miR-126 inhibits papillary thyroid carcinoma growth by targeting LRP6
Retraction in: /10.3892/or.2021.8097
- Authors:
- Published online on: July 31, 2015 https://doi.org/10.3892/or.2015.4165
- Pages: 2202-2210
Abstract
Introduction
Papillary thyroid carcinoma (PTC) is the most common malignant thyroid tumor, and its incidence has been steadily increasing in the last decades worldwide (1). The majority of patients with PTC have excellent prognosis and therapeutic response with a combination of radioiodine and levothyroxine after surgical resection (2), however, ~10% of patients present recurrence and metastasis in local/regional and distant sites within 10 years (3). It has been shown that poor prognosis was associated with certain clinical and pathological characteristics, such as, tumor size, extrathyroidal invasion, lymph node metastasis and advanced tumor-node-metastasis (TNM) stage (1,3). Therefore, further investigation into the progression of PTC initiation and pathogenesis, which contributes to the exploration of effective schemes for PTC diagnosis and therapy, is required.
MicroRNAs (miRNAs) are a class of highly conserved small (19–25 nucleotides) non-coding RNAs that regulate diverse cellular processes by binding to the 3′ untranslated region (3′UTR) of target messenger RNAs (mRNAs) (4,5). It has been shown that miRNAs play crucial roles in a vast range of biological processes, including cell proliferation, angiogenesis, cell cycle, migration and migration (6). Accumulating studies have demonstrated that miRNAs function as tumor promoter or suppressor (7), and are involved in tumor development, progression and metastasis (8). Considering the modulation of miRNAs during tumorigenesis, they are currently considered as novel diagnosis biomarkers or therapy agents for tumor diagnosis and therapy.
miR-126, an important functional miRNA, has been reported to be commonly downregulated in various types of cancers (9). Growing evidence shows that miR-126 plays crucial roles in regulating cellular growth, migration, invasion and apoptosis in several types of tumors, such as hepatocellular carcinoma (10), osteosarcoma (11), non-small cell lung (12) and gastric cancer (13), cervix (14), colorectal (15) and colon cancer (16). A previous study indicated that miR-126 expression was downregulated in thyroid carcinomas (PTC and follicular carcinoma) compared to benign tumors (17). However, the clinical significance and the details roles of miR-126 in PTC remain unknown. In the present study, we examined the expression patterns of miR-126 in PTC tissue comparing to corresponding adjacent normal tissue, and investigated the effect of miR-126 on PTC growth in vitro and in vivo. We identified low-density lipoprotein receptor-related protein 6 (LRP6) as a direct target of miR-126 and showed that overexpression of miR-126 in PTC cells inhibited cell proliferation, migration and invasion and suppressed tumor growth in a nude mouse model. These findings indicate that miR-126 may serves as a therapeutic target for PTC.
Materials and methods
Patients and specimens
The present study was approved by the Institutional Ethics Committee of Jilin University. Written consent was obtained from all patients prior to surgery. Thirty paired thyroid papillary cancer specimens and adjacent normal thyroid papillary tissues were collected from the Department of Pathology, China-Japan Union Hospital of Jilin University (Changchun, China) between September 2012 and December 2014. The corresponding adjacent normal tissues from the same patients with PTC were obtained 3 cm beyond the boundary of PTC tissues. The samples were immediately snap-frozen in liquid nitrogen and stored at −80°C until use. All samples were confirmed as thyroid papillary cancer by trained pathologists. No patients received chemotherapy or radiotherapy prior to surgery. Clinicopathological parameters including patient age, gender, tumor size, lymph node metastasis and TNM stage were collected and listed in Table I.
 | Table IAssociation between miR-126 expression and clinicopathological features of human papillary thyroid carcinoma (PTC). |
Cell lines and culture
Two human PTC cell lines, TPC-1 and K1, and human thyroid follicular epithelial cells (Nthy-ori 3-1) were obtained from the Type Culture Collection of the Chinese Academy of Sciences. All cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (both from Gibco, USA), penicillin (100 U/ml; Sigma-Aldrich, St. Louis, MO, USA) and streptomycin (100 μg/ml) (Enpromise, Hangzhou, China) at 37°C in a humidified chamber supplemented with 5% CO2. Cells at ~90% confluence were split at 1:2 ratio every 2–3 days.
RNA oligoribonucleotides, plasmids and transfection
miR-126 mimic and corresponding negative control (miR-NC) were purchased from RiboBio (Guangzhou, China). The 3′-UTRs of human LRP6 (pGL3-LRP6 Wt) containing the potential binding sites of miR-126 were PCR-amplified from genomic DNA and cloned separately into the pGL3 luciferase reporter plasmid (Promega, Madison, WI, USA), Mutation in the miR-126 binding site module of LRP6 (pGL3-LRP6 Mut) was introduced using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the manual instructions. Transfection was performed in TPC-1 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.
RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA of cells and tissues was extracted using TRIzol (Invitrogen) according to the manufacturer's instruction. Total RNA was reversely transcribed to cDNA using the PrimeScript RT reagent kit (Takara Bio, Japan), and quantitative PCR was performed using Fast SYBR-Green master mix (Applied Biosystems, Foster City, CA, USA) under ABI 7900 sequence detection system (Life Technologies, Grand Island, NY, USA). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control for mRNAs. The specific primers for GAPDH and LRP6 were as follows: GAPDH (sense), 5′-TCAACGACCACTTTGTCAAGCTCA-3′ and GAPDH (antisense, 5′-GCTGGTGGTCCAGGGGTCTT ACT-3′; LRP6 (sense), 5′-GCTCAGAGTCCCAGTTCCAG-3′ and LRP6 (antisense), 5′-TCCCTTCATACGTGGACACA-3′.
To quantify the miR-126 expression levels, the expression of small nuclear U6 was used as an internal control. The relative expression level of miR-126 and U6 was determined using mirVana qRT-PCR miRNA detection kit (Ambion, Austin, TX, USA) according to the manufacturer's instructions under ABI 7900 sequence detection system. The specific primers for miRNA-126 and U6 were purchased from Applied Biosystems. All experiments were performed in triplicate. Relative expression levels were calculated using the 2−ΔΔCt method.
Cell viability and colony formation assay
Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In brief, transfected cells (1×104) were cultured in 96-well plates and stained at the indicated time points (24, 48 and 72 h) with 100 μl sterile MTT (0.5 mg/ml; Sigma) for 4 h at 37°C, followed by removal of the culture medium and the addition of 150 μl dimethylsulfoxide (DMSO; Sigma-Aldrich), followed by the measurement of the absorbance at 490 mm. Three independent experiments were performed for colony formation assay, transfected cells were trypsinized and seeded into 6-well plates (1×103 cells/well). After 2 weeks, cells were fixed with 10% formaldehyde for 15 min, stained with 1.0% crystal violet (both from Sigma) for 5 min, and then counted and photographed under a light microscope (Olympus, Tokyo, Japan). The percentage colony formation was calculated by adjusting miR-Ctrl to 100%. All experiments were performed in triplicate.
Cell cycle and apoptosis assay
Cell cycle and apoptosis assay were performed on TPC-1 cells 48 h after transfection. For the cell cycle, transfected cells in a culture dish were harvested by trypsinization, washed in ice-cold phosphate-buffered saline (PBS), and fixed in 80% ice-cold ethanol. Bovine pancreatic RNase (2 μg/ml; Sigma-Aldrich) was added to the cells and cultured for 30 min incubation at 37°C, and then incubated in propidium iodide (10 μg/ml; Invitrogen) at 4°C for 30 min in the dark. Propidium iodide-stained cells (>10,000 cells) were analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). All experiments were performed in triplicate.
Cell apoptosis assay was performed using Annexin V/propidium iodide detection kit (KeyGene, Nanjing, China) by a FACSCalibur flow cytometer (BD Biosciences) according to the manufacturer's protocol. The apoptosis ratio was calculated using CellQuest 3.3 software (BD Biosciences).
Wound-healing assay
Transfected cells (2×104) were seeded into a 6 cm dish and cultured for 24 h at 37°C in a 5% CO2 incubator. After incubation, the linear wound of cellular monolayer was created by scratching confluent cell monolayer. The monolayer of scratched cells was washed by PBS to remove debris. After incubation for 24 h at 37°C with 5% CO2, area of migration was photographed under a microscope (Olympus) for evaluation.
In vitro invasion assay
The invasive capacity of TPC-1 cells was assessed using the BioCoat Invasion Chamber system (BD Biosciences, San Jose, CA, USA). Twenty-four hours after transfection, 2×104 transfected cells were diluted in serum-free RPMI-1640 medium and plated on the top of Matrigel invasion chambers coated with Matrigel (8-μm pore size; BD Biosciences). A volume of 2.5 ml of RPMI-1640 medium containing 10% FBS was added to the lower compartment. The invasion assay was performed for 24 h at 37°C in a 5% CO2 incubator. After incubation, non-invading cells were removed by scrubbing, and migrated cells present on the lower surface of the membrane were fixed in 70% ethanol for 30 min and stained with 2% crystal violet for 10 min on a glass slide. Cells from 10 random fields were counted under an IX51 inverted microscope (Olympus).
Luciferase reporter assay
TPC-1 cells (1×104) were seeded into a 24-well plate, when 50–70% confluent they were co-transfected with miR-126 mimic or miR-Ctrl and 100 ng plasmid pGL3-LRP6 Wt or pGL3-LRP6 Mut using Lipofectamine 2000 according to the manufacturer's protocol. Forty-eight hours after transfection, cells were collected and luciferase activity was measured using the Dual-Luciferase Reporter Assay system (Promega Corporation, Madison, WI, USA). The specific activity is expressed as the fold-change of the experimental group vs. the miR-Ctrl group. The tests were repeated in 3 independent experiments.
TCF/LEF transcriptional activity assay
Reporter plasmids containing wild-type (CCTTTGATC; TOPflash) or mutated (CCTTTGGCC; FOPflash) T cell factor (TCF)/LEF DNA binding sites were purchased from Upstate Biotechnology (Lake Placid, NY, USA). TPC-1 cells (1×104) were seeded into a 24-well plate, at 50–70% confluence they were co-transfected with miR-126 mimic or miR-Ctrl and 100 ng wild-type/mutated type plasmid using Lipofectamine 2000 according to the manufacturer's protocol, and cultured for 48 h, then luciferase activity was measured using the dual-luciferase reporter assay system.
Nude mouse xenograft assay
Twenty female BALB/c mice (18–20 g, 4–5 weeks old) were purchased from the Experimental Animal Center of Changchun Biological Institute (Changchun, China), and kept under specific pathogen-free (SPF) conditions. All experimental procedures involving animals were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health. Animal protocols were approved by the Institutional Animal Care and Use Committee of Jilin University (Changchun, China).
TPC-1 cells (2×106) stably expressing miR-126 or miR-Ctrl, were suspended in 100 μl PBS and then injected subcutaneously into the posterior flank of female BALB/c athymic nude mice. Tumor volumes in mice were measured with a slide caliper every week until the scarifice. Tumor volumes were calculated according to the formula: Volume (mm3) = 1/2 × width2 × length. Five weeks after injection, mice were sacrificed and tumors tissues were resected and weighed. Total protein of tumor tissues was extracted to measure the LRP6 protein expression level by western blotting.
Western blotting
The cells were lysed in ice-cold RIPA buffer (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 48 h post-transfection. The total proteins were quantitated using BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Total protein lysates (30 μg/each lane) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes (NC membranes; Invitrogen), blocked in 4% dry milk at room temperature for 1 h, and immunostained with primary antibodies at 4°C overnight using the following antibodies: anti-LRP6, anti-MMP-7, anti-CCND1, anti-c-Myc, anti-cyclin D1 and anti-β-actin (all from Santa Cruz, Biotechnology, Inc.). The anti-β-actin antibody (Santa Cruz, Biotechnology, Inc.) was used to normalize the protein input. After washing, membranes were incubated with corresponding secondary antibodies for 1 h at room temperature. The blots were detected, and visualized using an enhanced chemiluminescence kit (GE Healthcare) according to the manufacturer's instructions.
Statistical analysis
All data are expressed as mean ± standard deviation (SD) of at least 3 independent experiments or 3 samples. The difference between groups was analyzed using a Student's t-test. Statistical comparison of more than two groups was performed using one-way ANOVA followed by a Tukey's post hoc test. The association between miR-126 expression and clinicopathological variables was assessed by the Chi-square tests. Statistical analysis was performed with GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). P<0.05 was considered to indicate a statistically significant result.
Results
Expression of miR-126 is downregulated in PTC tissue and cell lines
The expression of miR-126 was detected in 30 pairs of human PTC tissue and adjacent normal tissues by real-time quantitative RT-PCR (qRT-PCR). As shown in Fig. 1A, the miR-126 was markedly decreased in PTC tissues samples when compared with adjacent normal tissues (P<0.01). Among these 30 cases, 27 cases revealed a relative lower level in PTC, which suggested that reduction of miR-126 was a frequent event in human PTC. To investigate the clinical relevance of miR-126 in HCC, we divided the 30 patients to high miR-126 expression group (n=16) and low miR-126 expression group (n=14) using the mean value (0.402±0.05) of relative expression levels in all 30 PTC tumors as a cut-off. It was found that the aberrant expression of miR-126 was correlated with lymph node metastasis (P<0.01), tumor size (P<0.01) and TNM stage (P<0.01), which are all indicators of poor prognosis (Table I). No significant association was found between the expression of miR-126 and age or gender in the PTC (Table I).
Furthermore, miR-126 expression was determined by RT-PCR in two PTC cell lines (TPC-1 and K1) and human thyroid follicular epithelial cells (Nthy-ori 3-1). Notably, miR-126 expression was downregulated in PTC cell lines compared to normal thyroid cells (Nthy-ori 3-1) (Fig. 1B). The data above suggest that miR-126 was decreased in both PTC tissues and cell lines. We selected the TPC-1 cells, which showed the lowest expression of miR-126, to conduct further experiments.
Overexpression of miR-126 inhibits cell proliferation, colony formation, induces cell cycle and apoptosis of PTC cells
To explore the potential role of miR-126 in PTC cell growth, we transfected TPC-1 cells with miR-126 mimics and then performed in vitro cell proliferation and colony formation, cell cycle and apoptosis assays at indicated times. qRT-PCR confirmed that TPC-1 cells transfected with miR-126 mimic significantly increased miR-126 expression level compared to cells transfected with miR-Ctrl (Fig. 2A). MTT and clone formation assays showed that overexpression of miR-126 in TPC-1 cells significantly inhibited cell proliferation (Fig. 2B) and colony formation (Fig. 2C). As proliferation directly associated with cell cycle distribution, the effect of miR-126 on cell cycle progression was evaluated in TPC-1 cells. As expected, the percentage of G1 phase cells increased, and the percentage of S phase cells decreased in TPC-1 cells transfected with miR-126 mimic compared to cells transfected with miR-Ctrl (Fig. 2D). In addition, cell apoptosis was investigated in TPC-1 cells transfected with miR-126 mimic or miR-Ctrl. The result showed that overexpression of miR-126 increased cell apoptosis relative to miR-Ctrl group (Fig. 2E).
Overexpression of miR-126 inhibits cell migration and invasion of PTC cells
The above results showed that miR-126 downregulation was associated with lymph node metastasis in patients with PTC, therefore, to investigate whether miR-126 affect metastasis in vitro, migration and invasion assays were performed in TPC-1 cells transfected with miR-126 mimic or miR-Ctrl by wound healing and Transwell assays, respectively. Consistent with the clinical data, overexpression of miR-126 in TPC-1 cells significantly inhibited cell migration (Fig. 3A) and invasion (Fig. 3B). Collectively, these results suggested that miR-126 efficiently inhibits migration and invasion of PTC cells.
LRP6 is a direct target of miR-126
To explore the mechanism underlying the growth inhibition by miR-126 in PTC cells, we used publicly available algorithms (TargetScan6.2 and miRanda) to help identify the miR-126 targets in human PTC. The results showed that there is a miR-126 binding site in LRP6 mRNA 3′UTR (Fig. 4A). As predicted, qRT-PCR and western blotting showed that ectopic miR-126 in TPC-1 cells downregulated LRP6 expression on mRNA (Fig. 4B) and protein levels (Fig. 4C). To further confirm whether LRP6 is a direct target of miR-126, we constructed pGL3-LRP6 Wt or pGL3-LRP6 Mut plasmid, then transfected pGL3-LRP6 Wt or pGL3-LRP6 Mut plasmid into TPC-1 cells, along with miR-126 mimic or miR-Ctrl for luciferase assay evaluation. Luciferase assay further revealed that TPC-1 cells transfected with miR-126 mimic repressed wild-type LRP6-3′UTR reporter activity (P<0.01), while miR-126 mimic had no inhibition effect on the mutant LRP6-3′UTR reporter activity (Fig. 4D), indicting the direct regulation of miR-126 in the 3′UTR of LRP6 mRNA.
Upregulation of miR-126 deactivates Wnt/β-catenin signaling
It has been shown that LRP6 is closely correlated with Wnt/β-catenin signaling activity; therefore, we further examined whether miR-126 was involved in the regulation of Wnt/β-catenin signaling. As shown in Fig. 5A, the β-catenin reporter assay revealed that ectopic miR-126 markedly decreased the TCF/LEF activities in TPC-1 cells (Fig. 5A). In addition, we also examined the protein expression levels of downstream genes in the Wnt/β-catenin signaling pathway, including cyclin D1, MMP-7, CCND1 and c-Myc. Western blot assay showed that overexpression of miR-126 obviously downregulated the protein expression in TPC-1 cells (Fig. 5B). These data suggest that upregulation of miR-126 deactivates Wnt/β-catenin signaling and decreases TCF/LEF transcriptional activity.
miR-126 suppresses PTC cell tumorigenicity in vivo
Based on the observed decreases in migratory, invasive and proliferative behavior in TPC-1 cells transfected with miR-126 in vitro, we further examined miR-126 biofunction in suppression of HCC cell tumorigenicity in vivo by inoculating nude mice with tumor cells. The results showed that overexpression of miR-126 can significantly suppress the tumor growth of TPC-1 xenografts in nude mice (Fig. 6A) and decreases tumor volume (Fig. 6B) and tumor weight (Fig. 6C) compared to miR-Ctrl group, indicating the suppressive function of miR-126 on PTC cell tumorigenicity in vivo. Furthermore, we also determined LRP6 expression of tumor tissue. The result of western blotting showed that LRP6 protein expression was downregulated in the xenograft tumors of miR-126 mimic group compared to the xenograft tumors of miR-Ctrl group (Fig. 6D). These data indicated that miR-126 suppressed PTC cell tumorigenicity in vivo by targeting LRP6.
Discussion
Dysregulation of miRNAs frequently occurs in various types of human cancers, and aberrant expression of miRNAs may contribute to initiation, promotion and progression of human cancers by affecting the expression of multiple target genes (18–20). Thus, a comprehensive understanding of the relationship between specific miRNAs and tumor development is crucial for the diagnosis and therapy of tumors. A number of miRNAs were reported to be involved in cell proliferation, migration and invasion of papillary thyroid carcinoma (PTC) (21,22). For instance, Huang et al (23) reported that forced expression of miR-219-5p suppressed PTC cell proliferation and migration and promoted apoptosis by targeting the estrogen receptor (ER)α. Zhu et al (24) showed that miR-182 in PTC promotes cell proliferation and invasion through direct suppression of close homolog of LI (CHL1).
Li et al (25) found that downregulation of miR-181b expression causes cellular growth inhibition, promoting cellular apoptosis by targeting CYLD. Chou et al (26) reported that upregulation of miR-146b significantly promotes cell migration and invasiveness and increases resistance to chemotherapy-induced apoptosis in PTC. Data from the present study provide evidence that the miR-126 expression levels was significantly downregulated in PTC tissue and PTC cell lines, the aberrant expression of miR-126 was correlated with lymph node metastasis, tumor size and TNM stage, and that upregulation of miR-126 in PTC cells inhibited proliferation, colony formation, migration and invasion, induced cell apoptosis and suppressed tumor growth in a nude mouse model.
miRNA-126 (miR-126), located within the 7th intron of EGFL7 which resides on human chromosome 9, plays a crucial role in controling angiogenesis by binding to several transcripts (27,28). Accumulating evidence shows that miR-126 expression is commonly downregulated in various types of cancer, act as a tumor suppressor and inhibits cancer cell growth, adhesion, migration and invasion through suppressing a range of important gene targets (9–16). Recently, several novel targets of miR-126 have been confirmed, such as SOX2 (10), VEGF-A (13), CXCR4 (16), PI3KR2 (29), Crk (29), IκBα (30), IRS-1 (31) and ADAM9 (32). In addition, lipoprotein receptor-related protein 6 (LRP6) has also been reported to be a target gene of miR-126 in human hepatocellular carcinoma (33). In the present study, we found that LRP6 was a target gene of miR-126 in human thyroid cancer.
LRP6, a member of the expanding low-density lipoprotein (LDL) receptor family, is found to be expressed in different types of human tissues (34). It is well known that LRP6 is one of the Wnt-co-receptors, which activates the transcription of Wnt/β-catenin target genes through promoting β-catenin translocation into the nucleus (35,36). In addition, LRP6 was reported to be upregulated in various types of human cancer, including lung, colon, breast, liver cancer, and to promote the progression of tumors through regulation of the Wnt/β-catenin signaling pathway (37). Deregulation of the Wnt/β-catenin signaling pathway is common in various types of tumors and activation of this pathway is thought to be an early event in tumorigenesis, and plays essential roles during tumor progression (34). Notably, LRP6 has been found to be regulated by several miRNAs such as miR-202 (38), miR-183 (39), miR-610 (40), miR-30e (41) and miR-577 (42). Consistent with these results, in the present study we found that LRP6 is a target of miR-126 by luciferase assay, and that upregulation of miR-126 decreased the expression of LRP6 on mRNA and protein levels. We also found that ectopic miR-126 markedly decreased the TCF/LEF activities in TPC-1 cells. Our results also showed that inhibition of LRP6 by miR-126 resulted in downregulation of the cyclin D1, MMP-7, CCND1 and c-Myc genes, which are the downstream target genes of the Wnt/β-catenin signaling pathway. These results indicated that the tumor suppressive function of miR-126 may be correlated with modulation of the Wnt/β-catenin signaling pathway through targeting LRP6.
In summary, the present study demonstrated that miR-126 is downregulated in PTC tissue and cell lines, and its expression level was significantly associated with lymph node metastasis, TNM stage and tumor size. Overexpression of miR-126 in PTC cells drastically inhibited cell proliferation, colony formation, migration and invasion, induced cell apoptosis and cell cycle arrest at G1 stage in vitro, as well as suppressed tumor growth in vivo. Moreover, we identified LP6 as a crucial target gene of miR-126, and found that miR-126 inhibited LP6 expression and deactivated Wnt/β-catenin signaling pathway, which contributed to suppress PTC growth. These findings suggested that miR-126 could be a potential target for the treatment of PTC.
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