HG-MUT, FOXO6-WT, or FOXO6-MUT) were transfected into human hepatoblastoma cells using Lipofectamine 3000 according to the manufacturer’s instructions (Thermo Fisher Scientific). After 72 hours of incubation, the cells were harvested by passive lysis buffer (Promega). Luciferase activity was determined using a dual luciferase assay kit (Promega).

Western blotting

Cells were harvested using radioimmunoprecipitation assay lysis buffer and centrifugated at 13,500 × g for 20 minutes at 4°C. Supernatants were collected in Eppendorf tubes, and protein concentrations determined using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Equal amounts of protein samples were subjected to sodium dodecyl sulfate– polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. Membranes were incubated with the primary antibody at 4°C overnight, then incubated with the secondary antibody for 2 hours at room temperature. Primary antibodies were: anti-FOXO6 (dilution 1:1000), anti-E-cadherin (dilution 1:1000), anti-N-cadherin (dilution 1:1000), and anti-vimentin (dilution 1:1000); secondary antibodies were: anti-rabbit (dilution 1:2000), and anti-mouse (dilution 1:2000). All antibodies were from Abcam (Cambridge, MA, USA). Protein blots were visualized using ECL chemiluminescence with GAPDH as a loading control.

Colony forming assay

After transfection, HuH-6 and HepG2 cells (1 × 103 per well) were seeded in 6-well plates and grown for 2 weeks in a humidified incubator at 37°C with 5% CO2. They were then washed with phosphate-buffered saline (Corning Inc.), fixed with 4% paraformaldehyde (Sigma-Aldrich) and stained with 0.5% crystal violet (Solarbio Science & Technology, Beijing, China) for 10 minutes. Colonies with more than 50 cells were counted using a microscope (Olympus, Tokyo, Japan).

Tumor xenograft model

Thirty-six 4-week-old female nude mice (Shanghai Laboratory Animal Center of Chinese Academy of Sciences, Shanghai, China) were raised in a specific pathogen-free animal room and allowed access to feed and water ad libitum. We established a HUH-6 cell line stably expressing shRNA-MIR210HG (MIR KD). We first designed the MIR210HG interference sequence and ligated this into the pGPU6/GFP/Neo expression vector to construct the shRNA-MIR210HG plasmid and negative control plasmid. The shRNA-MIR210HG plasmid was transfected into HUH-6 cells using Lipofectamine 3000, then cells were screened with G418 resistance for 14 days to obtain a cell line stably expressing shRNA-MIR210HG. A total of 3 × 105 MIR KD or HUH-6 cells were subcutaneously injected into the right back of mouse flanks. Tumors were removed 32 days after transplant and their diameters were measured.

Bioinformatics analysis

We used RegRNA2.0 (http://regrna2.mbc.nctu.edu.tw/) to identify miRNAs capable of binding MIR210HG.

We also used the TargetScanHuman 6.2 database (http://www.targetscan.org/vert_61/) to predict potential mRNA targets of miR-608.

Research statement

This study followed guidelines for REporting recommendations for tumour MARKer prognostic studies (REMARK). All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (2011).

Statistical analysis

Data are presented as means ± S.E.M from at least three independent experiments. Comparisons between groups were analyzed using the Student’s t test with SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered to be statistically significant.

Results

MIR210HG is upregulated in hepatoblastoma tissues and cells

RT-qPCR analysis showed that MIR210HG expression was significantly upregulated in 30 pairs of hepatoblastoma tissues compared with adjacent healthy liver tissues (p < 0.001; Figure 3a). Moreover, MIR210HG expression was also significantly higher in hepatoblastoma cells compared with non-malignant L-02 hepatocytes (p < 0.01; Figure 3b). These results indicate that MIR210HG is a potential regulator involved in hepatoblastoma progression.

Figure 3.

MIR210HG expression in hepatoblastoma tissues and cell lines as determined by RT-qPCR. (a) Comparing the expression of MIR210HG between paired hepatoblastoma cancer tissue and adjacent healthy liver tissues (n = 30). (b) Comparing the expression of MIR210HG between hepatoblastoma cell lines (SSMC-7721, HuH-6, and HepG2) and normal liver cells. **p < 0.01, ***p < 0.001.

miR, microRNA; RT-qPCR, quantitative reverse transcription polymerase chain reaction.

Knockdown of MIR210HG suppressed cell proliferation, migration, and invasion in HuH-6 and HepG2 cells

We found that MIR210HG expression was higher in HuH-6 and HepG2 cells than in SSMC-7721 cells (Figure 3b). Here, to investigate the function of MIR210HG in hepatoblastoma in vitro, we designed a MIR210HG shRNA which we transfected into hepatoblastoma cells. RT-qPCR revealed that MIR210HG shRNA transfection significantly reduced MIR210HG expression in HuH-6 and HepG2 cells (p < 0.001; Figure 4a). The CCK-8 assay showed that silencing of MIR210HG significantly suppressed cell viability in HuH-6 (p < 0.05) and HepG2 (p < 0.01) cells (Figure 4b), while the colony formation assay showed similarly significant suppression of proliferation in HuH-6 and HepG2 cells (p < 0.01; Figure 4c, d). Moreover, the Transwell assay revealed that MIR210HG silencing significantly suppressed the migration (p < 0.01) and invasion (p < 0.001) of HepG2 and HuH-6 cells compared with the sh-NC transfected group (Figure 4e–h).

Figure 4.

Knockdown of MIR210HG suppressed proliferation, migration, and invasion in HuH-6 and HepG2 cells. (a) RT-qPCR analysis of MIR210HG expression in HuH-6 and HepG2 cells transfected with sh-NC or sh-MIR210HG lncRNA vectors. (b) CCK-8 analysis of HuH-6 or HepG2 cells expressing the corresponding vector. (c, d) HuH-6 and HepG2 cells show reduced proliferation after MIR210HG depletion using the colony formation assay. (e, f) The Transwell assay showing reduced migration in HuH-6 and HepG2 cells after depleting MIR210HG. (g, h) The Transwell assay showing reduced invasion in HuH-6 and HepG2 cells after depleting MIR210HG. *p < 0.05, **p < 0.01, ***p < 0.001.

miR, microRNA; RT-qPCR, quantitative reverse transcription polymerase chain reaction; sh, short hairpin; lnc, long non-coding; CCK, cell counting kit.

Knockdown of MIR210HG inhibited tumor growth in vivo

To understand the role of MIR210HG in hepatoblastoma in vivo, we used a mouse HUH-6 cell xenograft model to investigate the effect of MIR210HG on tumor growth. Tumor size was measured every 8 days (Figure 5) and shown to be reduced in the MIR210HG knockdown group compared with the control group, suggesting that MIR210HG influences tumor growth in vivo.

Figure 5.

MIR210HG influenced tumor growth in vivo. (a) Tumors removed 32 days after inoculation. (b) Tumor volume following inoculation with sh-MIR210HG or control vectors.

miR, microRNA.

MIR210HG regulated miR-608 expression in HuH-6 and HepG2 cells

Fifteen miRNAs were predicted to bind with MIR210HG. We selected miR-608 for further study because it is a known tumor suppressor.[11-13] Bioinformatics was used to predict the seed region of miR-608 in mutated and wild-type MIR210HG. The miR-608 fragment 5′-UGCCUCGACAGGGUUGUGGUGGGGA-3′ paired well with 5′-GAGGAGGGTCCCAGCGCCGCCCT-3′ located in MIR210HG (Figure 1a). RT-qPCR showed that MIR210HG significantly decreased the expression of miR-608 in HuH-6 and HepG2 cells (p < 0.001; Figure 1b). To avoid non-specific binding, we mutated the miR-608 binding site of MIR210HG from 5′-GAGGAGGGTCCCAGCGCCGCCCT-3′ to 5′-GAGGAGGGACGCUGCGGCGCGT-3′, to generate RLuc-MIR210HG-Mut. MIR210HG cDNA was integrated downstream of the luciferase gene (RLuc-MIR210HG-WT) and transfected into HuH-6 and HepG2 cells with different miRNA mimics (Figure 1c, d). RLuc-MIR210HG-WT expression was significantly reduced by the miR-608 mimic in HepG2 (p < 0.001) and HuH-6 cells (p < 0.01), but the miR-608 mimic had no effect on RLuc-MIR210HG-mut expression. These data suggest that miR-608 directly regulates MIR210HG expression in HuH-6 and HepG2 cells.

miR-608 inhibition abolished the effect of sh-MIR210HG in HuH-6 and HepG2 cells

To determine whether miR-608 abolished the function of sh-MIR210HG in HepG2 cells, we designed an miR-608 inhibitor to inhibit its binding with sh-MIR210HG. The CCK-8 assay showed that silencing of miR-608 significantly promoted cell viability in HuH-6 and HepG2 cells (p < 0.001; Figure 6a, b). The colony formation assay also showed that silencing of miR-608 significantly promoted cell proliferation in HuH-6 (p < 0.01; Figure 6c, e) and HepG2 cells (p < 0.001; Figure 6d, f). Moreover, the Transwell assay revealed that miR-608 silencing significantly promoted the migration and invasion of HuH-6 (p < 0.01; Figure 6g, i, k, m) and HepG2 cells (p < 0.001; Figure 6h, j, l, n) compared with control groups. Collectively, these results show that inhibiting the function of miR-608 abolished the effect of sh-MIR210HG in HuH-6 and HepG2 cells.

Figure 6.

Transfection with a miR-608 inhibitor abolished the effect of sh-MIR210HG. (a, b) CCK-8 analysis of HuH-6 or HepG2 cells expressing the corresponding vector. (c–f) HuH-6 and HepG2 cells show reduced proliferation after MIR210HG depletion, while this is partially restored when MIR210HG levels are increased. (g–j) The Transwell assay showing reduced migration in HuH-6 and HepG2 cells after depleting miR-608, while this is partially restored when MIR210HG levels are increased. (k–n) The Transwell assay showing invasion in HuH-6 and HepG2 cells. **p < 0.01, ***p < 0.001, ns, not significant.

miR, microRNA; CCK, cell counting kit.

MIR210HG regulated the expression of FOXO6 in HuH-6 and HepG2 cells

Among the predicted mRNA targets of miR-608, we focused on the transcription factor FOXO6, whose expression is promoted in many cancer cells such as those of gastric cancer, non-small cell lung cancer, and HCC. To determine whether FOXO6 is a downstream target of miR-608, the wild-type or mutant 3′-untranslated region (UTR) of FOXO6 was cloned into the luciferase coding region of the pGL3 vector and transfected into HuH-6 and HepG2 cells with miR-608 mimics or a negative control mimic (Figure 2a). miR-608 overexpression significantly reduced the luciferase reporter activity of wild-type, but not mutant, FOXO6 (p < 0.001; Figure 2b, c).

To investigate whether miR-608 affects the relative concentration of FOXO6, we used RT-qPCR to measure FOXO6 mRNA expression. miR-608 knockdown significantly increased mRNA expression of FOXO6 (p < 0.01), while miR-608 overexpression significantly decreased FOXO6 mRNA levels (p < 0.001) (Figure 2d, e). Western blotting showed that miR-608 mimics significantly downregulated protein levels of FOXO6 in HuH-6 and HeG2 cells (p < 0.05), whereas miR-608 knockdown increased FOXO6 protein levels in HuH-6 and HepG2 cells (p < 0.05 in HepG2 cells; Figure 2f–i). These results suggest that miR-608 combines with FOXO6 to reduce its expression in HuH-6 and HepG2 cells.

FOXO6 abolished the effect of sh-MIR210HG in HuH-6 and HepG2 cells

To determine whether the MIR210HG–miR608–FOXO6 interaction could regulate hepatoblastoma cells in vitro, we constructed a pcDNA3.1-FOXO6 expression vector. The CCK-8 assay showed that FOXO6 overexpression significantly promoted cell viability in HuH-6 and HepG2 cells (p < 0.01; Figure 7a, b), while the colony formation assay showed that it significantly promoted proliferation in HuH-6 and HepG2 cells (p < 0.01; Figure 7c–f). Moreover, the Transwell assay revealed that FOXO6 overexpression significantly increased migration and invasion of HuH-6 (p < 0.001; Figure 7g, i, k, m) and HepG2 cells (p < 0.001; Figure 7h, j, l, n), abolishing the effect of sh-MIR210HG. Western blotting showed that sh-miR210HG significantly increased the protein expression of E-cadherin (p < 0.05) and significantly decreased that of N-cadherin (p < 0.01) and vimentin (p < 0.05) in HuH-6 cells, whereas this was partially restored when FOXO6 levels were increased (Figure 8a–d). These results suggest that the MIR210HG–miR608–FOXO6 network is critical for hepatoblastoma cell function.

Figure 7.

Transfecting pcDNA3.1-FOXO6 abolished the effect of sh-MIR210HG. (a, b) CCK-8 analysis of HuH-6 or HepG2 cells expressing the corresponding vector. (c–f) HuH-6 and HepG2 cells showing increased proliferation after FOXO6 overexpression, while this is partially restored when MIR210HG levels are decreased. (g–j) The Transwell assay showing increased migration of HuH-6 and HepG2 cells after FOXO6 overexpression, while this is partially restored when MIR210HG levels are decreased. (k–n) The Transwell assay showing increased invasion of HuH-6 and HepG2 cells after FOXO6 overexpression, while this is partially restored when MIR210HG levels are decreased. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant.

FOXO6, forkhead box O6; miR, microRNA; CCK, cell counting kit.

Figure 8.

The function of the MIR210HG–miR-608–FOXO6 axis in HUH-6 cell growth. (a–d) Western blots detecting the expression of E-cadherin, N-cadherin, and vimentin, using GAPDH as the internal control. *p < 0.05, **p < 0.01, ns, not significant.

miR, microRNA; FOXO6, forkhead box O6; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Discussion

Hepatoblastoma is a common liver malignancy in children that is not associated with hepatitis virus infection or hepatocirrhosis. The survival rates of patients with unresectable metastasis is notably lower than those with completely resected tumors. Therefore, understanding the mechanism of hepatoblastoma and developing novel biomarkers is important for clinical diagnosis.

The abnormal expression of lncRNAs has been identified in several human cancers, and lncRNAs have been reported to regulate a series of pathological processes, including cell growth, differentiation, and migration by functioning as tumor suppressors or oncogenes that regulate gene expression. The lncRNA MIR210 host gene (MIR210HG) is transcribed by gene ENSG00000247095.2, and was recently found to be associated with tumorigenesis. For example, Ruan et al. used the Cancer Genome Atlas database to analyze lncRNAs dysregulated in human colon cancers and found that MIR210HG was up-regulated in colon cancer compared with healthy colon tissues. Moreover, Wang et al reported higher miR210HG expression in HCC tumor tissues compared with paracancerous tissues, indicating MIR210HG as a marker for poor prognosis in HCC. However, the expression and function of MIR210HG in hepatoblastoma are unclear.

In this study, RT-qPCR was used to show the significantly increased expression of MIR210HG in 30 pairs of hepatoblastoma tissues compared with adjacent healthy liver tissues; similar results were observed in hepatoblastoma cells and normal liver cell lines. We also found that miR210HG knockdown significantly suppressed tumor growth in vivo and significantly suppressed hepatoblastoma cell viability, proliferation, invasion, and migration in vitro.

Recent work has shown that lncRNAs act as competitive endogenous RNAs, which adsorb miRNAs and regulate physiological and pathological processes. Here, we used the bioinformatics database RegRNA 2.0 to predict the target miRNA of MIR210HG. miRNAs negatively regulate gene expression through binding mRNAs, and miR-608 was previously shown to regulate the expression of FOXO6, TEAD2, and other target proteins to influence cancer progression.[15,18] We found that MIR210HG could bind miR-608 and reduce its expression in HepG2 cells. We also showed that miR-608 knockdown promoted hepatoblastoma cell viability, proliferation, invasion, and migration in vitro. These results indicate that MIR210HG binds miR-608 to regulate the progression of hepatoblastoma.

miR-608 was recently reported to act as an inhibitor in non-small cell lung cancer cells by regulating the expression of TEAD2. Additionally, Wang et al. found that miR-608 is associated with the risk of HCC in a large-scale population. Here, we found that FOXO6 mRNA is a potential target of miR-608. The downregulation of FOXO6 has been shown to inhibit proliferation, invasion, and glycolysis in colorectal cancer cells, while FOXO6 was also found to play an important role in HCC, especially in epithelial–mesenchymal transition (EMT). EMT is critical in the development of cancer through its effects on cancer cell proliferation, migration, and invasion, and the decreased expression of FOXO6 in breast cancer was previously reported to promote EMT, migration, and proliferation in cancer cells. In this work, we found that decreased expression of MIR210HG increased the protein expression of E-cadherin and decreased that of N-cadherin and vimentin, whereas this was partially restored when FOXO6 expression was increased or miR-608 levels decreased. These results indicate that EMT in human hepatoblastoma cells is regulated by the MIR210HG–miR608–FOXO6 axis.

No target drugs for MIR210HG, miR-608, or FOXO6 are currently in experimental or clinical trial stages, suggesting there is still relatively little research about their roles in cancers, so these should be further studied. In our future work, we aim to investigate the expression levels of MIR210HG, miR-608, and FOXO6 in the serum of patients with hepatoblastoma to determine whether the MIR210HG–miR-608–FOXO6 axis has an indicative effect on the prognosis of hepatoblastoma patients.

Conclusions

In this study, we demonstrated that MIR210HG was highly expressed in hepatoblastoma tissues and cell lines, and that the knockdown of MIR210HG inhibited hepatoblastoma development through binding to miR-608 and upregulating FOXO6. Our results suggest that the MIR210HG–miR-608–FOXO6 axis could be a novel target for hepatoblastoma treatment.