Abstract
Abstract
Osteosarcoma is the most common and highly aggressive bone neoplasm often occurs among adolescents and young people. In despite of the major advances in the treatment, the overall survival of osteosarcoma patients remains poor. Long non-coding RNAs (lncRNAs) have been identified as key regulators involved in tumorigenesis and progression of osteosarcoma. However, the exact roles and mechanisms of lncRNAs in osteosarcoma remain unclear. In this study, the functions and underlying molecular mechanisms of a novel lncRNA, ITGB2 antisense RNA1 (ITGB2-AS1) were investigated in osteosarcoma. The expression levels of ITGB2-AS1 were up-regulated in osteosarcoma tissue samples and cell lines determined by qRT-PCR assay. Osteosarcoma patients with high ITGB2-AS1 expression had a significantly poor prognosis. In addition, knockdown of ITGB2-AS1 inhibited the proliferation and induced apoptosis of osteosarcoma cells using MTT assay and flow cytometry detection. Furthermore, wound healing and transwell invasion assay revealed that depletion of ITGB2-AS1 suppressed the migration and invasion abilities of osteosarcoma cells. Molecular mechanism study indicated that ITGB2-AS1 inhibition impaired Wnt/β-catenin signalling pathway in osteosarcoma cells. Taken together, our study suggested that ITGB2-AS1 played critical roles in the development and progression of osteosarcoma via Wnt/β-catenin signalling, indicating that ITGB2-AS1 might be a valuable diagnostic biomarker and potential anticancer therapeutic target for osteosarcoma.
Introduction
Osteosarcoma is a rare tumour entity especially affecting children and young adults, accounting for approximately 20% of all primary bone malignancies [1,2]. Osteosarcoma is characterized by highly aggressive potency and rapidly metastasizes, and the 5-year survival rate of osteosarcoma patients with metastases remains below 30% [3]. Currently, surgery combined with chemotherapeutic treatment is the mainstay therapy strategy for primary osteosarcoma [4]. Although improvements have been made in treatment approaches for osteosarcoma in recent years, the prognosis remains unsatisfactory partly due to unknown of the underlying mechanisms of osteosarcoma [5,6]. Therefore, it is urgent to explore the underlying mechanisms involved in osteosarcoma development and progression which could facilitate to discover novel diagnosis molecular biomarkers and effective therapeutic targets for osteosarcoma patients to increase the survival rate.
Long non-coding RNAs (lncRNAs) are transcripts with longer than 200 nucleotides, while without protein-coding potential [7]. More and more evidences indicate that lncRNAs are involved in various cellular and biological processes, including proliferation, cell-cycle progression, and cell differentiation [8]. Abnormal expression of lncRNAs had been discovered in various cancer types such as prostate cancer, breast cancer and osteosarcoma [9]. For example, ARLNC1 knockdown suppressed prostate cancer growth in vitro and in vivo through regulating androgen receptor (AR) signalling [10]. Wang et al. [11] identified EPIC1 as an oncogenic lncRNA which interacted with myc and promoted cell-cycle progression in breast cancer. Moreover, lncRNA PANDA was discovered to positively regulate the proliferation of osteosarcoma cells by repressing the transcription of p18, a cyclin-dependent kinase inhibitor [12]. However, more lncRNAs involved in progression of osteosarcoma need to be further investigated to facilitate potential prognostic markers and therapeutic targets discovery.
LncRNA ITGB2-AS1 (ITGB2-AS1), located in chromosome 21q22.3, is a newly identified lncRNA. Up to date, only a study by Liu et al. [13] reported that ITGB2-AS1 expression was significantly up-regulated in breast cancer. However, the expression and biological function of ITGB2-AS1 in osteosarcoma have not been investigated. In the present study, the expression of ITGB2-AS1 was found to be up-regulated in both osteosarcoma tissues and cell lines, and the high ITGB2-AS1 expression levels predicted poor overall survival (OS) of osteosarcoma patients. Moreover, the effects of ITGB2-AS1 on cell proliferation, migration and invasion in osteosarcoma were investigated. We further demonstrated that ITGB2-AS1 served as a critical regulator in development and progression of osteosarcoma through modulating Wnt/β-catenin signalling pathway. Our present study reported, for the first time, that targeting ITGB2-AS1 may be a novel therapeutic strategy for osteosarcoma.
Materials and methods
Osteosarcoma tissue samples
Human osteosarcoma tumour tissues and matched normal bone tissues were obtained from 172 patients at the Department of Orthopedics, The Affiliated Huai’an No. 1 People’s Hospital of Nanjing Medical University. All the osteosarcoma tissues were confirmed pathologically from the samples obtained from surgery. None of the patients had received chemotherapy or radiation therapy prior to the surgery. All the collected tissues were immediately snap-frozen in liquid nitrogen and stored at –80 °C for further analysis. Clinical information of osteosarcoma patients was collected and summarized in Table 2. All the patients were given written informed consent and the study was approved the Research Ethics Committee of The Affiliated Huai’an No. 1 People’s Hospital of Nanjing Medical University.
Cell lines and cell culture
Human osteosarcoma cancer cell lines (MG63, U2OS, HOS and Saos-2) and osteoblast hFOB1.19 were all purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). The cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Life Technologies, Pudong, Shanghai, China) containing 10% fetal bovine serum (FBS, Life Technologies, Carlsbad, CA) and 100 U/ml penicillin (Meilune Technology, Dalian, Liaoning, China), and 100 µg/ml streptomycin (Meilune Technology, Dalian, Liaoning, China). All cells were maintained in a humidified incubator with 5% CO2 at 37 °C.
Construction of recombinant lentivirus and cell infection
The plasmids construction and lentivirus package were carried out through standard molecular cloning protocols and lentivirus package methods by GeneChem Co., Ltd. (Shanghai, China). All the sequences are provided in Table 1. For lentivirus infection, MG63 and U2OS cells were seeded onto 12-well plates at a density of 1 × 104 cells/well. After the cell confluence reaching to 60%, 100 μl lentivirus (virus titer, 109 TU/ml) of each group (shControl, lncRNA ITGB2-AS1-shRNA1 and lncRNA ITGB2-AS1-shRNA2) were added into each well. Medium were subsequently changed after 24 h and the cells were incubated for another 48 h. The cells were then observed under an inverted fluorescence microscope (Nikon, Tokyo, Japan). The DNA plasmids or microRNA mimics were transfected by using transfection reagent Lipofectamine 3000 (Invitrogen, Grand Island, NY) according to the manufacturer’s instructions.
Table 1. The primers for PCR.
Quantitative real-time PCR (qRT-PCR)
Total RNAs from osteosarcoma tissues and cell lines were isolated by TRizol Reagent (Invitrogen) following the manufacturer’s protocols. For qRT-PCR, the first-strand cDNA was reversely transcribed from 10 ng total RNA using a PrimeScript RT reagent kit (Takara, Shiga, Japan). RT-qPCR was performed using the Faststart Universal SYBR Green Master (Roche, Pudong, Shanghai, China). GAPDH was used as an endogenous control. The primer sequences are provided in Table 1. The relative expression fold change of mRNAs was calculated by the 2−ΔΔCT method.
Western blot analysis
All cells were washed twice with phosphate-buffered saline (PBS) solution and the cell lysates were harvested by RIPA buffer (Beyotime, Shanghai, China) with Complete Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations were assessed using bicinchoninic acid (BCA) assay kit (Beyotime). The proteins were separated by 10% SDS-PAGE and subsequently transferred to PVDF membrane (Millipore, Billerica, MA). The membranes were probed with primary antibodies: anti-caspase-9 antibody (#ab32539, Abcam, Cambridge, MA, USA), anti-caspase-3 antibody (#ab13585, Abcam), anti-E-cadherin antibody (#ab15148, Abcam), anti-vimentin antibody (#ab20346, Abcam), anti-N-cadherin antibody (#ab18203, Abcam), anti-β-catenin antibody (#51067–2-AP, Protein Tech, Pudong, Shanghai, China), anti-cyclin D1 antibody (#60186–1-Ig, Protein Tech), anti-c-myc antibody (#ab39688, Abcam) and anti-GAPDH antibody (#ab181602, Abcam). After being washed by three times using TBST, the membrane was further incubated with secondary antibodies for 1 h at room temperature and detected by enhanced chemiluminescent (ECL) kit (Beyotime).
Cell proliferation assay
Cell proliferation was determined by MTT assay (Beyotime). Briefly, after plating cells in 96-well plates (1 × 103 cells/well) and incubating overnight, 20 μl MTT (0.5 mg/ml) was then added into each well and incubated for 4 h at 37 °C. Then, the supernatant was discarded and added dimethyl sulfoxide (100 µl/well) into each well to dissolve the precipitated formazan. Finally, the absorbance was measured at 490 nm on a microplate reader (Thermo Scientific, Hudson, NH). All the experiments were repeated at least three times.
Colony formation assay
Cells were plated in six-well plates (1000 cells/well) and incubated in medium at 37 °C. Two weeks later, the cells were washed twice with PBS and fixed in methanol for 30 min. Then, the cells were stained with 1% crystal violet dye (Beyotime) and the colonies were counted after the excess crystal violet was washed out with PBS.
Wound□healing migration assay
An equal amount (70 μl) of osteosarcoma MG63 or U2OS cell suspensions (5 × 105 cells/ml) were added into each reservoir of the cell insert µ-dish (35 mm high culture-insert ibiTreat; ibidi, GmbH, Germany) and incubated overnight at 37 °C. Then, the culture inserts were removed and serum-free culture medium containing 0.2% bovine serum albumin (BSA) was added into the dishes. The gap between two cell layers was documented at 0 and 24 h with a microscope (Nikon, Tokyo, Japan). The results were analyzed by software Image-Pro Plus 6.0 (Media Cybernetics Inc., Bethesda, MD).
Transwell invasion assay
The invasion capabilities of MG63 and U2OS cells were determined using the Matrigel invasion assay (Millipore). Cells (200 µl; 2.5 × 105 cells/ml) were seeded in the upper chamber and the lower chamber was filled with 600 μl medium 10% FBS. After 16 h, cells on the upper surface were wiped using a cotton swab. The invasive cells attached to the lower surface were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet (Beyotime) for 10 min. Then, the cells were counted and taken photos after washing out the excess crystal violet.
Flow cytometry analysis of apoptosis
Apoptosis assay was conducted using an Annexin-VFITC apoptosis detection kit (Beyotime). Briefly, cells (1 × 106) were harvested and washed twice with ice-cold PBS. Then the cells were resuspended in 100 μl flow cytometry binding buffer and stained with Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) regents. The apoptosis cells were subsequently analyzed by FACSCalibur flow cytometer (BD Biosciences, CA).
Luciferase assay
TOP/FOP Flash luciferase reporter plasmid systems (Biovector, Beijing, China) were applied to determine the activities of the WNT signalling pathway. Osteosarcoma cells incubated in 24-well plates were co-transfected with the TOP/FOP Flash luciferase reporter plasmid and Renilla TK-luciferase vector (Promega, Madison, WI). Then, the Renilla luciferase activities of the osteosarcoma cells were detected by the dual luciferase reporter assay system (Promega).
Statistical analysis
All statistical analyses were performed using the SPSS for Windows statistical software, version 16.0 (SPSS, Chicago, IL) and GraphPad Prism 5.0 (Graphpad Software, La Jolla, CA). Data are presented as means ± SEM. Statistical comparisons between two groups were evaluated by one-way ANOVA or Student’s t-test. The OS was estimated using the Kaplan–Meier method. The Cox proportional hazards model for univariate and multivariate survival analysis was used to assess predictors related to survival. A value of p < .05 was considered statistically significant.
Results
LncRNA ITGB2-AS1 was up-regulated in osteosarcoma tissues and correlated with poor prognosis
To investigate the functions of lncRNA ITGB2-AS1 in osteosarcoma, the expression levels of ITGB2-AS1 were evaluated using qRT-PCR assay. The data showed that ITGB2-AS1 appeared to have higher expression levels in osteosarcoma tissues than in the non-tumour tissues (Figure 1(A)). Similarly, the expression levels of ITGB2-AS1 in osteosarcoma cell lines including Saos-2, U2OS, MG63 and HOS were also up-regulated compared with the osteoblast hFOB1.19 cells (Figure 1(B)). In addition, the association between the ITGB2-AS1 expression and clinicopathological parameters of patients with osteosarcoma was analyzed. We found that increased ITGB2-AS1 expression levels were significantly associated with osteosarcoma differentiation (p < .01), tumour-node-metastasis (TNM) stage (p = .004) and metastasis (p = .014) while other parameters such as age (p = .3) and tumour size (p = .135) in osteosarcoma patients was not significant (Table 2). Then, the results of Kaplan–Meier assay revealed that patients with high ITGB2-AS1 expression had a significantly poorer prognosis than those with low expression (Figure 1(C)). Finally, univariate and multivariate analysis of prognostic parameters suggested that differentiation (p = .006), TNM stage (p = .009), metastasis (p = .008) and ITGB2-AS1 expression levels (p = .006) were independent unfavourable prognostic factors in osteosarcoma patients (Table 3). Therefore, our data indicated that increased ITGB2-AS1 expression was associated with the progression and development of osteosarcoma.
Published online:
27 September 2018Figure 1. Expression levels of ITGB2-AS1 in osteosarcoma tissues and cell lines. (A) Relative expression of ITGB2-AS1 in osteosarcoma tissues and paired normal bone tissues (n = 172). (B) Relative expression of ITGB2-AS1 in osteoblast cells (hFOB 1.19) and osteosarcoma cell lines including Saos-2, U2OS, MG63 and HOS cells. (C) Low expression of ITGB2-AS1 exhibited better OS in osteosarcoma patients (n = 172; p = .0044). The values were means ± SEM; *p < .05, **p < .01.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/ianb20/2018/ianb20.v046.sup03/21691401.2018.1511576/20210106/images/medium/ianb_a_1511576_f0001_c.jpg"})
Figure 1. Expression levels of ITGB2-AS1 in osteosarcoma tissues and cell lines. (A) Relative expression of ITGB2-AS1 in osteosarcoma tissues and paired normal bone tissues (n = 172). (B) Relative expression of ITGB2-AS1 in osteoblast cells (hFOB 1.19) and osteosarcoma cell lines including Saos-2, U2OS, MG63 and HOS cells. (C) Low expression of ITGB2-AS1 exhibited better OS in osteosarcoma patients (n = 172; p = .0044). The values were means ± SEM; *p < .05, **p < .01.
Table 2. Correlation between ITGB2-AS1 expression and different clinicopathological features in patients with osteosarcoma.
Table 3. Univariate and multivariate analysis of prognostic parameters in osteosarcoma patients by cox regression analysis.
Knockdown of ITGB2-AS1 suppresses cell proliferation and promotes apoptosis of osteosarcoma cells
To examine the roles of ITGB2-AS1 in osteosarcoma progression, MG63 and U2OS cells were infected with shControl, ITGB2-AS1-shRNA1 or ITGB2-AS1-shRNA2 lentivirus, respectively. The lentivirus knockdown efficiency of ITGB2-AS1 was confirmed by qRT‐PCR and the data showed that lentivirus-mediated RNA interference obviously impaired the expression of ITGB2-AS1 in both the MG63 and U2OS osteosarcoma cells (Figure 2(A,B)). Then, MTT assay was further employed to assess the effects of ITGB2-AS1 on cell proliferation. As the data showed, the proliferation rates of MG63 and U2OS cells infected with ITGB2-AS1-shRNA lentivirus were obviously decreased compared with that of the control group (Figure 2(C,D)). Additionally, colony formation assay also demonstrated that knockdown the expression of ITGB2-AS1 reduced the cell colony number of both the MG63 and U2OS cells (Figure 2(E–G)). Besides, flow cytometry analysis was carried out to explore whether ITGB2-AS1 was involved in osteosarcoma cell apoptosis. The results revealed that knockdown of ITGB2-AS1 significantly increased the apoptotic rate of the osteosarcoma cells (Figure 2(H,I)). Moreover, western-blot assay confirmed that suppression of ITGB2-AS1 dramatically increased the expression of caspase 3 and caspase 9 in both MG63 and U2OS osteosarcoma cells (Figure 2(J,K)). Overall, our data indicated that ITGB2-AS1 facilitated proliferation and inhibited apoptosis in osteosarcoma.
Published online:
27 September 2018Figure 2. Suppression of ITGB2-AS1 inhibited osteosarcoma cell proliferation and induced apoptosis. (A–B) Relative expression levels of ITGB2-AS1 in MG63 and U2OS cells. (C–D) MTT assay determined the proliferation rates of MG63 and U2OS cells. (E–G) The colony formation assays and quantitative analysis of MG63 and U2OS cell colony number. (H–I) Flow cytometry analysis of MG63 and U2OS cell apoptosis. (J–K) Western-blot analysis of the apoptotic molecular in MG63 and U2OS cells. The values were means ± SEM; *p < .05, **p < .01.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/ianb20/2018/ianb20.v046.sup03/21691401.2018.1511576/20210106/images/medium/ianb_a_1511576_f0002_c.jpg"})
Figure 2. Suppression of ITGB2-AS1 inhibited osteosarcoma cell proliferation and induced apoptosis. (A–B) Relative expression levels of ITGB2-AS1 in MG63 and U2OS cells. (C–D) MTT assay determined the proliferation rates of MG63 and U2OS cells. (E–G) The colony formation assays and quantitative analysis of MG63 and U2OS cell colony number. (H–I) Flow cytometry analysis of MG63 and U2OS cell apoptosis. (J–K) Western-blot analysis of the apoptotic molecular in MG63 and U2OS cells. The values were means ± SEM; *p < .05, **p < .01.
Depression of ITGB2-AS1 inhibits migration and invasion of osteosarcoma cells
The effects of ITGB2-AS1 on the migration of MG63 and U2OS osteosarcoma cells were also evaluated using wound healing migration assays. The results validated that down-regulation of ITGB2-AS1 markedly suppressed the cell migratory capability of MG63 and U2OS osteosarcoma cells compared with the control group (Figure 3(A–D)). In addition, transwell invasion assays revealed that ITGB2-AS1 knockdown significantly inhibited the invasion of MG63 and U2OS osteosarcoma cells (Figure 3(E–G)). Besides, the underlying molecular mechanisms of ITGB2-AS1 affecting osteosarcoma cells migration and invasion abilities were further investigated through detecting the expression of epithelial-mesenchymal transition (EMT) related markers by western-blot assay. The data revealed that both N-cadherin and vimentin were down-regulated while the epithelial marker E-cadherin was upregulated in the ITGB2-AS1 silenced MG63 and U2OS osteosarcoma cells (Figure 3(H,I)). Therefore, our data suggested that ITGB2-AS1 played a critical role in osteosarcoma migration and invasion.
Published online:
27 September 2018Figure 3. Effects of ITGB2-AS1 knockdown on the migration and invasion of osteosarcoma. (A–B) Wound healing assay was used to measure the migration capabilities of MG-63 cells. (C–D) Wound healing assay was used to measure the migration capabilities of U2OS cells. (E–G) Transwell invasion assay was used to measure the migration capabilities of MG63 and U2OS cells. (H–I) Protein expression levels of critical EMT markers were detected in ITGB2-AS1 knockdown MG63 and U2OS cells by western blot analysis. The values were means ± SEM; *p < .05, **p < .01.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/ianb20/2018/ianb20.v046.sup03/21691401.2018.1511576/20210106/images/medium/ianb_a_1511576_f0003_c.jpg"})
Figure 3. Effects of ITGB2-AS1 knockdown on the migration and invasion of osteosarcoma. (A–B) Wound healing assay was used to measure the migration capabilities of MG-63 cells. (C–D) Wound healing assay was used to measure the migration capabilities of U2OS cells. (E–G) Transwell invasion assay was used to measure the migration capabilities of MG63 and U2OS cells. (H–I) Protein expression levels of critical EMT markers were detected in ITGB2-AS1 knockdown MG63 and U2OS cells by western blot analysis. The values were means ± SEM; *p < .05, **p < .01.
ITGB2-AS1 inhibition decreases Wnt/β-catenin signalling pathway in osteosarcoma cells
To investigate whether Wnt/β-catenin signalling pathway was involved in the modulation of the effects of ITGB2-AS1 on osteosarcoma development and progression, TOP/FOP flash reporter assays were conducted in MG63 cells. The data suggested that the relative luciferase activities of ITGB2-AS1 silence cells were dramatically decreased compared with the control group, which indicated that the activation of the Wnt/β-catenin signalling pathway was suppressed when impairing ITGB2-AS1 expression (Figure 4(A)). Additionally, qRT-PCR assay was further utilized to determine the effects of ITGB2-AS1 on β-catenin expression and several critical downstream genes involved in the Wnt/β-catenin signalling pathway such as cyclin D1 and c-myc in osteosarcoma. The data demonstrated that knockdown of ITGB2-AS1 in MG63 and U2OS cells significantly reduced the mRNA expression levels of β-catenin, cyclin D1 and c-myc (Figure 4(B,C)). Consistent with the qRT-PCR results, the protein expression levels of β-catenin, cyclin D1 and c-myc evaluated by western-blot assay were also decreased in ITGB2-AS1 silenced MG63 and U2OS cells (Figure 4(D,E)). Taken together, our results suggested that that ITGB2-AS1 played an essential role in regulation the activity of Wnt/β-catenin signalling pathway in osteosarcoma development and progression.
Published online:
27 September 2018Figure 4. The effects of ITGB2-AS1 on the activity of Wnt/β-catenin signaling in osteosarcoma. (A) Luciferase reporter assay using TOP flash vectors was applied to determine β-catenin transcription factor/lymphoid enhancer binding factor (TCF/LEF) promoter activity in MG63 and U2OS cells. (B-C) qRT-PCR showed that depression of ITGB2-AS1 inhibited the β-catenin, cyclin D1 and c-myc expression in MG63 and U2OS cells. (D–E) Western blot analysis. The values were means ± SEM; *p < .05, **p < .01.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/ianb20/2018/ianb20.v046.sup03/21691401.2018.1511576/20210106/images/medium/ianb_a_1511576_f0004_b.jpg"})
Figure 4. The effects of ITGB2-AS1 on the activity of Wnt/β-catenin signaling in osteosarcoma. (A) Luciferase reporter assay using TOP flash vectors was applied to determine β-catenin transcription factor/lymphoid enhancer binding factor (TCF/LEF) promoter activity in MG63 and U2OS cells. (B-C) qRT-PCR showed that depression of ITGB2-AS1 inhibited the β-catenin, cyclin D1 and c-myc expression in MG63 and U2OS cells. (D–E) Western blot analysis. The values were means ± SEM; *p < .05, **p < .01.
Discussion
Osteosarcoma has become the most common primary malignant bone neoplasm in children and adolescents worldwide [14]. Due to the high capability of invasion and metastasis, patients with osteosarcoma often have poor prognosis [15]. Therefore, to identify novel molecular targets and uncovering the underlying molecular mechanisms of osteosarcoma development and progression is facilitated to find effective therapeutic approaches. Up to date, more and more studies focused on the potential of lncRNAs as a treatment targeting. In the present study, we found that ITGB2-AS1 was highly expressed in osteosarcoma and its high expression was associated with the low OS of osteosarcoma patients. Moreover, we demonstrated that ITGB2-AS1 played a critical role in osteosarcoma cells proliferation, migration and invasion via modulating Wnt/β-catenin signalling pathway.
Recently, accumulating evidence has suggested that lncRNAs served as key regulators in various cellular processes of osteosarcoma [16]. For instance, lncRNA HULC was highly expressed in osteosarcoma and its knockdown suppressed cancer cells proliferation, migration, invasion, and inhibited apoptosis by modulating miR-122 [17]. LncRNA ATB was also found to be highly expressed in osteosarcoma and accelerate osteosarcoma cells proliferation, migration and invasion by suppressing miR-200s [18]. LncRNA NBAT1 was confirmed to be a tumour suppressor because its overexpression suppressed growth and metastasis of osteosarcoma cells [19]. Besides, previous study had revealed that ectopic expression of ITGB2-AS1 could promote the migration and invasion of breast cancer cells by enhancing the expression of ITGB2, indicating that ITGB2-AS1 served as a tumour promoter in breast cancer [13]. However, to our best knowledge, the expression, clinical significance and biological function of ITGB2-AS1 have not been investigated. In our study, the functions of ITGB2-AS1 were also explored in osteosarcoma. Cell proliferation and colony formation assays proved that knockdown of ITGB2-AS1 suppressed the osteosarcoma proliferative ability. Furthermore, flow cytometry apoptosis detection assay suggested that the suppression of ITGB2-AS1 induced osteosarcoma cells apoptosis by increasing the expression of caspase 3 and caspase 9. In addition, depletion of ITGB2-AS1 inhibited the migration and invasion of osteosarcoma cells through regulating EMT related markers including N-cadherin and vimentin. Overall, our data indicated that ITGB2-AS1 excreted crucial roles in modulating the development and progression of osteosarcoma.
The Wnt signalling pathway plays essential roles in embryo development and adult tissue homeostasis [20]. Emerging evidence has revealed that tumorigenesis and tumour metastasis was highly associated with aberrant activation of the Wnt signalling pathway [21]. Particularly, previous studies demonstrated that dysregulation of lncRNAs led to constitutively active Wnt signalling in cancer growth and metastasis process [22]. For instance, lncRNA ZEB1-AS1 promoted colorectal cancer cell proliferation by regulating Wnt/β-catenin signalling [23]. Additionally, reports showed that lncRNA LSINCT5 interacted with NCYM, which activated Wnt/β-catenin signalling, to promote bladder cancer progression [24]. Besides, lncRNA H19 promoted epithelial-mesenchymal transition (EMT) of colorectal cancer cells through acting on Wnt signalling pathway [25]. In this study, we also evaluated the effects of ITGB2-AS1 on Wnt/β-catenin signalling pathway in osteosarcoma and found that ITGB2-AS1 inhibition impaired Wnt/β-catenin signalling suggestion of an activation effect ITGB2-AS1 on Wnt/β-catenin signalling in osteosarcoma.
In conclusion, we discovered that ITGB2-AS1 was overexpressed in osteosarcoma and associated with prognosis of osteosarcoma patients. Furthermore, we further provided evidence that ITGB2-AS1 functioned as an oncogenic lncRNA through modulating Wnt/β-catenin signalling pathway, which may provide valuable diagnostic and therapeutic strategies for osteosarcoma.
| Primer name | Primer sequence |
|---|---|
| ITGB2-AS1 Fwd | 5′-AGGAGATGGAACGAGGAAA-3′ |
| ITGB2-AS1 Rev | 5′-TTAGTGGTCTGCGAAGGTG-3′ |
| β-catenin Fwd | 5′-CATTACAACTCTCCACAACC-3′ |
| β-catenin Rev | 5′-CAGATAGCACCTTCAGCAC-3′ |
| Cyclin D1 Fwd | 5′-GCCACATACGGATGAGGACC-3′ |
| Cyclin D1 Rev | 5′-CACTTGACGAAGCAGCACCA-3′ |
| c-myc Fwd | 5′-GATTCTCTGCTCTCCTCGAC-3′ |
| c-myc Rev | 5′-TCCAGACTCTGACCTTTTGC-3′ |
| GAPDH Fwd | 5′-GTCAACGGATTTGGTCTGTATT-3′ |
| GAPDH Rev | 5′-AGTCTTCTGGGTGGCAGTGAT-3′ |
| Clinicopathological features | Number | ITGB2-AS1 expression | p Value | |
|---|---|---|---|---|
| High | Low | |||
| Age (years) | ||||
| <20 | 75 | 40 | 35 | .300 |
| ≥20 | 97 | 44 | 53 | |
| Gender | ||||
| Male | 116 | 61 | 55 | .195 |
| Female | 56 | 23 | 33 | |
| Tumour size (cm) | ||||
| <5 | 94 | 46 | 58 | .135 |
| ≥5 | 68 | 38 | 30 | |
| Differentiation | ||||
| Well + moderate | 96 | 36 | 60 | .000 |
| Poor | 76 | 48 | 28 | |
| TNM stage | ||||
| I + II | 101 | 40 | 61 | .004 |
| III + IV | 61 | 44 | 27 | |
| Metastasis | ||||
| No | 112 | 47 | 65 | .014 |
| Yes | 60 | 37 | 23 | |
| Variable | Univariate analysis | Multivariate analysis | ||||
|---|---|---|---|---|---|---|
| RR | 95% CI | p Value | RR | 95% CI | p Value | |
| Age | 1.432 | 0.678–2.421 | .355 | – | – | – |
| Gender | 1.689 | 0.832–2.667 | .227 | – | – | – |
| Tumour size | 1.537 | 0.933–2.231 | .166 | – | – | – |
| Differentiation | 3.345 | 1.563–5.564 | .001 | 2.783 | 1.326–4.789 | .006 |
| TNM stage | 2.893 | 1.347–4.336 | .005 | 2.428 | 1.137–4.023 | .009 |
| Metastasis | 3.554 | 1.632–5.321 | .003 | 2.678 | 1.237–4.332 | .008 |
| ITGB2-AS1 expression | 3.345 | 1.465–5.457 | .001 | 3.035 | 1.237–4.667 | .006 |
Acknowledgements
We thank all donors for their donations. We thank Prof. Lin Zhang and Dr Ming Wang from Department of molecular biology teaching and research, Nanjing Medical University for their technical support. We thank Ph. D Yun Long from Department of Pathology, The Affiliated Huai’an No. 1 People’s Hospital of Nanjing Medical University, for their technical support. We thank the Eastern Hepatobiliary Surgical Hospital and Institute, The Second Military University, Shanghai, for their generous help.
Disclosure statement
No potential conflict of interest was reported by the authors.
References
- Luetke A, Meyers PA, Lewis I, et al. Osteosarcoma treatment – where do we stand? A state of the art review. Cancer Treat Rev. 2014;40:523–532. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Isakoff MS, Bielack SS, Meltzer P, et al. Osteosarcoma: current treatment and a collaborative pathway to success. JCO. 2015;33:3029–3035. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Mirabello L, Troisi RJ, Savage SA. Osteosarcoma incidence and survival rates from 1973 to 2004: data from the surveillance, epidemiology, and end results program. Cancer. 2009;115:1531–1543. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Kansara M, Teng MW, Smyth MJ, et al. Translational biology of osteosarcoma. Nat Rev Cancer. 2014;14:722–735. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Anderson ME. Update on survival in osteosarcoma. Orthop Clin North Am. 2016;47:283–292. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Kopp F, Mendell JT. Functional classification and experimental dissection of long noncoding RNAs. Cell. 2018;172:393–407. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Kazemi T, Younesi V, Jadidi-Niaragh F, et al. Immunotherapeutic approaches for cancer therapy: an updated review. Artif Cells Nanomed Biotechnol. 2015;44:1–779. [Taylor & Francis Online], [Web of Science ®], [Google Scholar]
- Bhan A, Soleimani M, Mandal SS. Long noncoding RNA and cancer: a new paradigm. Cancer Res. 2017;77:3965–3981. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Huarte M. The emerging role of lncRNAs in cancer. Nat Med. 2015;21:1253–1261. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Zhang Y, Pitchiaya S, Cieślik M, et al. Analysis of the androgen receptor-regulated lncRNA landscape identifies a role for ARLNC1 in prostate cancer progression. Nat Genet. 2018;50:814–824. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Wang Z, Yang B, Zhang M, et al. lncRNA epigenetic landscape analysis identifies EPIC1 as an oncogenic lncRNA that Interacts with MYC and promotes cell-cycle progression in cancer. Cancer Cell. 2018;33:706–720. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Kotake Y, Goto T, Naemura M, et al. Long noncoding RNA PANDA positively regulates proliferation of osteosarcoma cells. Anticancer Res. 2017;37:81–85. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Liu M, Gou L, Xia J, et al. LncRNA ITGB2-AS1 could promote the migration and invasion of breast cancer cells through up-regulating ITGB2. Int J Mol Sci. 2018;19:1866. [Crossref], [Web of Science ®], [Google Scholar]
- Botter SM, Neri D, Fuchs B. Recent advances in osteosarcoma. Curr Opin Pharmacol. 2014;16:15–23. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Ferrari S, Serra M. An update on chemotherapy for osteosarcoma. Expert Opin Pharmacother. 2015;16:2727–2736. [Taylor & Francis Online], [Web of Science ®], [Google Scholar]
- Li Z, Yu X, Shen J. Long non-coding RNAs: emerging players in osteosarcoma. Tumour Biol. 2016;37:2811–2816. [Crossref], [PubMed], [Google Scholar]
- Kong D, Wang Y. Knockdown of lncRNA HULC inhibits proliferation, migration, invasion, and promotes apoptosis by sponging miR-122 in osteosarcoma. J Cell Biochem. 2018;119:1050–1061. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Han F, Wang C, Wang Y, et al. Long noncoding RNA ATB promotes osteosarcoma cell proliferation, migration and invasion by suppressing miR-200s. Am J Cancer Res. 2017;7:770–783. [PubMed], [Web of Science ®], [Google Scholar]
- Yang C, Wang G, Yang J, et al. Long noncoding RNA NBAT1 negatively modulates growth and metastasis of osteosarcoma cells through suppression of miR-21. Am J Cancer Res. 2017;7:2009–2019. [PubMed], [Web of Science ®], [Google Scholar]
- Logan CY, Nusse R. The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol. 2004;20:781–810. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Zhan T, Rindtorff N, Boutros M. Wnt signaling in cancer. Oncogene. 2017;36:1461–1473. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Huo X, Han S, Wu G, et al. Dysregulated long noncoding RNAs (lncRNAs) in hepatocellular carcinoma: Implications for tumorigenesis, disease progression, and liver cancer stem cells. Mol Cancer. 2017;16:165. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Lv SY, Shan TD, Pan XT, et al. The lncRNA ZEB1-AS1 sponges miR-181a-5p to promote colorectal cancer cell proliferation by regulating Wnt/beta-catenin signaling. Cell Cycle. 2018;1–10. [Web of Science ®], [Google Scholar]
- Zhu X, Li Y, Zhao S, et al. LSINCT5 activates Wnt/β-catenin signaling by interacting with NCYM to promote bladder cancer progression . Biochem Biophys Res Commun. 2018;502:299–306. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Ding D, Li C, Zhao T, et al. LncRNA H19/miR-29b-3p/PGRN axis promoted epithelial-mesenchymal transition of colorectal cancer cells by acting on Wnt signaling. Mol Cells. 2018;41:423–435. [PubMed], [Web of Science ®], [Google Scholar]