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.

Keywords: LncRNAs; Wnt/β-catenin pathway; melatonin; osteosarcoma; therapeutic methods.

FIGURE 1

The effects of melatonin on the cell growth and proliferation of OS cells. (A–C) The roles of melatonin in the cell survival of Saos‐2 (A), MG63 (B) and U2OS (C) was measured by trypan blue assay. (D–F) The cell viability in Saos‐2 (D), MG63 (E) and U2OS (F) treated with different concentrations of melatonin was determined by MTT assay, respectively. (G–I) The cell proliferation ability was measured by colony formation assay in Saos‐2 (G), MG63 (H) and U2OS (I) in the presence of melatonin. Scale bar, 175 μm. Significant difference relative to control group was presented as *p < 0.05; **p < 0.01 and ***p < 0.001

FIGURE 2

The influence of melatonin on the migration and invasion of OS cells. (A–C) The effects of melatonin on the migration ability of OS cells were detected by wound healing assay. (D–F) The functions of melatonin in the metastasis of OS cells were determined by attachment assay and detachment assay. (G‐I) The roles of melatonin in the cell invasion were assessed by Transwell assay. Scale bar = 200 μm. Significant difference relative to control group was presented as *p < 0.05; **p < 0.01 and ***p < 0.001

FIGURE 3

Expression level of lncRNA JPX in OS cell lines. (A‐C) The expression level of lncRNA LUADT1, ZDHHC8P1, JPX, LINP1, AGAP2‐AS1 was measured by real‐time qPCR analysis in Saos‐2 (A), MG63 (B) and U2OS (C) cells after treatment with 1.5 mM melatonin. (D) Expression level of lncRNA JPX in OS cell lines (Saos‐2, MG63 and U2OS) and human osteoblasts. Significant difference relative to control group was presented as *p < 0.05; **p < 0.01 and ***p < 0.001

FIGURE 4

LncRNA JPX enhanced the proliferation and metastasis of OS cells. (A) The expression level of lncRNA JPX was examined in OS cells transfected with lncRNA JPX and lncRNA NC. (B) The cell viability of OS cells transfected with lncRNA JPX was evaluated by MTT assay. (C) The proliferative ability of OS cells treated with lncRNA NC and lncRNA JPX was examined by using colony formation assay. (D) The cell migration elevated by lncRNA JPX was detected with wound healing assay. (E) Transwell assay was conducted to detect the cell invasion ability of OS cells after transfected with lncRNA JPX. (F) Attachment assay and detachment assays were applied to analyse the effects of lncRNA JPX on the metastasis of OS cells. Scale bar, 250 μm. Significant difference relative to control group was presented as *p < 0.05; **p < 0.01 and ***p < 0.001

FIGURE 5

Knockdown of lncRNA JPX suppressed the proliferation and metastasis of OS cells. (A) LncRNA JPX was knockdown in OS cells by specific shRNA (shRNA JPX). The transfection efficiency was examined with real‐time qPCR analysis. (B) MTT assay was carried out to determine the cell viability in OS cells transfected with shRNA NC or shRNA JPX. (C) Colony formation assay was performed to test the proliferative ability of OS cells after knockdown of lncRNA JPX. (D) Wound healing assay was then utilized to assess the migration of OS cells after shRNA JPX transfection. (E) Invasion ability was examined in OS cells in shRNA JPX group by using transwell assay. (F) Attachment and detachment assays were conducted to detect the metastasis ability of OS cells transfected with shRNA JPX for 48 h. Scale bar, 250 μm. Significant difference relative to control group was presented as *p < 0.05; **p < 0.01 and ***p < 0.001

FIGURE 6

Melatonin inhibited the biological functions of OS cells by suppressing lncRNA JPX. (A) The reduced cell viability caused by melatonin treatment was reversed by overexpression of lncRNA JPX. (B) The decreased cell proliferation induced by melatonin was blocked by lncRNA JPX transfection. (C) Melatonin‐induced decrease in the invasion ability of OS cells was turned over in the presence of lncRNA JPX. (D) The attenuated migration of OS cells, which was caused by 1.5 mM melatonin, was partially reversed after lncRNA JPX transfection. (E and F) The attachment assay and detachment assays of OS cells in the presence of melatonin and lncRNA JPX. Scale bar = 250 μm. Significant difference relative to control group was presented as *p < 0.05; **p < 0.01 and ***p < 0.001

FIGURE 7

Wnt/β‐catenin signalling pathway was involved in lncRNA JPX mediated tumour progression in OS cells. (A) The protein levels of Wnt/β‐catenin signalling target genes were measured by western blot in MG63 cells transfected with lncRNA NC or lncRNA JPX. (B) The protein expression of the core factors of Wnt/β‐catenin signalling pathway was examined by western blot assay in MG63 cells treated with Wiki4. (C) The proliferative ability of MG63 cells treated with lncRNA JPX or lncRNA JPX + Wiki4 was examined with colony formation assay. (D) The invasion increased by lncRNA JPX was decreased in the presence of Wiki4. (E) 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