Anticancer effects of melatonin via regulating lncRNA JPX‐Wnt/β‐catenin signalling pathway in human osteosarcoma cells

Abstract Osteosarcoma (OS) is a type of malignant primary bone cancer, which is highly aggressive and occurs more commonly in children and adolescents. Thus, novel potential drugs and therapeutic methods are urgently needed. In the present study, we aimed to elucidate the effects and mechanism of melatonin on OS cells to provide a potential treatment strategy for OS. The cell survival rate, cell viability, proliferation, migration, invasion and metastasis were examined by trypan blue assay, MTT, colony formation, wound healing, transwell invasion and attachment/detachment assay, respectively. The expression of relevant lncRNAs in OS cells was determined by real‐time qPCR analysis. The functional roles of lncRNA JPX in OS cells were further examined by gain and loss of function assays. The protein expression was measured by western blot assay. Melatonin inhibited the cell viability, proliferation, migration, invasion and metastasis of OS cells (Saos‐2, MG63 and U2OS) in a dose‐dependent manner. Melatonin treatment significantly downregulated the expression of lncRNA JPX in Saos‐2, MG63 and U2OS cells. Overexpression of lncRNA JPX into OS cell lines elevated the cell viability and proliferation, which was accompanied by the increased metastasis. We also found that melatonin inhibited the OS progression by suppressing the expression of lncRNA JPX via regulating the Wnt/β‐catenin pathway. Our results suggested that melatonin inhibited the biological functions of OS cells by repressing the expression of lncRNA JPX through regulating the Wnt/β‐catenin signalling pathway, which indicated that melatonin might be applied as a potentially useful and effective natural agent in the treatment of OS.


| INTRODUC TI ON
Osteosarcoma (OS), characterized by a higher prevalence in adolescents and children, has been regarded as the most common bone malignancy with a high fatality rate. 1,2 As a highly malignant bone tumour, OS originates from bone marrow mesenchymal stem cells (BMSCs), characterized by the presence of malignant mesenchymal spindle cells and production of malignant osteoid or immature bone. 3,4 Unfortunately, OS usually occurs in the extremities and easily metastasizes to lung and predominately affects the rapid growth of bones in children and adolescents. 5 The five-year survival rate of patients with OS is ~60%-70% and is as low as 20%-28% in patients with metastases. 6,7 Currently, with the development of various treatment methods for OS, including surgery, radiotherapy and chemotherapy, the rate of metastasis is still about 40%, and the clinical outcome has not been markedly improved. 8 Thus, a variety of novel agents need to be developed and applied for the treatment of OS as adjuvant therapeutic strategies.
Melatonin (N-acetyl-5-methoxytryptamine) is a type of pineal indolamine and naturally occurring derivative of the amino acid tryptophan with diverse biological activities, which is commonly observed in the nature and occurs in unicellular organisms, fungi, plants and animals. [9][10][11] Melatonin is initially extracted and identified in the bovine pineal tissues. And now, a vast number of studies indicate that this indolamine could be synthesized in the brain, retina, gastrointestinal tract, thymus and skin, and is mainly produced by the pineal gland at night. 12 Melatonin exerts numerous biological functions, such as sleep induction, biological rhythms modulation, anti-apoptotic signalling function, vaso-regulation, anti-tumour action, antioxidant properties, cytoprotective effects and immunomodulation. 13,14 According to the extensive involvement of melatonin in various fundamental biological functions, it is anticipated that melatonin possesses beneficial effects in a variety of disorders and diseases, including insomnia, Alzheimer's disease, osteoporosis, Parkinson's disease, fatty liver disease, Huntington's disease, Amyotrophic lateral sclerosis, migraine and headache manifestations and gastrointestinal diseases. [15][16][17][18][19][20] As an autocoid, a chronobiotic, a sleep-inducing agent, an immune modulator and a biological adjusting agent, melatonin is also regarded as an anti-tumour agent, which has significant beneficial roles in the prevention and treatment of diverse cancer types. 21,22 A series of novel studies clarified the roles of melatonin in multiple cancers due to its anti-metastatic potential, drug sensitivity restoration, apoptosis induction, growth inhibition and anti-angiogenic and anti-invasive actions. 23,24 However, the functions of melatonin in OS have not been fully clarified and an accurate mechanism of how melatonin properly orchestrates such functions needs to be further investigated.
Long non-coding RNAs (lncRNAs) are defined as an important kind of the non-coding RNA family, which is more than 200 nucleotides (nt) in length. [25][26][27] An increasing number of studies have revealed that lncRNAs participate in various biological processes during life, including transcriptional modification, modulation of chromatin architecture, cellular growth, differentiation, development, RNA processing and cell cycle control. [28][29][30][31] The relationship between lncRNAs and cancers has been recently revealed. LncRNAs are aberrantly expressed in a variety of cancers, and they have displayed great potential as powerful tumour markers. 32 Human OS cell lines were cultured in high glucose DMEM (Hyclone, USA) supplemented with 10% FBS and 1% penicillin-streptomycin.
All the cells were cultured in a 37℃, 5% CO 2 humidified incubator (Thermo, USA). When the density reached 60% confluence, the drug treatment and transfection were carried out.

| Lentiviral transduction
LncRNA JPX-overexpressing lentivirus, lncRNA JPX-specific shRNA (shRNA JPX) and scramble negative control (NC) were purchased from GenePharma Co., Ltd. (Shanghai, China). The cells were transfected with lncRNA JPX and shRNA JPX using polybrene (Cyagen Biosciences, USA) according to the manufacturer's instructions. After 48 h, the cells were utilized for additional analysis. The expression level of lncRNA JPX was then confirmed by real-time qPCR analysis.

| Trypan blue assay
In brief, OS cells were seeded into the six-well plates at a density of

| MTT assay
Cell viability was examined using MTT assay. The cells were seeded in a 96-well (NEST, China) at a density of 5 × 10 4 /ml. Then, the cells were exposed to melatonin or/and lncRNA JPX for 48 h. After 48 h, the medium was discarded and the cells were maintained in 0.5 mg/ ml MTT solution at 37℃ temperature for 4 h. Then, the medium was replaced by 150 µl DMSO solution. The optical density (OD) values at a wavelength of 490 nm were detected by using a microplate reader (TECAN, Switzerland).

| Colony formation assay
Cell proliferation was detected using colony formation assay. The cells at a density of 5 × 10 2 /ml were plated in six-well plates (NEST, China) and allowed to grow. The medium was changed every three days. After a 14-day incubation, the colonies were fixed with the methanol for 15 min and observed by staining with 1% crystal violet (Biosharp, China) for 30 min at room temperature and washed again.
At last, the colonies were observed and counted under a microscope (Olympus, Japan).

| Wound healing assay
The cell migration was assessed by wound healing assay. The cells were seeded into six-well plates at a density of 1 × 10 6 /well. The sterile pipette tips were applied to scratch cell layers. After washing with PBS three times to remove the floating cells, the cells were photographed and the wound closing process was observed using an inverted light microscope (Olympus, Japan) at 0 and 48 h, respectively. The images were captured and used for the measurement of the wound width.

| Transwell assay
Cell invasion ability was monitored using transwell assay. 24-well transwell chambers (Corning, USA) and 8µm-sized pore membranes coated with matrigel were used to perform invasion assay. A total of  For cell detachment assay, the cells were culture in 24-well plates at a density of 5 × 10 4 cells/well. After 24 h, 0.05% trypsin was added into the cells for 3 min to detach the cells. Then, the high glucose DMEM was applied to inactivate the trypsin and the detached cells were collected. The remaining cells were treated with 0.25% trypsin and the cells were counted. The percentage of the detached cells to total cells was obtained.

| Analysis of expression by real-time qPCR analysis
The harvested cells were subjected to total RNA isolation by using TRIzol (Invitrogen, USA) according to the instructions. The quality and purity of RNAs were examined using NanoDrop machine (Thermo, USA). The preparation of the cDNA was conducted following the instructions described in the cDNA synthesis kit manual (Applied Biosystems, USA). The cDNAs were amplified by PCR analysis by using SYBR reagents (Roche, Switzerland). And fold changes were analysed by relative quantification (2 −ΔΔCt ) method. The sequences of primers used for real time qPCR analysis were shown in Table 1.

| Protein extraction and western blot
Quantitative analysis on the protein expression level was conducted by western blot analysis according to the previous study. 40

| Statistical analysis
All the experiments were performed at least three times independently. Data were presented as mean ± SD. Statistical analysis was performed using GraphPad Prism software (Graphpad, USA).
Statistical significance was assessed by the Student's t-test or oneway multivariate analysis of variance (ANOVA). The data conformed to the normal distribution. p < 0.05 were considered significant difference.  Figure 1G-I, the results of colony formation assays showed that the colony number was progressively decreased after melatonin treatment, which declared that the proliferation ability was obviously suppressed in the presence of melatonin. These results confirmed the suppressive effects of melatonin on the cell growth and proliferation in OS cells.

| Melatonin induces the reduced metastasis in OS cells
To investigate whether melatonin affected the invasive and metastatic ability of OS cells, three OS cell lines were treated with different concentrations of melatonin (0.1, 0.5, 1, 1.5 and 2 mM) for 48 h, and the wound healing assay, attachment/detachment assays and transwell assay were applied. As presented in Figure 2A-C, the results from the wound healing assay showed that, compared to the control group, the dose-dependent decrease in migration was observed in Saos-2, MG63 and U2OS cells treated with melatonin.
Melatonin at 1, 1.5 and 2 mM exerted the most optimal effects on the migration and disrupted the migration ability of these three cell lines (

| LncRNA JPX is upregulated in OS cells and suppressed by melatonin treatment
It has been widely reported that lncRNAs participate in the development of many cancer types. [41][42][43][44] In order to determine whether The results of the wound healing assay showed that the percentage of migrated cells in lncRNA JPX group was higher than that in lncRNA NC group, indicating that upregulation of lncRNA JPX favoured the migration of OS cells ( Figure 4D). We next tested whether lncRNA JPX was also able to facilitate the cell invasion of OS cells. As expected, the percentage of invasive cells was significantly increased in lncRNA JPX overexpressed cells, which suggested that the increase of lncRNA JPX promoted the invasion of OS cells ( Figure 4E). Besides, in Saos-2, MG63 and U2OS cells, overexpression of lncRNA JPX greatly increased the metastasis of these OS cells compared with that in lncRNA NC group ( Figure 4F). Collectively, these results indicated that lncRNA JPX played a positive role in the progression of OS cells.

| Knockdown of lncRNA JPX negatively regulates the cell growth and metastasis of OS cells
To  Figure 5C). Besides, scratch assay displayed that the migration of OS cells was significantly inhibited after lncRNA JPX knockdown ( Figure 5D). Furthermore, the decreased invasion ability of OS cells was also observed after knockdown of lncRNA JPX ( Figure 5E).
Furthermore, the knockdown of lncRNA JPX markedly reduced the metastasis of OS cells in comparison with the cells from shRNA NC group ( Figure 5F). In short, the above data revealed that the low expression of lncRNA JPX effectively suppressed the cell growth and metastasis of OS cells.

| Melatonin regulates the tumour growth and metastasis of OS cells by inhibiting the expression of lncRNA JPX
To further determine whether melatonin exerted its function through downregulation of lncRNA JPX, OS cells were treated with 1.5 mM melatonin or 1.5 mM melatonin+lncRNA JPX, respectively.
As presented in Figure 6A, the functions of melatonin on the cell viability of OS cells could be reversed when the cells were cotransfected with lncRNA JPX. Additionally, the results from colony formation assay showed that the inhibitory roles of melatonin in the proliferation of OS cells could be blocked by overexpression of lncRNA JPX ( Figure 6B). As revealed in Figure 6C Figure 7B). Using colony formation assay, we found that lncRNA JPX accelerated the proliferation of MG63 cells, but exhibited no influence on the proliferation ability in MG63 cells after Wiki4 treatment ( Figure 7C). Accordingly, transwell assay showed that lncRNA JPX promoted the invasion of MG63 cells, but the action was abolished by Wiki4 treatment ( Figure 7D). Furthermore, the wound healing assay also confirmed Compared with the cells from the control group, melatonin markedly decreased the number of colonies, and overexpression of lncRNA JPX elevated the reduced number of colonies, which was caused by melatonin ( Figure 7F). And this action was abolished in the presence of Wiki4 ( Figure 7F). Besides, transwell assay suggested that overexpression of lncRNA JPX could increase the decreased number of invasive cells induced by melatonin treatment ( Figure 7G). However, the treatment of Wiki4 could weaken this effect ( Figure 7G). In addition, in Wiki4-treated cells, overexpression of lncRNA JPX exerted no effects on the migration of MG63 cells, which was pretreated with melatonin ( Figure 7H). Taken together, melatonin exerted its function by suppressing the expression of lncRNA JPX via regulating the Wnt/β-catenin pathway in OS cells.

| DISCUSS ION
OS is a type of malignant tumour that begins in the BMSCs that form bones. Long bone is the most common site, like legs and arms.
However, it could also start in any bones. Teenagers and young adults are the most affected population, but it could also affect younger children and older adults. The global incidence is about 1-3/ million population. 50 Surgery is usually performed to remove the tumour and metastasis, while chemotherapy is applied simultaneously. have not yet been ascertained. In this study, we found that mela- The migratory ability of MG63 cells treated with lncRNA JPX or lncRNA JPX + Wiki4 was detected by wound healing assay (F-H) The roles of Wnt/β-catenin signalling in the inhibition of lncRNA JPX by melatonin. Scale bar = 250 μm. Significant difference relative to control group was presented as *p < 0.05; **p < 0.01 and ***p < 0.001

| CON CLUS ION
In this study, melatonin was proved to inhibit the proliferation, invasion and migration in a dose-dependent manner of OS cells. LncRNA JPX-Wnt/β-catenin axis was confirmed to be the downstream pathway of melatonin inhibiting the biological behaviours of OS cells. Taken together, these data concluded that melatonin inhibited the biological functions of OS cells via regulating lncRNA JPX-Wnt/β-catenin signalling pathway. This study clarified the new molecular mechanism of OS and provided a novel treatment target in the following clinical investigation.

ACK N OWLED G M ENTS
The authors are grateful to all staff who contributed to this study.

CO N FLI C T O F I NTE R E S T
No potential conflicts of interest were disclosed.