Identification and functional study of osteosarcoma metastasis marker genes
- Authors:
- Published online on: July 1, 2015 https://doi.org/10.3892/ol.2015.3444
- Pages: 1848-1852
Abstract
Introduction
Metastasis is a complex process describing the spread of cancerous cells from the primary tumor site to other organs. Osteosarcoma is the most common primary bone tumor amongst children and young adults (1). The most prevalent locations for osteosarcoma are the distal femoral, femur and proximal tibial metaphyses (2). It was reported that in ~50% of cases, osteosarcoma was localized to the knee region (3). Patients with bone metastases experience severe bone pain, spinal cord compression and osteolysis, which reduce the structural integrity of bone and results in increased risk of fracture (4). Osteosarcoma progression results in cell migration from the primary tumor, invading the lung tissue and establishing metastatic nodules in the lungs (5). Various novel improvements have been established for the treatment of patients with osteosarcoma, but metastatic disease diagnosis has not been well-studied (6,7). This is due to the poor understanding of the molecular basis of osteosarcoma metastasis genes. The identification of candidate genes in the osteosarcoma metastasis process is significant, so that these may be utilized as molecular markers for diagnosis of the disease in the initial stages. In addition, targeted therapy of key metastatic molecules by selectively blocking the migratory and invasive ability of the osteosarcoma cells may present a potential strategy for the inhibition of tumor metastasis (5). The present study aimed to identify osteosarcoma metastasis genes and examine the functionality of their target genes.
Materials and methods
Patients and tumor sample collection
Osteosarcoma samples were collected from patients at the Sichuan Cancer Hospital (Chengdu, China) through an institutional review board approval committee. In addition, the project was approved by the ethics committee of Sichuan Cancer Hospital, and written informed consent was obtained from the patients. A total of 31 samples were included in the present study, which were obtained from 31 patients, including 19 males and 12 females. The age of the patients ranged from 8 to 23 years. The collected samples were separated into two categories: Osteosarcoma without metastasis and osteosarcoma with metastasis. Control serum and bone samples and distal femur biopsy specimens, which had been previously collected and stored at the hospital from healthy organ donors were used. Complete information regarding the patients and samples is presented in Table I. Immediately following collection, the tumor specimen samples were snap frozen in liquid nitrogen and stored at −80°C prior to RNA extraction. All biopsy tumor specimens were confirmed to contain >90% tumor cells. In addition, blood samples were also collected from the 31 patients and stored accordingly for additional experiments.
RNA extraction
Total RNA was extracted from the two categories of samples using the TRIzol reagent method (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions. Osteosarcoma samples were homogenized in TRIzol reagent using 1 ml per 50–100 mg tissue. RNA concentrations and quality were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA), and the RNA integrity was assessed by agarose gel electrophoresis (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Purification of poly A+ messenger RNA (mRNA) from the total RNA was conducted using the Qiagen Oligotex mRNA Mini kit (Qiagen, Venlo, Netherlands) according to the manufacturer's instructions. The isolated poly A+ mRNA was stored at −80°C in a deep freezer for further experiments.
Complementary DNA (cDNA) synthesis and subtraction
The purified mRNA from the two groups of patients was subjected to cDNA subtraction analysis using the DsDD cDNA Subtraction and cloning kit (294–62001; Wako Pure Chemical Industries, Ltd., Osaka, Japan). The experiments were performed according to the manufacturer's instructions, which selectively amplifies differentially expressed sequences. These sequences were specific for osteosarcoma metastasis and were subsequently cloned using the DsDD cDNA Subtraction and cloning kit, as per the manufacturer's protocol. The osteosarcoma metastasis clones were sequenced and the sequence data were subjected to Basic Local Alignment Search Tool (BLAST; http://blast.ncbi.nlm.nih.gov/Blast.cgi) analysis with the Nucleotide and Expressed Sequence Tag databases by performing the sequence alignment.
Immunoblotting
Protein samples were prepared from the two groups of patient specimens and controls, and resolved by 8% SDS-PAGE (Bio-Rad Laboratories, Inc.). The resolved protein samples from the SDS-PAGE gel were transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc.), blocked with 4% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) for 1 h and incubated with monoclonal mouse anti-ezrin (catalog no. E8897; dilution, 1:3,000) and polyclonal rabbit anti-β4 integrin (catalog no. SAB4501588; dilution, 1:8,000) primary antibodies (Sigma-Aldrich) overnight at 4°C. For loading controls, monoclonal mouse anti-β-actin (catalog no. ab6276; dilution, 1:12,000) and anti-transferrin (catalog no. ab769; dilution, 1:12,000) antibodies were used (Abcam, Cambridge, UK). The nonspecific binding of the primary antibody was washed out with a 1X TBST buffer (Sigma-Aldrich). Horseradish peroxidase-conjugated polyclonal goat anti-mouse IgG secondary antibody (catalog no. ab97023; Abcam) was used at a dilution of 1:10,000 (incubation, 1 h at room temperature). The washed membrane was developed with the diaminobenzidene/H2O2 substrate (Amresco LLC, Solon, OH, USA) according to the manufacturer's instructions to produce a brown-colored product, which was detected on the membranes. Experiments were repeated at least twice to confirm the results.
Results
Observation of osteosarcoma samples
Osteosarcoma is the eighth most common cancer among children (1). Accurate diagnosis of osteosarcoma metastasis is an important requirement for effective treatment of the disease prior to metastasis. To date, molecular markers for the diagnosis of osteosarcoma metastasis are not well established. Identification of osteosarcoma metastasis genes will facilitate the design of novel molecular markers for the early detection of osteosarcoma metastasis in its initial stage. Therefore, the present project was designed to identify osteosarcoma metastasis genes. Samples were collected from 31 patients and grouped into two categories: Osteosarcoma without metastasis and osteosarcoma with metastasis. The complete information regarding the patients and samples are presented in Table I. All biopsy tumor specimens were observed and confirmed by histological analysis by a pathologist and specialized scientist, by the observation of >90% tumor cells.
Identification of osteosarcoma metastasis genes
In order to identify the osteosarcoma metastasis genes, samples from osteosarcoma patients with and without metastasis were subjected to RNA extraction followed by cDNA subtraction using a DsDD cDNA subtraction kit. In the cDNA subtraction experiment, samples from patients with osteosarcoma without and with metastasis functioned as a driver cDNA library and tester cDNA library, respectively. The tester and driver cDNA libraries were critical for the identification of osteosarcoma metastasis genes using cDNA subtraction protocols. According to the manufacturer's instructions, the differentially expressed specific transcripts were amplified through polymerase chain reaction. Subsequently, the specific transcripts were cloned and sequenced. In total, 184 cDNA subtracted clones were obtained. The sequenced data were analyzed by BLAST of the subtracted cDNA sequences with the EST and Nucleotide databases of the National Center for Biotechnology Information. Data pertaining to 12 of the differentially expressed specific transcripts (osteosarcoma metastasis genes), selected based upon interest, expression pattern and literature survey for further analysis, are presented in Table II.
Validation of osteosarcoma metastasis genes
In order to validate the cDNA subtraction data and study the functional aspects of the identified osteosarcoma metastasis genes, immunoblot analysis was performed for the selected targets. Based upon the results of the literature survey, ezrin and β4 integrin were selected for the functional studies as effective osteosarcoma metastasis genes. The tissue samples from osteosarcoma patients without and with metastasis were subjected to protein sample preparation, followed by immunoblotting with anti-ezrin and anti-β4 integrin antibodies. Analogous experiments were performed on the control tissues. As shown in Fig. 1A, the immunoblotting results illustrated that enhanced ezrin and β4 integrin expression was detected in samples from osteosarcoma with metastasis patients, compared with the limited expression in samples of osteosarcoma without metastasis. Similar results were obtained for all 31 samples analyzed.
To further evaluate the expression pattern of ezrin and β4 integrin in the serum samples of patients with osteosarcoma with and without metastasis, as well as the controls, protein samples were prepared and immunoblotting was performed. The data are presented in Fig. 1B, which exhibits the limited expression of ezrin and β4 integrin detected in serum samples from osteosarcoma with metastasis patients. Limited expression of ezrin and β4 integrin was also detected in three osteosarcoma patients without metastasis (Patient IDs OS00152, OS00191 and OS00207). This was in comparison to the total lack of ezrin and β4 integrin expression in the serum samples of the remaining osteosarcoma patients without metastasis and the control samples.
Discussion
Osteosarcoma metastasis is one of the most dangerous diseases among the youth population (1). Various novel improvements have been established for the treatment of patients with osteosarcoma; however the diagnosis of metastasis has not been well studied (6,7). In order to detect osteosarcoma metastasis at an early stage to facilitate the effective treatment and management of the disease, the identification of genes underlying osteosarcoma metastasis is essential. In the present study, a novel technique, namely specific cDNA subtraction, was used for the identification of osteosarcoma metastasis genes. These studies emphasize the important contribution of the identification of osteosarcoma metastasis genes to the diagnosis of metastatic osteosarcoma at its initial stages.
Following cDNA subtraction, a total of 184 clones were identified, which were subsequently sequenced and analyzed using bioinformatics tools. Putative and partial sequences were also detected in the 184 clones. Among the presented 12 transcripts, ezrin and β4 integrin were selected for further analysis, based upon interest, expression pattern and literature survey. The data from the cDNA subtraction analysis demonstrated the expression of ezrin and β4 integrin in the samples from osteosarcoma patients with metastasis. In order to validate these results, immunoblot analysis was performed. The results confirmed that the two genes, ezrin and β4 integrin, were osteosarcoma metastasis genes.
To further verify these data, serum samples were prepared from patients with osteosarcoma with and without metastasis and controls and immunoblot analyses were performed. Ezrin and β4 integrin expression was detected in the samples of osteosarcoma with metastasis patients, confirming that these were osteosarcoma metastasis genes. By contrast, limited expression of ezrin and β4 integrin was also detected in certain osteosarcoma without metastasis patients. This may indicate that these patients are under risk of osteosarcoma metastasis at initial stage. Except in these three patients, no expression of ezrin and β4 integrin was observed in samples from osteosarcoma patients without metastasis. These results suggested that the presence of ezrin and β4 integrin may be utilized for the diagnosis of osteosarcoma metastasis in initial stages.
Ezrin is a member of the ezrin/radixin/moesin (ERM family), which shares the common membrane-binding N-terminal FERM domain with band-4.1 family members (21,22). Ezrin has a role in the metastatic spread of not only osteosarcoma, but also mammary and pancreatic adenocarcinomas (23–25). Notably, in the present study, the expression of ezrin in the serum of patients with osteosarcoma with metastasis and the limited expression in patients with osteosarcoma without metastasis highlighted the fact that ezrin may be employed as a marker for osteosarcoma metastasis. Further studies are essential for the development of ezrin as a novel marker for the detection of osteosarcoma metastasis at early stages.
Integrins consist of non-covalently associated α and β subunits. These heterodimers regulate key biological processes, including cell adhesion, signaling, migration, proliferation, survival, angiogenesis, oncogenesis and metastasis (26–30). To date, 18 α subunits and 8 β subunits have been identified. The subunit α6-β4 (known as β4 integrin) was originally identified as a tumor-associated antigen. It was also demonstrated that the expression and function of β4 integrin was implicated in the progression of various types of cancer (31–34). Of note, the results of the present study confirmed the presence of β4 integrin expression in patients with osteosarcoma with metastasis, which indicated that β4 integrin may be an osteosarcoma metastasis gene. In addition, limited expression of β4 integrin was observed in certain samples of patients with osteosarcoma without metastasis, which may indicate those patients under risk of osteosarcoma metastasis, highlighting the efficacy of this potential marker.
In conclusion, the results of the present study indicated that ezrin and β4 integrin were highly expressed in patients with osteosarcoma metastasis. Based upon the cDNA subtraction analysis followed by immunoblotting experiments, it was concluded that these two transcripts were osteosarcoma metastasis genes. Further studies are required to develop β4 integrin and/or ezrin as novel markers for the detection of osteosarcoma metastasis in the initial stages.
Acknowledgements
The present study was financially supported by Sichuan Province Applied Basic Research Project (no. 2013JY0152). The authors would like to thank the patients, whose support provided the backbone for the project. The authors also wish to thank the institutional review board approval committee and ethics committee for facilitating the successful completion of this project.
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