Long non-coding RNA (lncRNA) maternally expressed gene 3 (MEG3) is a tumor suppressor in several cancers, such as glioma, prostate cancer and esophageal cancer. However, the role of MEG3 in hepatocellular carcinoma (HCC) and the related molecular mechanisms are not well understood. The present study aimed to determine the biological function of MEG3 in regulating HCC cell viability, apoptosis and migration. In addition, the interaction between MEG3, microRNA (miR)-9-5p and Midkine (MDK), and the activation of the phosphoinositide-dependent kinase (PDK)/AKT pathway in HCC cell line MHCC-97L were examined. Luciferase reporter assays, reverse transcription-quantitative PCR and western blotting were used to determine the interaction between MEG3, miR-9-5p and MDK and the activation of the PDK/AKT pathway. Cell viability was determined by the CCK8 assay and the cell cycle analysis using flow cytometry analysis. Cell apoptosis was examined by flow cytometry analysis and caspase 3/9 activity. Wound healing assays and western blotting were used to investigate cell migration. The present study demonstrated that MEG3 suppressed HCC cell viability and migration, and induced cell apoptosis. In addition, it was also found that MEG3 targets the miR-9-5p/MDK axis and modulates the PDK/AKT pathway in HCC. In conclusion, the findings of the present study demonstrated that lncRNA MEG3 affects HCC cell viability, apoptosis and migration through its targeting of miR-9-5p/MDK and regulation of the PDK/AKT pathway. The MEG3/miR-9-5p/MDK axis may be a potential therapeutic target in HCC.
Hepatocellular carcinoma (HCC), a malignant tumor arising from the liver, accounts for 80% of primary liver cancer (
Long non-coding RNAs (lncRNAs), are a class of non-coding transcripts with lengths exceeding 200 nucleotides, which participate in a variety of physiological and pathological processes, including embryogenesis, tumorigenesis and some cardiovascular diseases (
Midkine (MDK) was initially found as a growth factor in retinoic acid-induced embryonic tumor cells (
The present study aimed to investigate the effect of MEG3 on HCC cell viability, apoptosis and migration. In addition, the interaction between MEG3, miR-9-5p and MDK, and the activation of PDK/AKT pathway in HCC cells was also assessed. The present study provides new insights into the molecular mechanisms of MEG3 in HCC and suggests a novel therapeutic target for HCC.
MHCC-97L cell line (cat. no. BNCC337741) was purchased from Beijing Beina Chuanglian Institute of Biotechnology. The cells were maintained in RPMI-1640 medium (Nanjing KeyGen Biotech. Co. Ltd.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific Inc.) and 100 U/ml penicillin G sodium and 0.1 mol/ml streptomycin sulfate, at 37°C for 48 h in a humidified atmosphere of 5% CO2.
miR-9-5p mimic (3.75 µl, 25 nM), miR-9-5p negative control (NC; 3.75 µl, 25 nM), lncRNA MEG3 overexpression vector (2.5 µg, 2.5 µg/ml) and lncRNA MEG3 NC (2.5 µg, 2.5 µg/ml) were purchased from General Biosystems, Inc. The sequences of miR-9-5p mimic and the corresponding NC were: miR-9-5p mimic sense, 5′-UCUUUGGUUAUCUAGCUGUAUGA-3′, antisense, 5′-UCAUACAGCUAGAUAACCAAAGA-3′; NC: Sense, 5′-UCACAACCUCCUAGAAAGAGUAGA-3′, antisense, 5′-UCUACUCUUUCUAGGAGGUUGUGA-3′. Cell transfection was conducted using Lipofectamine 3000® transfection reagent (Thermo Fisher Scientific Inc.) according to the manufacturer's instructions. Cells were incubated with the complexes at 37°C for 6 h, then the transfection media was replaced with culture medium. 48 h later, the cells were subjected to subsequent experimentation. Cells in the control group were untransfected cells.
Total RNA was extracted from MHCC-97L cells using the Ultrapure RNA kit (CoWin Biosciences), and then reverse-transcribed into cDNA using a HiFiScriptcDNA synthesis kit (CoWin Biosciences). Reverse transcription was performed at 50°C for 15 min, and then at 85°C for 5 min. Primers used for amplification were: MEG3 forward, 5′-CATACAAAGCAGCCACTCAC-3 and reverse, 5′-GGGATCCTTCCATTCAGGAC-3′; GAPDH forward, 5′-CAATGACCCCTTCATTGACC-3 and reverse, 5′-GAGAAGCTTCCCGTTCTCAG-3′; miR-9-5p forward, 5′-TCTTTGGTTATCTAGCTGTATGA and reverse, 5′-CCAGCTATGCGCCATTAGCAA-3′; U6 forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3 and reverse, CGCTTCACGAATTTGCGTGTCAT-3′ (CoWin Biosciences). RT-qPCR assays were performed using the UltraSYBR Mixture (CoWin Biosciences). Data were analyzed using the 2−ΔΔCq method (
Total proteins were extracted from MHCC-97L cells using RIPA Cell Lysis Buffer (Applygen Technologies Inc.) and then protein concentrations were quantified using the bicinchoninic acid (BCA) method (Thermo Fisher Scientific Inc.). A total of 50 µg of protein was separated using 10% SDS-PAGE gels and transferred to PVDF membranes (EMD Millipore). After blocking overnight at 4°C in 5% BSA, the PVDF membranes were incubated with the primary antibodies, including Rabbit Anti-caspase-3 (1:5,000; cat. no. ab32351), rabbit anti-caspase-9 (1:2,000; cat. no. ab202068), rabbit `nti-MDK (1:1,000; cat. no. ab52637), rabbit anti-PDK1 (1:2,000; cat. no. ab207450) (all Abcam), rabbit anti-AKT (1:500; cat. no. bs-2720R; BIOSS), rabbit anti-matrix metalloproteinase-1 (MMP-1) (1:1,000; cat. no. bs-4597R; BIOSS), Rabbit Anti-phosphorylated (p)-AKT (cat. no. bs-2720R; 1:1,000; BIOSS), Rabbit Anti p-PDK1 (cat. no. AF3018; 1:1,000; Affinity Biosciences) and Mouse Monoclonal Anti-GAPDH (cat. no. TA-08; 1:2,000; OrigeneTechnologies, Inc.) at 4°C overnight. Horseradish peroxidase-labeled secondary antibodies, including Goat Anti-Mouse IgG (H+L) (cat. no. ZB-2305) and Goat Anti-Rabbit IgG (H+L) (cat. no. ZB-2301) (both OrigeneTechnologies, Inc.), were used at 1:2,000 dilution and the membranes were incubated at room temperature for 2 h. GAPDH was used as the internal loading control. Signals were detected with the SuperSignal® west pico chemiluminescent substrate (Thermo Fisher Scientific Inc.), and band density was determined by ImageLab software v.5.2 125104 (Bio-Rad Laboratories Inc.).
Wild-type lncRNA MEG3 3′-UTR and MDK 3′-UTR containing the putative binding sites of miR-9-5p (
Cells were seeded at 1×104 cells/well in 96-well plates and maintained in RPMI-1640 medium at 37°C in an atmosphere of 5% CO2 for 48 h. Cell viability was measured using a CCK8 Kit (Nanjing KeyGen Biotech. Co. Ltd.) according to the manufacturer's instructions. Cells were incubated with the CCK8 reagent (10 µl) at 37°C for 1 h. Absorbance at 450 nm was measured using a Tecan Safire II Microplate Reader (Tecan Group Ltd.).
FCM analysis of the cell cycle of MHCC-97L cells was performed using a Cell Cycle Staining kit [MultiSciences (Lianke) Biotech Co., Ltd.] according to the manufacturer's instructions. The cell suspension was centrifuged at 978 × g for 3 min, and the cells were subsequently fixed in ethanol at 4°C for 2 h. After washing with PBS for 3 times, 1 ml of DNA staining solution (PI containing RNase A) was added to the tubes. The cells were incubated at room temperature in the dark for 30 min, and the cell cycle was analyzed using the NovoCyte 2060R flow cytometer (ACEA Biosciences, Inc.) with NovoExpress software v.1.2.5 (ACEA Biosciences, Inc.).
Apoptosis rate of MHCC-97L cells was determined by FCM using an Annexin V-FITC/propidium iodide PI Apoptosis kit [MultiSciences (Lianke) Biotech Co., Ltd.]. Briefly, 1×106 cells were washed with cold PBS twice, and re-suspended in 300 µl binding buffer. After incubation with 3 µl Annexin V-FITC and 3 µl PI-PE at room temperature for 10 min in the dark, the cells were mixed with 200 µl binding buffer, and cell apoptosis rate was analyzed using a NovoCyte 2060R flow cytometer (ACEA Biosciences, Inc.) with NovoExpress software v.1.2.5 (ACEA Biosciences, Inc.). Early and late apoptosis were both analyzed.
MHCC-97L cells were grown in FBS free medium to ~100% confluence in 6-well plates following transfection. The confluent monolayers were scratched by using a 10 µl pipette tip, and then washed with PBS 3 times. Images were taken by an inverted fluorescence microscope (MF53; Guangzhou Mingmei Photoelectric Co., Ltd.) immediately or 24 h after wounding and migration was quantified. Migration rate was calculated as the migration distance divided by the migration time.
Statistical analyses were performed using SPSS v.19.0 (IBM, Corp.). Al experiments were performed at least 3 times. Experimental data were presented as the mean ± SD, and the statistical significance was assessed by one-way analysis of variance followed by the post hoc least significant difference (LSD) test. P<0.05 was considered to indicate a statistically significant difference.
In preliminary experiments, the expression of lncRNA MEG3 was examined in 5 HCC cell lines and it was found that MHCC-97L had the lowest lncRNA MEG3 expression (data not shown). Therefore, the MHCC-97L cell line was chosen for subsequent gain-off-function experiments. lncRNA MEG3 overexpression vector was transfected into MHCC-97L cells to investigate the effect of lncRNA MEG3 on HCC cell viability, apoptosis and migration. As demonstrated in
Luciferase reporter assays were used to investigate whether lncRNA MEG3 targets miR-9-5p. As shown in
lncRNA MEG3 effect on miR-9-5p expression was determined by RT-qPCR analysis. As shown in
The pathogenesis of HCC is complex and numerous studies have demonstrated that lncRNAs are involved in the initiation, development and metastasis of HCC (
lncRNAs can serve as a kind of ceRNA to regulate target genes through interacting with miRNAs (
In the present study, the MHCC-97L cell line was chosen for gain-off-function experiments. Indeed, using at least one other HCC cell line would make the present study more convincing. It is a limitation of the present study that only 1 cell line was used. lncRNA and miRNA may also have negative feedback regulation. Another limitation of the present study is that it only demonstrated the effect of lncRNA MEG3 overexpression on miR-9-5p, without reverse validation. The MHCC-97L cell line is representative of HCC cell lines in previous studies (
In conclusion, the present study demonstrated that MEG3 affects HCC cell viability, apoptosis and migration through its targeting of miR-9-5p/MDK and regulation of the PDK/AKT pathway. The present study provided new insights into the molecular mechanisms of MEG3 in HCC, and suggested that the MEG3/miR-9-5p/MDK axis is a potential therapeutic target in HCC.
Not applicable.
This study was supported by the First-Class Discipline Construction Project in Guizhou Province [Chinese Pharmacy; grant no. (2017)008], Science and Technology Plan Project of Guizhou Province [grant no. (2017)1073], and Department of Science and Technology Qian Ke He Platform Talent of Guizhou [grant no. (2017) 5655].
The data sets generated and analyzed during the present study are available from the corresponding author on reasonable request.
DW and QL designed the study, analyzed the data and prepared the manuscript. DW, ZM and DM conducted the experiments. All authors were substantially involved in the research, acquisition of data, analysis and manuscript preparation. DW and QL confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Effect of lncRNA MEG3 on HCC cell viability. (A) Expression of lncRNA MEG3 in the cells transfected with the lncRNA MEG3 overexpression vector determined by RT-qPCR (n=5). (B) Cell viability determined by CCK8 assay. n=5. (C and D) Cell cycle analysis determined by flow cytometry. Cells in the control group were untransfected cells (n=3). *P<0.01 compared with the lncRNA MEG3 NC group. lncRNA, long non-coding RNA; MEG3, maternally expressed gene 3 (MEG3); HCC, hepatocellular carcinoma; NC, negative control; PI, propidium iodide; RT-q, reverse transcription-quantitative.
Effect of lncRNA MEG3 on HCC cell apoptosis determined by flow cytometry. Cells in the control group were untransfected cells (n=3). *P<0.01 compared with the lncRNA MEG3 NC group. lncRNA, long non-coding RNA; MEG3, maternally expressed gene 3 (MEG3); HCC, hepatocellular carcinoma; NC, negative control; PI, propidium iodide.
Effect of lncRNA MEG3 on HCC cell migration determined by wound healing assay. Cells in the control group were untransfected cells (n=3). *P<0.01 compared with the lncRNA MEG3 NC group. lncRNA, long non-coding RNA; MEG3, maternally expressed gene 3 (MEG3); HCC, hepatocellular carcinoma; NC, negative control.
Interaction between lncRNA MEG3, miR-9-5p and MDK determined by the dual luciferase reporter assay. (A) lncRNA MEG3 targets miR-9-5p. *P<0.01 compared with the lncRNA MEG3+miR-9-5p mimic NC group. (B) miR-9-5p targets MDK (n=3). *P<0.01 compared with the MDK+miR-9-5p mimic NC group. lncRNA, long non-coding RNA; MEG3, maternally expressed gene 3 (MEG3); NC, negative control; miR, microRNA; MDK, Midkine.
Effect of lncRNA MEG3 on the expression of miR-9-5p, MDK, apoptosis and migration-related proteins, and PDK/AKT pathway activation. (A) Effect of lncRNA MEG3 on miR-9-5p expression. (B) Western blot images. (C) Relative protein expression of MDK, caspase-3, caspase-9 and MMP1 determined by western blotting. (D) Effect of lncRNA MEG3 on PDK/AKT signaling. Cells in the control group were untransfected cells (n=3). *P<0.01 compared with the lncRNA MEG3 NC group. lncRNA, long non-coding RNA; MEG3, maternally expressed gene 3 (MEG3); HCC, hepatocellular carcinoma; NC, negative control; miR, microRNA; MDK, Midkine; PDK1, phosphoinositide-dependent kinase 1, p, phosphorylated; MMP-1, matrix metalloproteinase 1.