MicroRNA-744-5p suppresses tumorigenesis and metastasis of osteosarcoma through the p38 mitogen-activated protein kinases pathway by targeting transforming growth factor-beta 1

Abstract

Osteosarcoma (OS) is the most common malignant bone tumor in children and adolescents. Accumulating evidence has revealed that microRNAs (miRNAs) play a crucial role in the progression of OS. In this study, we found that miR-744-5p was the least expressed miRNA in patients with OS by analyzing GSE65071 from the GENE EXPRESSION OMNIBUS (GEO) database. Through real-time quantitative PCR (qRT-PCR), western blotting, colony formation assay, 5-Ethynyl-2-Deoxyuridine (EdU) incorporation assay, transwell migration, and invasion assays, we demonstrated its ability to inhibit the proliferation, migration, and invasion of OS cells in vitro. According to the luciferase reporter assay, transforming growth factor-β1 (TGFB1) was negatively regulated by miR-744-5p and reversed the effects of miR-744-5p on OS. Subcutaneous tumor-forming animal models and tail vein injection lung metastatic models were used in animal experiments, and it was found that miR-744-5p negatively regulated tumor growth and metastasis in vivo. Furthermore, rescue assays verified that miR-744-5p regulates TGFB1 expression in OS. Further experiments revealed that the p38 MAPK signaling pathway is involved in the miR-744-5p/TGFB1 axis. Generally, this study suggests that miR-744-5p is a negative regulator of TGFB1 and suppresses OS progression and metastasis via the p38 MAPK signaling pathway.

Keywords: Osteosarcoma; TGFB1; miR-744-5p; p38 MAPK signaling pathway.

Graphical abstract

Figure 1.

miR-744-5p is downregulated in osteosarcoma cell lines and clinical tissues. (a) Volcano plot demonstrated the expressive diversity of miRNAs between OS and normal tissues from GSE65071. (b) The cluster heat map showed the upregulated and downregulated miRNAs in GSE65071. (c) The top 10 downregulated miRNAs are listed. (d) The relative expression of miR-744-5p was remarkably suppressed in OS cell lines. (e) Expression of miR-744-5p was significantly downregulated in OS clinical tissues than in para-carcinoma tissues. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2.

miR-744-5p is closely related to the clinical features of patients with OS. (a) Lower expression of miR-744-5p was found in the middle and advanced stage of OS compared to the early stage. (b) Lower expression of miR-744-5p was related to larger tumors. (c) Lower expression of miR-744-5p was found in more patients with metastasis. (d) Log-Rank test demonstrated that patients with higher miR-744-5p expression had a better prognosis. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3.

Overexpression of miR-744-5p suppressed OS cells EMT, migration and invasion in vitro. (a) miR-744-5p mimics were successfully transfected into 143B and MG-63 cell lines. (b-e) Colony formation and EdU assays demonstrated that overexpression of miR-744-5p suppressed the proliferation of OS cells. (f-i) Transwell migration and invasion assays showed that overexpressed miR-744-5p remarkably inhibited the migratory and invasive ability of OS cells. (j-l) WB indicated that overexpression of miR-744-5p suppressed the expression level of metastasis-related proteins in OS cells. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4.

miR-744-5p suppressed xenograft tumor growth and pulmonary metastasis in vivo. (a-c) miR-744-5p mimics suppressed the tumor growth in nude mice, and the volume and weight were smaller and lighter compared to the miR-744-5p-NC group. (d, e) Higher expression of E-cadherin and lower expression of Ki-67, N-cadherin and vimentin were found in the Lv-miR-744-5p group according to IHC. (f, g) The HE staining demonstrated that less OS cells were found in the lungs of nude mice of the Lv-miR-744-5p group, indicating that miR-744-5p suppressed pulmonary metastasis. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5.

TGFB1 expression was upregulated in OS cell lines and tissues and was a target of miR-744-5p. (a) miRDB database demonstrated 111 genes targeted to miR-744-5p, and the MAPK signaling pathway was the most relative one according to the KEGG pathway enrich the analysis. (b, c) Among all candidate genes in the MAPK axis, TGFB1 was significantly downregulated by miR-744-5p both in 143B and MG-63 cells at the same time. (d, e) The WT-TGFB1-3’-UTR and MUT-TGFB1-3’-UTR were synthesized. Overexpressed miR-744-5p significantly suppressed the luciferase activity of WT-TGFB1-3’-UTR but had no effect on MUT-TGFB1-3’-UTR in 143B and MG-63 cells. (f-h) WB showed that miR-744-5p downregulated the expression level of TGFB1 and p-P38. (i) Higher expression of TGFB1 was found in OS cell lines, especially in 143B and MG-63 cells. (j) Expression of TGFB1 was significantly upregulated in OS clinical tissues than para-carcinoma tissues. (k) TGFB1 expression level was negatively related to miR-744-5p in OS tissues. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6.

TGFB1 is related to the clinical features of patients with OS. (a) Higher expression of TGFB1 was found in the middle and advanced stage of OS compared to the early stage. (b) Higher expression of TGFB1 was related to larger tumors. (c) Higher expression of TGFB1 was found in more patients with metastasis. (d) Although there was no statistical difference in overall survival between the two groups, patients with a lower expression level of TGFB1 tend to have a better prognosis. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 7.

miR-744-5p downregulated MAPK signaling pathway through inhibiting TGFB1 expression. (a) TGFB1 was successfully transfected into 143B and MG-63 cell lines. (b-f) Colony formation and EdU assays demonstrated that miR-744-5p suppressed the proliferation of OS cells, and overexpression of TGFB1 could reverse the effect. (g-j) Transwell migration and invasion assays indicated that overexpressed miR-744-5p significantly suppressed the migratory and invasive ability of OS cells, and overexpressed TGFB1 could abolish the influence. (k-n) Western blotting assays showed that miR-744-5p downregulated metastasis-related, MAPK-related and TGFB1 proteins in OS cells, while TGFB1 own contrary functions. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 8.

miR-744-5p suppressed tumor growth and pulmonary metastasis through TGFB1 in vivo. (a-c) miR-744-5p inhibited metastasis and growth of xenograft tumors, and TGFB1 reversed the effects of miR-744-5p. (d, e) IHC showed that TGFB1 upregulated the expression level of Ki-67, N-cadherin and vimentin, which were suppressed in the miR-744-5p mimics group. (f, g) TGFB1 promoted the decreased pulmonary metastasis caused by miR-744-5p. * p < 0.05, ** p < 0.01, *** p < 0.001.