miR-141-3p suppresses proliferation and promotes apoptosis by targeting GLI2 in osteosarcoma cells

Retraction in: /10.3892/or.2023.8576

  • Authors:
    • Nan Wang
    • Pengcheng Li
    • Wei Liu
    • Ningning Wang
    • Zhi Lu
    • Jianzhou Feng
    • Xiandong Zeng
    • Jiujie Yang
    • Yong Wang
    • Wei Zhao
  • View Affiliations

  • Published online on: December 12, 2017     https://doi.org/10.3892/or.2017.6150
  • Pages: 747-754
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Abstract

MicroRNAs (miRNAs) have been reported as key regulators in numerous diseases including osteosarcoma. The function of microRNA-141-3p (miR-141-3p) and whether this function is achieved by regulation of GLI family zinc finger 2 (GLI2) in osteosarcoma remain unclear. In the present study, we found decreased expression of miR-141-3p, but increased expression of GLI2 in osteosarcoma tissues and cell lines. In addition, we demonstrated a negative correlation between miR-141-3p and GLI2. Furthermore, we revealed that elevation of miR-141-3p resulted in a marked inhibition of proliferation and promotion of apoptosis as well as an obviously decrease in GLI2 in osteosarcoma cell lines. Furthermore, we determined that GLI2 is a target of miR-141-3p by a constructed luciferase assay. In addition, we showed that miR-141-3p could negatively regulate GLI2 and its downstream parathyroid hormone-related protein 1 (PTHRP1). Finally, through a series of antisense experiments we confirmed that the effect of miR-141-3p on proliferation and apoptosis was achieved through the GLI2 pathway in osteosarcoma cells. The findings of the present study may provide a new target for treating osteosarcoma.

Introduction

Osteosarcoma is the most prevalent primary malignant bone tumor in childhood and adolescence. It was reported that the incidence of osteosarcoma is approximately 1–3/1000,000 individuals per year worldwide (1,2). Although combined therapy including surgical methods and multi-chemotherapy have achieved great progress, the overall five-year survival rate of osteosarcoma patients remains low (3,4). Therefore, discovering new targets is urgent for clinical and basic research.

GLI family zinc finger 2 (GLI2) is a transcription factor in the Hedgehog-Gli signaling pathway. Recently, GLI2 was extensively reported as a key regulator in various diseases including osteosarcoma (5,6). Yang et al revealed that the silencing of GLI2 by siRNA decreased osteosarcoma cell proliferation and viability (7). Nakamura et al found that arsenic trioxide suppressed proliferation via downregulation of GLI2 expression in osteosarcoma cells (8). Nagao et al found that GLI2 was aberrantly elevated in human osteosarcoma biopsy specimens and that knockdown of GLI2 by RNA interference (RNAi) inhibited osteosarcoma growth (9). Accordingly, identification of an endogenous molecule to regulate GLI2 is urgent in the targeted therapy of osteosarcoma.

MicroRNAs (miRNAs), 22–25 nt in length, are a group of small non-coding RNAs which are crucial in various biological processes including cell growth, cell apoptosis, cell cycle control, cell differentiation and cell migration/invasion. miR-141-3p has been found to be extensively involved in diverse malignant tumors such as gastric cancer, hepatocellular carcinoma, prostate cancer, renal cell and esophageal carcinoma (1014). Qiu et al reported that miR-141-3p inhibited human stromal stem cell proliferation by targeting cell division cycle 25A (CDC25A) (15). Li et al revealed that miR-141-3p promoted cell proliferation via targeted binding to Krüppel-l-like factor 9 (KLF9) in prostate cancer (12). To date, the function of miR-141-3p and whether it regulates GLI2 in osteosarcoma remain unknown.

In the present study, we detected the expression level of miR-141-3p in osteosarcoma tissues and its function in osteosarcoma cell proliferation and apoptosis. In addition, we demonstrated the target binding effect between miR-141-3p and the 3′ untranslated region (3′UTR) of GLI2. In addition, we found that miR-141-3p suppressed cell proliferation and promoted apoptosis via the GLI2 pathway in osteosarcoma cells. The present study may provide a better understanding of miR-141-3p in osteosarcoma.

Materials and methods

Patients and tissue samples

Twenty-eight cases of osteosarcoma and paired para-tumor tissues were collected during tumorectomy at the Central Hospital Affiliated to Shenyang Medical College between December 2010 and December 2016. All 28 cases had a definite pathological diagnosis and the clinical stages of these patients were determined according to the TNM classification of the International Union Against Cancer (UICC). Written informed consent was obtained from the patients whose tissues were used in this research. The Institute Research Medical Ethics Committee of Central Hospital Affiliated to Shenyang Medical College granted approval for the present study.

Cell culture and cell transfection

Human osteosarcoma cell lines MG-63, MNNG/HOS and SW1353 were obtained from the Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences, Shanghai, China) and were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Carlsbad, CA, USA), and the human osteoblast cell line hFOB 1.19 was cultured in DMEM/F12 (both from Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS; Sigma, St. Louis, MO, USA), 100 IU/ml penicillin and 100 mg/ml streptomycin (Baomanbio, Shanghai, China). All cell lines were cultured at 37°C in a humidified atmosphere containing 5% CO2. When the cultured osteosarcoma cells grew to 80% confluency, 50 nM of the miR-141-3p mimics, mimic control, miR-141-3p inhibitor, inhibitor control or the constructed plasmids was correspondingly transfected into the cultured cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. All miRNA oligonucleotides were purchased from GenePharma (Shanghai, China).

Immunohistochemistry and in situ hybridization assay

All the procedures were carried out as previously described (16). In brief, tissue slides (4-µm thick) were firstly incubated with a goat anti-GLI2 antibody (concentration of 5 µg/ml; cat. no. ab223651; Abcam, Cambridge, UK) at 4°C overnight, then subsequently incubated with biotinylated secondary antibodies (dilution, 1:1,000; cat. no. ab6885; Abcam) at 37°C for 30 min. Followed by streptavidin-horseradish peroxidase complex incubation and diaminobenzidine tetrahydrochloride (DAB) staining, and hematoxylin (both from Abcam) counterstain. All slides were assessed by two experienced pathologists who were ignorant of the patient clinical pathology and other information independently. GLI2 expression level was evaluated as previously described (17).

In situ hybridization staining was performed on fresh paraffin sections. Briefly, the tissue slides were mixed with 5′-digoxigenin LNA-modified-DANCR (Exiqon A/S, Vedbaek, Denmark) using the IsHyb in situ Hybridization kit (BioChain Institute, Inc., Newark, CA, USA) according to the manufacturer's protocol.

Reverse transcription and quantitative real-time PCR

All the procedures were carried out as previously described (17). In brief, total RNA was extracted by TRIzol (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized using the PrimeScript RT reagent kit (Takara, Dalian, China). The expression of miR-141-3p was detected using a TaqMan miRNA assay kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions and calculated using RNU6B small nuclear RNA as an endogenous control by the 2−ΔΔCt method. All of the reactions were run in triplicate.

Western blot analysis

Total cellular and tissue proteins were extracted by RIPA lysis buffer (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Sample were subjected to 10% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane and then blocked for 1 h at room temperature. Each membrane was incubated with primary antibodies at 4°C overnight and then secondary antibodies (dilution, 1:2,000; cat. no. ab205718; Abcam) at room temperature for 1 h the next day. Target proteins were probed with specific antibodies, GLI2, PTHRP1 and GAPDH. The details of the antibodies mentioned above were the following: rabbit anti-GLI2 (concentration of 1 µg/ml; cat. no. ab167389; Abcam); rabbit anti-PTHRP1 (dilution, 1:1,000; cat. no. ab32064; Abcam) and rabbit anti-GAPDH antibodies (dilution, 1:10,000; cat. no. ab181602; Abcam).

EdU (5-ethynyl-2-deoxyuridine) assay

Osteosarcoma cells were pre-transfected with different miR-141-3p plasmids for 72 h. The EdU incorporation assay was performed according to the manufacturer's protocol using EdU detection kits (RiboBio Co., Ltd., Guangzhou, China). The nuclei were observed under laser scan confocal microscopy, and the quantitative data are expressed as the percentage of EdU-positive nuclei relative to the total number of nuclei counted.

Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay

Cell apoptosis was determined using the TUNEL assay as previously described (18). Briefly, MG-63 and MNNG/HOS cells were firstly seeded on coverslips respectively, and were then fixed using 4% paraformaldehyde for 30 min, followed by permeabilizing with 0.1% Tritons X-100 for 2 min on ice. Furthermore, the cells were labeled using TUNEL kit (KeyGen Biotech, Nanjing, China) according to the manufacturer's protocol. The apoptotic index was calculated using the following formula: Apoptotic index = (total number of apoptotic cells/total number of cells) × 100%.

Plasmid construction

The GLI2 fragment containing miR-141-3p (hsa-miR-141-3p, miRBase accession no. MIMAT0000432) binding site was amplified and cloned into the pmirGLO vector (Promega, Madison, WI, USA) to synthetize a wild-type reporter plasmid pmirGLO-GLI2-wt. The putative binding site of miR-141-3p in GLI2 was mutated using the QuikChange Site-Directed Mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA) to gain a wild-type reporter plasmid pmirGLO-GLI2-mut. The above plasmids were used for the following luciferase reporter assays. Similarly, the GLI2 fragment containing the miR-141-3p binding site was amplified and cloned into the KpnI and XhoI restriction sites (Promega) of the pcDNA3.1 vector to synthesize a wild-type GLI2 overexpression plasmid pcDNA3.1-FGF-18-wt while a mutant type GLI2 overexpression plasmid pcDNA3.1-GLI2-mut was synthetized using QuikChange Site-Directed Mutagenesis kit. These two plasmids were used to construct GLI2 overexpression cell models.

Dual-luciferase reporter assay

MG-63 and MNNG/HOS cells were seeded in a 24-well plate and co-transfected with the constructed reporter vectors and miR-141-3p mimics or the negative control using Lipofectamine 2000 according to the manufacturer's instructions. After 36 h, luciferase activity was measured by a Dual-Luciferase Reporter Assay system according to the manufacturer's instructions (Promega).

Statistical analysis

All data were analyzed using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). All experiments were repeated three times and all data from three independent experiments are expressed as mean ± SD. The relationship between miR-141-3p and GLI2 expression was tested with Spearman correlation analysis. Differences in miR-141-3p and GLI2 expression in the different groups of tissues and cell lines were analyzed by the Wilcoxon signed rank test. A two-sided P-value of <0.05 was considered to be statistically significant.

Results

Expression of GLI2 is upregulated but expression of miR-141-3p is downregulated in osteosarcoma tissues and cell lines

We firstly detected miR-141-3p expression in 28 cases of osteosarcoma tissues and paired para-tumor tissues. As displayed in Fig. 1A and B, the expression of miR-141-3p in osteosarcoma tissues was markedly lower than that in the para-tumor tissues (P<0.01). Secondly, we determined GLI2 expression in the tissue samples above by means of real-time PCR, western blotting and IHC. In addition, the outcomes are shown in Fig. 1C-E. An obviously elevated GLI2 was presented in osteosarcoma tissues when compared with that in para-tumor tissues (P<0.01). Thirdly, we detected miR-141-3p expression in the normal human osteoblastic cell line hFOB 1.19, and in the osteosarcoma cell lines MG-63, MNNG/HOS and SW1353 using real-time PCR. As shown in Fig. 1F, the expression of miR-141-3p in hFOB 1.19 cells was higher than that in the osteosarcoma cell lines MG-63, MNNG/HOS and SW1353 (P<0.01). Expression of GLI2 demonstrated a reverse trend compared with miR-141-3p expression as determined by real-time PCR and western blotting (P<0.01) (Fig. 1G and H). Finally, the correlation analysis revealed an obviously inverse correlation between miR-141-3p and GLI2 (Spearman correlation analysis, r=0.7934, P<0.0001) (Fig. 1I). The detailed information of the patients is not shown. In brief, our findings showed upregulation of GLI2, but downregulation of miR-141-3p in the osteosarcoma tissues and cell lines.

Overexpression of miR-141-3p inhibits proliferation and promotes apoptosis and decreased GLI2 expression in osteosarcoma cells

Since miR-141-3p was decreased in osteosarcoma tissues and cell lines as demonstrated in the above experiments, we aimed to determine the mechanism of action in osteosarcoma. As revealed in Fig. 2A and B, overexpression of miR-141-3p by transfection of miR-141-3p mimics notably inhibited proliferation ability in the osteosarcoma MG-63 and MNNG/HOS cells. In addition, elevation of miR-141-3p promoted apoptosis in the MG-63 and MNNG/HOS cells (Fig. 2C and D). Furthermore, we investigated the expression level changes in GL2 and found that upregulation of miR-141-3p led to a decrease in GLI2 expression at the post-transcriptional level (Fig. 2E and F).

miR-141-3p targets GLI2 and its downstream pathway in osteosarcoma cells

Since elevated miR-141-3p regulated osteosarcoma cell proliferation, apoptosis and the expression of GLI2, and miRNAs are known to regulate hundreds of mRNA targets, resulting in changing of the cellular phenotype, we wondered whether the function miR-141-3p was implemented through the GLI2 pathway. Firstly, we theoretically predicted the GLI2 3′ untranslated region (3′UTR) of GLI2 contained the binding sites for miR-141-3p using UCSC Genome Browser (Fig. 3A). Secondly, we verified that an increase in and an decrease in miR-141-3p could regulate GLI2 expression correspondingly at the post-transcriptional level (Fig. 3B and C). Meanwhile, as shown in Fig. 3D, we found that the expression of parathyroid hormone-related protein 1 (PTHRP1), an acknowledged downstream protein of GLI2, was also negatively regulated by miR-141-3p. Thirdly, we constructed reporter plasmids containing wild-type and mutant GLI2 3′UTR (Fig. 3E). Finally, we executed a luciferase reporter assay to determine the potential target binding effect between miR-141-3p and GLI2 3′UTR. In addition, the outcomes demonstrated that the fluorescence in co-transfection of miR-141-3p mimics and pmirGLO-GLI2-3′UTR-wt group was markedly weakened compared to the mimic control and pmirGLO-GLI2-3′UTR-wt co-transfection group. However, when the theoretical miR-141-3p binding sites in GLI2 3′UTR were mutated (co-transfection of mimic control/miR-141-3p mimics and pmirGLO-GLI2-3′UTR -mut), the difference was dismissed (Fig. 3F). These findings verified that miR-141-3p targets GLI2 and its downstream pathway.

GLI2 abrogates the suppressive effect of miR-141-3p on osteosarcoma cell proliferation

We verified the expression levels of miR-141-3p and GLI2 in osteosarcoma tissues and cell lines, and identified the mechanism of action of miR-141-3p on osteosarcoma cell proliferation and apoptosis and confirmed that GLI2 is a target of miR-141-3p. It is well known that miRNA could regulate target gene expression post-transcriptionally. Hence, we wondered whether miR-141-3p suppressed osteosarcoma proliferation via the GLI2 pathway. We constructed wild-type and mutant-type GLI2 overexpression plasmids pcDNA3.1-GLI2-wt and pcDNA3.1-GLI2-mut which containing wild-type and mutant-type miR-141-3p binding sites, respectively. Then we executed the antisense experiments to further determine whether the effect of miR-141-3p on osteosarcoma cell proliferation and apoptosis was achieved via the GLI2 pathway. As demonstrated in Fig. 4A, overexpression of miR-141-3p (transfection of miR-141-3p mimics) led to an obviously decrease in GLI2 expression at the post-transcriptional level, but the inhibition effect was prominently rescued by pcDNA3.1-GLI2-mut, but was not reversed by pcDNA3.1-GLI2-wt. In addition, the re-executed proliferation and apoptosis assays confirmed that it was pcDNA3.1-GLI2-mut, but not pcDNA3.1-GLI2-wt which reversed the suppressive effect of miR-141-3p on proliferation and the promoting effect on apoptosis in osteosarcoma cells (Fig. 4B and C).

In brief, all the outcomes above confirmed that the effect of miR-141-3p on proliferation and apoptosis was achieved through the GLI2 pathway in osteosarcoma cells.

Discussion

GLI2 is a transcription factor with highly conserved C2H2-Zn finger DNA-binding domains and is extensively reported as a representative Krüppel-like factor family (19). In addition, GLI2 is known as an effector molecule or a primary transcriptional activator downstream of the Hedgehog pathway (20). Research has demonstrated that GLI2 is a key regulator in numerous malignant tumors (21,22). Nagao et al reported that GLI2 acts as an oncogene in regards to the proliferation and metastasis in osteosarcoma (5,9). PTHRP1 is known as a downstream factor of GLI2 and is involved in numerous types of tumors including osteosarcoma (2328). Ho et al found that knockdown of PTHR1 decreased the invasion and growth and increased tumor differentiation in osteosarcoma cells (29). In the present study, we detected the expression of GLI2 in osteosarcoma tissues and cell lines and revealed an elevated GLI2 in osteosarcoma as previously reported. In addition, we found that transfection of mutant GLI2 overexpression plasmid-pcDNA3.1-GLI2-mut markedly reversed the inhibitory effect of miR-141-3p on osteosarcoma cell proliferation. The findings of our research verified again that GLI2 acts as an oncogene in osteosarcoma.

miR-141-3p is located at human chromosome 12p13.31 and is comprehensively involved in various tumors (10,11,30). In addition, miR-141-3p regulates cell proliferation and apoptosis according to previous research. Jiang et al reported that silencing of miR-141-3p abrogated the effects of propofol on proliferation, neuronal differentiation and migration in neural stem cells (NSCs) (31). Li et al found that miR-141-3p appears to be a novel oncogene miRNA and that upregulation of miR-141-3p promoted prostate cancer cell proliferation by targeting Krüppel-like factor 9 (KLF9) (12). Jin et al revealed that inhibition of miR-141-3p induced a higher apoptosis percentage in EC9706R cells (10). In the present study, we revealed a decreased expression level of miR-141-3p in osteosarcoma tissues and cell lines. Meanwhile, we confirmed that downregulation of miR-141-3p promoted proliferation and inhibited apoptosis through a loss of function test in osteosarcoma cells. These expression and functional experiments indicated that miR-141-3p functions as an anti-oncogene in osteosarcoma cells. Several methods including FITC Annexin V and flow cytometry, TUNEL assay and electron microscopy are extensively applied for the detection of apoptosis (3234). In the present study, we used TUNEL assay to evaluate the condition of apoptosis. To date, miRNAs are widely reported as key manipulators in various diseases via post-transcriptionally regulation of their target gene function (3539). In our research, through the online predictive software and the constructed luciferase assay, we elucidated that miR-141-3p could bind to GLI2 mRNA 3′UTR. In addition, we demonstrated that upregulation and downregulation of miR-141-3p mediated GLI2 and its downstream PTHRP1 expression correspondingly. Furthermore, through the antisense experiment we showed that the effects of miR-141-3p on osteosarcoma cell proliferation and apoptosis were achieved through the GLI2 pathway.

The tumorigenesis of osteosarcoma is a very complicated biological process involving diverse mechanisms. Our findings indicated that the miR-141-3p/GLI2 axis can be a potential target for the molecular-targeted treatment of osteosarcoma.

Acknowledgements

The present study was supported by grants from the National Natural Science Foundation of China (no. 81502333), the PhD Startup Research Foundation of Liaoning Province (no. 201601225), the Natural Science Foundation of Liaoning Province (nos. 20170540872 and 2015020377), and the Technological Innovation Fund of Shenyang Technology Division (no. F15-139-9-07).

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February-2018
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Wang N, Li P, Liu W, Wang N, Lu Z, Feng J, Zeng X, Yang J, Wang Y, Zhao W, Zhao W, et al: miR-141-3p suppresses proliferation and promotes apoptosis by targeting GLI2 in osteosarcoma cells Retraction in /10.3892/or.2023.8576. Oncol Rep 39: 747-754, 2018
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Wang, N., Li, P., Liu, W., Wang, N., Lu, Z., Feng, J. ... Zhao, W. (2018). miR-141-3p suppresses proliferation and promotes apoptosis by targeting GLI2 in osteosarcoma cells Retraction in /10.3892/or.2023.8576. Oncology Reports, 39, 747-754. https://doi.org/10.3892/or.2017.6150
MLA
Wang, N., Li, P., Liu, W., Wang, N., Lu, Z., Feng, J., Zeng, X., Yang, J., Wang, Y., Zhao, W."miR-141-3p suppresses proliferation and promotes apoptosis by targeting GLI2 in osteosarcoma cells Retraction in /10.3892/or.2023.8576". Oncology Reports 39.2 (2018): 747-754.
Chicago
Wang, N., Li, P., Liu, W., Wang, N., Lu, Z., Feng, J., Zeng, X., Yang, J., Wang, Y., Zhao, W."miR-141-3p suppresses proliferation and promotes apoptosis by targeting GLI2 in osteosarcoma cells Retraction in /10.3892/or.2023.8576". Oncology Reports 39, no. 2 (2018): 747-754. https://doi.org/10.3892/or.2017.6150