MicroRNA-144 acts as a tumor suppressor by targeting Rho-associated coiled-coil containing protein kinase 1 in osteosarcoma cells

Retraction in: /10.3892/mmr.2021.12344

  • Authors:
    • Jianjun Huang
    • Ying Shi
    • Hui Li
    • Meisongzhu Yang
    • Guohong Liu
  • View Affiliations

  • Published online on: June 16, 2015     https://doi.org/10.3892/mmr.2015.3937
  • Pages: 4554-4559
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Abstract

MicroRNAs (miRs) have been demonstrated to be associated with multiple processes in the development and progression of human malignancies. Previous studies have observed aberrant downregulation of miR‑144 in several types of cancer, including osteosarcoma. However, the function of miR‑144 and the underlying mechanism in osteosarcoma remain to be elucidated. The present study indicated that miR‑144 was markedly downregulated in osteosarcoma tissues and cell lines compared with that in the normal controls. Restoration of miR‑144 significantly inhibited cell proliferation, migration and invasion of MG‑63 osteosarcoma cells. In addition, Rho‑associated coiled‑coil containing protein kinase 1 (ROCK1) was identified as a novel target of miR‑144 in MG‑63 osteosarcoma cells. Furthermore, knockdown of ROCK1 suppressed the proliferation, migration and invasion of MG‑63 osteosarcoma cells to a similar extent to the effects of miR‑144 overexpression. In addition, the mRNA expression of ROCK1 was increased in osteosarcoma tissues and was negatively correlated with the expression of miR‑144. In conclusion, the results of the present study suggested that miR‑144 acts as a tumor suppressor by targeting ROCK1 in osteosarcoma.

Introduction

Osteosarcoma is the most common primary malignant tumor of bone (1). Despite previous advances in surgical resection combined with chemotherapy and radiotherapy, the prognosis of patients with osteosarcoma remains poor (1). Therefore, the identification of novel effective targets for the treatment of osteosarcoma is urgently required.

MicroRNAs (miRs) are a type of endogenous small RNA. They can bind to the complementary sites in the 3′-untranslated region (UTR) of their target mRNAs and inhibit their expression at the post-transcriptional level by inducing translational suppression or directly cleaving their target mRNAs (2). It has been widely demonstrated that aberrant upregulation or down-regulation of miRs is tightly associated with the development and progression of various human malignancies, and therefore, these miRs function as oncogenes or tumor suppressors (3).

miR-144 has been revealed to be involved in multiple types of human malignancy (4,5). In addition, miR-144 was reported to act as a tumor suppressor in certain types of cancer, including bladder (4), non-small cell lung (6) and colorectal (7) cancer. A previous study suggested that miR-144 may also be involved in the development and progression of osteosarcoma (8). However, the detailed role of miR-144 and its targets in osteosarcoma remain to be elucidated.

Rho-associated coiled-coil containing protein kinase 1 (ROCK1), a downstream effector of Rho, has been demonstrated to act as an oncogene in several types of cancer (9,10). It has been suggested that ROCK1 is involved in tumor growth and metastasis in osteosarcoma (11,12). However, the regulatory mechanism of ROCK1 in osteosarcoma cells remains to be fully elucidated.

The present study aimed to investigate the detailed function of miR-144 in osteosarcoma in vitro. In addition, the underlying molecular mechanisms involved were investigated. The results suggested that miR-144 suppresses the malignant phenotypes of osteosarcoma cells by directly inhibiting the expression of ROCK1.

Materials and methods

Osteosarcoma tissues collection

The present study was approved by the Ethics Committee of Jishou University (Jishou, China). Osteosarcoma tissue (n=20) and their matched normal adjacent tissue was obtained from the Department of Orthopedics, People's Hospital of Xiangxi Autonomous Prefecture, Jishou University (Jishou, China). Informed consent was obtained from all patients. The tissues were stored in liquid nitrogen shortly following surgical resection.

Cell culture

The human osteosarcoma cell lines Saos-2, MG-63 and U2OS, and the human osteoblast cell line hFOB 1.19 were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in RPMI-1640, Dulbecco's modified Eagle's medium (DMEM) or DMEM/F12 medium (all from Invitrogen Life Technologies, Carlsbad, CA, USA), respectively, supplemented with 10% fetal bovine serum (FBS; Invitrogen Life Technologies) at 37°C in a humidified atmosphere containing 5% CO2.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

The total RNA was isolated using TRIzol reagent (Invitrogen Life Technologies), according to the manufacturer's instructions. Reverse transcription was performed using the Takara RNA PCR kit (Takara Bio Inc., Shiga, Japan) according to the manufacturer's instructions. RT-qPCR was performed using a Hairpin-it™ miRNA qPCR Quantitation kit (Gene Pharma, Shanghai, China) for miRNA detection, or the SYBR green qPCR assay kit (Invitrogen Life Technologies) for mRNA detection, on a Light Cycler 480 (Roche, Basel, Switzerland). GAPDH and U6 small nuclear RNA were used as internal controls. The primers used were as follows: ROCK1 forward, 5′-AGGAAGGCGGACATATTAGTCCCT-3′ and reverse, 5′-AGACGATAGTTGGGTCCCGGC-3′; GAPDH forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′. The PCR cycling conditions were as follows: 95°C for 10 min, 40 cycles of denaturation at 95°C for 15 sec then annealing/elongation at 60°C for 60 sec. The 2-ΔΔCT method was used for relative quantification of the differences in the expression levels of each target.

Transfection

ROCK1 small interfering (si)RNA and control siRNA were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). miR-144 mimics and scrambled small RNA oligonucleotide were synthesized by Gene Pharma. The cells were cultured to 80% confluence and were transfected with these small RNA oligonucleotides using Lipofectamine 2000 (Invitrogen Life Technologies), according to the manufacturer's instructions.

Luciferase activity assay

Targetscan software (release 6.2) was used to predicate the putative target genes of miR-144 (13). As described previously (12), the full length 3′-UTR of ROCK1 was amplified by PCR from genomic DNA and cloned at the SacI and XhoI sites into the pmirGLO vector (Promega, Madison, WI, USA). The primers for the 3′-UTR of ROCK1 mRNA were as follows: Forward, 5′-GCACTAGTGTTCCATCTTCGGACGTTGA-3′ and reverse, 5′-ATACGCGTCTTCAACAGACCATGCTCCC-3′. The mutant 3′-UTR of ROCK1 mRNA was generated using a Quick Change mutagenesis kit (Stratagene, Heidelberg, Germany). Lipofectamine 2000 was used to co-transfect the cells with reporter vectors and miR-144 mimics or negative control. Following co-transfection for 48 h, the luciferase activity was determined using a dual luciferase reporter assay system (Promega) according to the manufacturer's instructions.

Proliferation assay

An MTT assay was used to quantify cell proliferation according to the manufacturer's instructions. Briefly, 104 cells/well were plated in a 96-well plate and incubated for 0, 12, 24 or 48 h at 37°C and 5% CO2. To assess cell proliferation, 10 µl MTT (5 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) was added to each well and the cells were subsequently incubated for 4 h at 37°C and 5% CO2. The supernatant was removed and 100 µl dimethylsulfoxide (Sigma-Aldrich) was added to dissolve the precipitate. The absorbance was measured at 492 nm using a microplate reader (3550; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Migration assay

A cell migration assay was performed using Transwell chambers. A cell suspension, containing 5×105 cells/ml without serum, was added into the upper chamber of the Transwells (BD Biosciences, Franklin Lakes, NJ, USA) and medium containing 10% FBS was added into the lower chamber. Following incubation for 24 h, the cells which had not migrated through the pores were removed using a cotton bud. The filters were subsequently fixed with 90% alcohol and stained with crystal violet (Sigma-Aldrich). Five random fields were counted under an inverted microscope (IX71; Olympus, Tokyo, Japan).

Invasion assay

A cell suspension, containing 5×105 cells/ml without serum, was added into the upper chamber of transwells pre-coated with Matrigel (BD Biosciences), and medium containing 10% FBS was added into the lower chamber. Following incubation for 24 h, the cells which had not migrated through the pores were removed using a cotton bud. The filters were subsequently fixed with 90% alcohol and stained with crystal violet. Five random fields were counted under an inverted microscope (Olympus).

Western blot analysis

The proteins in tissues or cells were isolated using radioimmunoprecipitation assay buffer (Sigma-Aldrich) and were quantified using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). The proteins (60 µg) were separated using 10% SDS-PAGE (Sigma-Aldrich) and were transferred onto a polyvinylidene difluoride (PVDF) membrane, which was subsequently incubated with Tris-buffered saline with Tween-20 (Sigma-Aldrich) containing 5% milk at room temperature for 3 h. The PVDF membrane was incubated with mouse anti-ROCK1 (1:100; cat. no. ab45171; Abcam, Cambridge, UK), and mouse anti-GAPDH (1:100; cat. no. ab181602; Abcam) primary antibodies at room temperature for 3 h. Following washing three times with phosphate-buffered saline (Sigma-Aldrich) with Tween-20, the membrane was incubated with the goat anti-mouse secondary antibodies (1:5,000; ab175773; Abcam) at room temperature for 40 min. Chemiluminescent detection was performed using an enhanced chemiluminescence kit (Pierce Biotechnology, Inc.). The relative protein expression was analyzed using ImagePro plus software 6.0 (Media Cybernetics, Rockville, MD, USA) and was represented as the density ratio normalized to GAPDH.

Statistical analysis

SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) was used to perform statistical analysis. Values are expressed as the mean ± standard deviation. Differences were assessed using two-tailed Student's t-test. The association between the expression levels of miR-144 and ROCK1 was assessed using two-tailed Pearson's correlation. P<0.05 was considered to indicate a statistically significant difference.

Results

Expression of miR-144 is downregulated in osteosarcoma tissues and cell lines

To investigate the function of miR-144 in osteosarcoma, the expression levels in human osteosarcoma tissues and their matched adjacent normal tissues were assessed using RT-qPCR. As shown in Fig. 1A, miR-144 was markedly downregulated in osteosarcoma tissues compared with that in adjacent normal tissues. In addition, it was demonstrated that miR-144 was downregulated in the three osteosarcoma cell lines Saos-2, MG-63 and U2OS compared with that in the normal osteoblast cell line hFOB 1.19 (Fig. 1B). Accordingly, the present study suggested that downregulation of miR-144 may be important in osteosarcoma.

Restoration of miR-144 inhibits cell proliferation, migration and invasion in osteosarcoma cells

The present study further investigated whether miR-144 affects osteosarcoma cell migration and invasion. MG-63 cells were transfected with miR-144 mimics or negative control, and cell migration and invasion assays were performed. As expected, transfection with miR-144 led to an increase in the expression of miR-144 in MG-63 cells (Fig. 2A). Furthermore, as shown in Fig. 2B–D, restoration of miR-144 caused a decrease in cell proliferation, migration and invasion of MG-63 cells. These findings indicated that miR-144 exerts a suppressive role in the regulation of osteosarcoma cell proliferation, migration and invasion.

miR-144 negatively regulates the protein expression of its target gene, ROCK1, in osteosarcoma cells

Potential targets of miR-144 were further investigated using Targetscan software. ROCK1 was predicated to be a putative target gene for miR-144, which has been demonstrated to regulate cell proliferation, migration and invasion (14). The effect of miR-144 on the protein expression of ROCK1 was assessed and it was demonstrated that the protein levels of ROCK1 were markedly reduced following the overexpression of miR-144 in MG-63 cells (Fig. 3A). Whether ROCK1 was a direct target gene of miR-144 was determined by performing a luciferase reporter assay in MG-63 cells (Fig. 3B–D). As shown in Fig. 3D, co-transfection with wild-type ROCK1-3′-UTR plasmid and the miR-144 mimics resulted in a significant decrease in luciferase activity. However, co-transfection with the mutant ROCK1-3′-UTR plasmid and miR-144 mimics revealed no change in luciferase activity. These results indicated that miR-144 negatively regulates the protein expression of ROCK1 by directly binding to the 3′-UTR of ROCK1, which can further cause translational inhibition in MG-63 cells.

siRNA-mediated inhibition of ROCK1 reduces cell proliferation, migration and invasion in osteosarcoma cells

Based on findings from the present study, it was speculated that ROCK1 may be involved in the regulatory role of miR-144 in osteosarcoma cell migration and invasion. To confirm this speculation, MG-63 cells were transfected with ROCK1 siRNA to knockdown the expression of ROCK1. As shown in Fig. 4A, the protein levels of ROCK1 were significantly reduced following transfection of MG-63 cells with ROCK1 siRNA. It was demonstrated that the downregulation of ROCK1 significantly inhibited MG-63 cell proliferation, migration and invasion (Fig. 4B–D). These results indicated that knockdown of ROCK1 inhibited osteosarcoma cell proliferation, migration and invasion, which resembled the suppressive effect of miR-144.

Expression of ROCK1 is upregulated in osteosarcoma

The mRNA expression levels of ROCK1 were determined in osteosarcoma tissues and their matched adjacent normal tissues, and the miR-1441 – ROCK1 correlation was investigated in osteosarcoma. As shown in Fig. 5A, the mRNA expression levels of ROCK1 were significantly increased in osteosarcoma tissues compared with those in their matched adjacent normal tissues. In addition, a significant inverse correlation was observed between the expression levels of ROCK1 and miR-144 (Fig. 5B). These findings suggested that the upregulation of ROCK1 is possibly due to the inhibition of miR-144, which may contribute to osteosarcoma cell migration and invasion.

Discussion

Deregulation of miRs, including miR-144, has been demonstrated to be involved in the development and progression of various types of human cancer (15,16). However, the detailed role of miR-144 and its target in osteosarcoma has remained to be elucidated. The present study demonstrated that miR-144 was downregulated in osteosarcoma tissues compared with that in their matched adjacent normal tissues. In fact, deregulation of miR-144 has been reported to be important in various types of cancer. Guo et al (4) demonstrated that downregulation of miR-144 increased bladder cancer cell proliferation and Guan et al (16) revealed that this promoted thyroid cancer cell invasion (16). Previously, 177 miRNAs were identified to be differentially expressed in osteosarcoma cell lines compared with that in normal bone, and miR-144 was further demonstrated to exhibit an intermediate expression in osteosarcoma tissues compared with that in osteoblasts and bone, which may reflect the differentiation level of osteosarcoma relative to the undifferentiated osteoblast and fully differentiated normal bone (8). However, the detailed role of miR-144 in osteosarcoma has remained to be elucidated. The present study demonstrated that restoration of miR-144 inhibits the proliferation, migration and invasion of osteosarcoma cells. As cell migration and local invasion are two key stages during cancer metastasis, these findings suggested that the inhibition of the expression of miR-144 may be involved in osteosarcoma growth and metastasis.

Theoretical prediction was used to further examine the potential target genes for miR-144 and revealed the putative target, ROCK1. ROCK1 is a serine/threonine kinase, belonging to the Rho family of guanine triphosphatase proteins (17). It has been well established that ROCK1 is involved in the reorganization of the actin cytoskeleton, which is important in cell migration and invasion (1719). In addition, the expression levels of ROCK1 were reported to be significantly upregulated in multiple types of human cancer, including bladder (14), prostate (20) and lung (21) cancer, suggesting that ROCK1 acts as an important oncogene in multiple types of cancer. A previous study investigated the role of ROCK1 in osteosarcoma. Liu et al (11) demonstrated that ROCK1 was upregulated in osteosarcoma tissues, consistent with the results of the present study, and high levels of ROCK1 were associated with poor clinical outcome of osteosarcoma (11). However, the regulatory mechanism by which the expression of ROCK1 is mediated has remained to be elucidated in osteosarcoma cells. The present study identified ROCK1 as a direct target of miR-144 and demonstrated that the overexpression of miR-144 inhibited the protein expression of ROCK1. In addition, knockdown of ROCK1 markedly suppressed osteosarcoma cell proliferation, migration and invasion, resembling the inhibitory effect of miR-144. These findings suggested that the inhibitory effect of miR-144 on osteosarcoma cell migration and invasion may be, at least in part, through directly targeting ROCK1. Similar mechanisms were demonstrated by other studies. For instance, Zhou et al (12) reported that miR-340 suppressed osteosarcoma tumor growth and metastasis by directly targeting ROCK1 and Wang et al (22) revealed that miR-335 inhibited migration and invasion by targeting ROCK1 in osteosarcoma cells (22). In addition, miR-145 was demonstrated to inhibit osteosarcoma cell proliferation and invasion by targeting ROCK1 (23). The present study demonstrated that high expression of ROCK1 was inversely correlated with miR-144 expression, highlighting the role of miR-144 in the regulation of cell proliferation, migration and invasion by targeting ROCK1 in osteosarcoma cells.

In conclusion, the findings of the present study demonstrated that the expression levels of miR-144 were markedly reduced in osteosarcoma tissues and cells compared with those in normal tissue, which may be associated with osteosarcoma growth and metastasis. In addition, restoration of miR-144 inhibited proliferation, migration and invasion of osteosarcoma cells, possibly by directly targeting ROCK1. Therefore, the present study highlighted the important functions of miR-144 and ROCK1 in osteosarcoma cells, suggesting that miR-144 and ROCK1 may be potential molecular targets for the treatment of osteosarcoma growth and metastasis.

Acknowledgments

This study was supported by a Research Project of the Education Department of Hunan Province (no. 11C1035), the Natural Science Fund Project of Hunan Province (no. 12JJ6074), the Scientific Research Project of the Health Department of Hunan Province (no. B2013-160) and the Talent Introduction Research of Jishou University (no. 201110).

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September-2015
Volume 12 Issue 3

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Spandidos Publications style
Huang J, Shi Y, Li H, Yang M and Liu G: MicroRNA-144 acts as a tumor suppressor by targeting Rho-associated coiled-coil containing protein kinase 1 in osteosarcoma cells Retraction in /10.3892/mmr.2021.12344. Mol Med Rep 12: 4554-4559, 2015
APA
Huang, J., Shi, Y., Li, H., Yang, M., & Liu, G. (2015). MicroRNA-144 acts as a tumor suppressor by targeting Rho-associated coiled-coil containing protein kinase 1 in osteosarcoma cells Retraction in /10.3892/mmr.2021.12344. Molecular Medicine Reports, 12, 4554-4559. https://doi.org/10.3892/mmr.2015.3937
MLA
Huang, J., Shi, Y., Li, H., Yang, M., Liu, G."MicroRNA-144 acts as a tumor suppressor by targeting Rho-associated coiled-coil containing protein kinase 1 in osteosarcoma cells Retraction in /10.3892/mmr.2021.12344". Molecular Medicine Reports 12.3 (2015): 4554-4559.
Chicago
Huang, J., Shi, Y., Li, H., Yang, M., Liu, G."MicroRNA-144 acts as a tumor suppressor by targeting Rho-associated coiled-coil containing protein kinase 1 in osteosarcoma cells Retraction in /10.3892/mmr.2021.12344". Molecular Medicine Reports 12, no. 3 (2015): 4554-4559. https://doi.org/10.3892/mmr.2015.3937