Circular RNAs (circRNAs), a type of non-coding RNAs derived from back-splicing, have been reported to function as gene expression regulators involved in tumor development of multiple human tumors. However, the clinical significance and underlying molecular mechanisms of circ_001569 in osteosarcoma still be unknown. In the study, we found that circ_001569 expression was significantly overexpressed in osteosarcoma tissues compared with adjacent noncancerous bone tissues. Higher circ_001569 expression significantly correlated with distant metastasis and advanced tumor stage of osteosarcoma patients. Gain-function and loss-function assays showed that circ_001569 knockdown significantly inhibited osteosarcoma cell proliferation and cell colon formation capacities. Moreover, upregulation of circ_001569 significantly promoted osteosarcoma cell resistance to cisplatin by activating Wnt/β-catenin signaling pathway. Thus, these results indicated that circ_001569 represented a novel potentially therapeutic target of osteosarcoma.
Osteosarcoma (OS) is one of the most common primary bone tumors and accounts for approximately 5–6% in all tumors of childhood and adolescence (
Circular RNAs are newly discovered endogenous non-coding RNAs characterized by their covalently closed loop structures without a 5′cap or a 3′Poly A tail (
In the present study, we found that circ_001569 was significantly overexpressed in osteosarcoma and correlated with distant metastasis, advanced tumor stage, and poor prognosis of osteosarcoma. Knockdown of circ_001569 significantly inhibited the proliferation ability of osteosarcoma. Moreover, upregulation of circ_001569 significantly promoted osteosarcoma cell resistance to cisplatin by activating Wnt/β-catenin signaling pathway. Thus, our results indicated that circ_001569 may serve as a novel potentially therapeutic target of osteosarcoma.
A total of 36 primary osteosarcoma tissue samples and adjacent non-cancerous bone tissue samples were obtained from the Department of Orthopedics, Affiliated Hospital of Weifang Medical University (Weifang, China) between January 2009 and February 2013. The patients included 23 males and 13 females, with a median age of 35.8 years and an age range of 10 to 56 years old. The histological grade was assessed according to the tumor-node-metastasis (TNM) system (
Two human osteosarcoma cell lines (MG-63 and U2OS) and a normal human osteoblastic cell line hFOB were obtained from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The hFOB cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The human OS cells were cultured in RPMI-1640 medium supplemented with 10% FBS (both Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin (Thermo Fisher Scientific, Inc.). Cells were incubated at 37°C with 5% CO2 in a humidified chamber.
siRNA targeting has-circ_001569 (5′-GCATCGTGCAGGACTGGAA-3′) and si-negative control (NC; 5′-UUCUCCGAACGUGUCACGUTT-3′) was constructed and purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). Cells were transiently transfected with 100 nmol/l oligoribonucleotides using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Human hsa_circ_001569 cDNA was synthesized and cloned into pLVX-IRESneo by GeneCopoeia, Inc. (Rockville, MD, USA). Cells were transfected with the oligonucleotides at room temperature at a final concentration of 20 nmol/l using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Cells were harvested following cell transfection at 48 h.
Total RNA was extracted from tissues and cells using TRIzol reagent (Thermo Fisher Scientific, Inc.). Reverse Transcription kit (Guangzhou RiboBio Co., Ltd.) was used to reverse RNA to cDNA, according to the manufacturer's protocols. RT-qPCR was performed using a SYBR Premix Ex TaqTM II kit (Takara Biotechnology Co., Ltd., Dalian, China) on an ABI7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec, 57°C for 20 sec and 72°C for 15 sec. The mRNA fold-change of circ_001569 was calculated by the 2−ΔΔCq method (
Cell proliferation capacity was evaluated using the Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer's protocol. Transfected cells were seeded onto 96-well plates (2,000 cells/well) and were incubated overnight at 37°C with 5% CO2 in a humidified chamber. The proliferation rate of osteosarcoma cells was detected every 24 h until 72 h. Each well was supplemented with 10 µl CCK-8 regent and cells were incubated for 2 h. Next, cell proliferation was measured at 450 nm using a Multiskan MK3 spectrophotometer.
The transfected osteosarcoma cells were plated at 500 cells/well onto 6-well plates. After cells were cultured at 37°C with 5% CO2 in a humidified chamber for two weeks, the cell colonies were fixed with 4% paraformaldehyde at room temperature for 20 min and stained with 0.1% crystal violet at room temperature for 20 min, and the images of cell colonies were captured and cell colonies were calculated.
Total protein was extracted using lysis buffer (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). A BCA kit (Tiangen Biotech Co., Ltd., Beijing, China) was used to detect the concentration of the proteins. Equal protein (30 µg) was separated on 8–12% SDS-PAGE gel and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Following blocking with 5% skimmed milk at room temperature for 1 h, the membrane was incubated with antibodies including p-GSK-3β (cat. no. 9336; 1:1,000), GSK-3β (cat. no. 9315; 1:2,000), β-catenin (cat. no. 9562; 1:1,000) and GAPDH (cat. no. 5174; 1:1,000; all Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C and then incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (cat. no. 10285-1-AP; 1:1,000; ProteinTech Groups, Inc., Chicago, IL, USA) at room temperature for 1 h. The membrane was detected with an enhanced chemiluminescence detection system (Nanjing KeyGen Biotech Co., Ltd.). GAPDH was used as an internal control.
The cells were transfected with si-NC and si-circ_001569 or pLVX-IRESneo-vector (vector) and pLVX-IRESneo-circ_001569 (circ_001569). Cell viability was detected using CCK-8, according to the manufacturer's protocol. The IC50 values were defined as the chemotherapeutic agent concentration producing 50% inhibition of cell proliferation. Absorbance was detected at 450 nm.
All data in the study are presented as the mean ± standard deviation and were analyzed using SPSS 19.0 software package (IBM Corp., Armonk, NY, USA). Student's t-test was applied to compare the differences between two groups. For comparisons among 3 or more groups, one-way analysis of variance, followed by the Student-Newman-Keuls post hoc test, was used. P<0.05 was considered to indicate a statistically significant difference.
To investigate the clinical significance of circ_001569 expression in osteosarcoma, the present study examined the expression of circ_001569 by RT-qPCR in 36 primary osteosarcoma tissues and adjacent non-cancerous bone tissues. As demonstrated in
To evaluate the effects of circ_001569 on cell proliferation, a loss-function assay was performed in U2OS cells and a gain-function assay was performed in MG-63 cells according to their expression of circ_001569 (
Combination of cisplatin with doxorubicin and methotrexate is frequently used for osteosarcoma treatment (
Activation of Wnt/β-catenin signaling is associated with chemotherapy resistance in osteosarcoma (
Next, the present study demonstrated whether circ_001569 overexpression enhanced the resistance of osteosarcoma to cisplatin, doxorubicin or methotrexate by activating the Wnt/β-catenin signaling pathway. As demonstrated in
Recent studies have reported that circRNAs serve as important regulators in cancer biological functions, including cell proliferation, migration, invasion and metastasis (
Previous studies have revealed that activation of Wnt/β-catenin signaling could enhance chemotherapy resistance to cisplatin, doxorubicin and methotrexate. For example, TWIST decreases osteosarcoma cell survival against cisplatin by decreasing the soluble β-catenin level through a PI3K-dependent manner (
In conclusion, the results of the present study demonstrated that circ_001569 expression was higher in osteosarcoma. Upregulation of circ_001569 promoted cell proliferation ability. Furthermore, it was demonstrated that circ_001569 promoted cell resistance to cisplatin in osteosarcoma by activating Wnt/β-catenin signaling. These results indicated that circ_001569 may be a potential therapeutic target of osteosarcoma.
Not applicable.
No funding was received.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
HZ and JY conceived and designed the study. JY, XL and YZ performed the experiments. JY, XL and YZ analyzed and interpreted the data. YZ wrote the manuscript.
Written informed consent was obtained from all patients and the study was approved by the Ethics Committee of Affiliated Hospital of Weifang Medical University.
All patients provided consent for the publication of their data.
The authors declare that they have no competing interests.
Expression of circ_001569 is higher in osteosarcoma. (A) The relative expression of circ_001569 was detected using RT-qPCR in 36 primary osteosarcoma tissue samples and adjacent non-cancerous bone tissue samples. (B) Association between circ_001569 expression and distant metastasis in osteosarcoma patients. (C) Association between circ_001569 expression and Tumor-Node-Metastasis stage in patients with osteosarcoma. (D) The relative expression of circ_001569 was detected using RT-qPCR in two human osteosarcoma cell lines (MG-63 and U2OS) and a normal human osteoblastic cell line, hFOB. Data are presented as the mean ± standard deviation. *P<0.05. RT-qPCR, reverse transcription-quantitative polymerase chain reaction.
Upregulation of circ_001569 expression enhances cell proliferation ability
Upregulation of circ_001569 promotes osteosarcoma cell resistance towards chemotherapy and activates Wnt/β-catenin signaling. (A) Inhibition of circ_001569 activity in U2OS cells resulted in a significant decrease of the IC50 value for cisplatin, doxorubicin or methotrexate, compared with the control group. (B) Overexpression of circ_001569 activity in MG-63 cells could enhance the IC50 value for cisplatin, doxorubicin or methotrexate. (C) The relative expression of p-GSK-3β, GSK-3β and β-catenin was detected using western blot analysis following transfection with si-NC or si-circ_001569 in U2OS cells. (D) The relative expression of p-GSK-3β, GSK-3β and β-catenin was detected using western blot analysis following transfection with empty vector or overexpressed circ_001569 plasmid in MG-63 cells. Data are presented as the mean ± standard deviation. *P<0.05. IC50, calculated 50% inhibition of growth; si, small interfering RNA; NC, negative control.
Upregulation of circ_001569 promotes resistance of osteosarcoma cells to cisplatin by activating the Wnt/β-catenin signaling pathway. (A) The Wnt/β-catenin inhibitor, XAV939, or (B) Wnt/β-catenin agonist, LiCl, could reduce or increase the IC50 value for cisplatin, doxorubicin or methotrexate. (C) The IC50 value for cisplatin, doxorubicin or methotrexate in U2OS cells was detected following treatment of the cells with si-NC, si-circ_001569 or Wnt/β-catenin agonist (LiCl) + si-circ_001569. (D) The IC50 value for cisplatin, doxorubicin or methotrexate in MG-63 cells was detected following the treatment of cells with vector, circ_001569, or Wnt/β-catenin inhibitor (XAV939) + circ_001569. Data are presented as the mean ± standard deviation. *P<0.05, #, not statistically significant. IC50, calculated 50% inhibition of growth; si, small interfering RNA; NC, negative control.
Association between circ_001569 expression and clinical characteristics in 36 patients with osteosarcoma.
circ_001569 expression | ||||
---|---|---|---|---|
Clinical characteristic | Total (n=36) | Lower (n=16) | Higher (n=20) | P-value |
Sex | 0.121 | |||
Female | 13 | 8 | 5 | |
Male | 23 | 8 | 15 | |
Age, years | 0.677 | |||
≤18 | 26 | 11 | 15 | |
>18 | 10 | 5 | 5 | |
Site | 0.799 | |||
Femur | 18 | 7 | 11 | |
Tibia | 10 | 5 | 5 | |
Humerus | 8 | 4 | 4 | |
Distant metastasis | 0.009 |
|||
Absent | 16 | 11 | 5 | |
Present | 20 | 5 | 15 | |
Tumor size, cm | 0.221 | |||
<5 | 14 | 8 | 6 | |
≥5 | 22 | 8 | 14 | |
TNM stage | 0.016 |
|||
I–IIA | 14 | 10 | 4 | |
IIB-III | 22 | 6 | 16 |
P<0.05.