MiR-506-3p Inhibits Epithelial-to-mesenchymal Transition And Autophagy In Osteosarcoma By Directly Targeting SPHK1

Objective: Osteosarcoma is a kind of common malignant tumor of bone in adolescents.For a long time in the past,its treatment effect has not been improved actually.Some patients will be accompanied by tumor metastasis,which will greatly hinder the treatment of patients.Therefore,the mechanism of metastasis related proteins in osteosarcoma is expected to provide a new direction and new therapeutic target for osteosarcoma.It is found that miR-506 is closely related to epithelial mesenchymal transformation of many kinds of cancer.Meanwhile,it is also involved in the regulation of osteosarcoma cell proliferation,invasion and apoptosis and other life processes.At the same time,it has been shown that SPHK1 is involved in regulating autophagy and affecting epithelial mesenchymal transformation of hepatoma cells,and has high expression in many kinds of tumors,which is closely related to the malignancy of tumor.Although it has been reported that miR-506 and SPHK1 are actively involved in the development and progression of cancer,the mechanism of miR-506 and SPHK1 in osteosarcoma autophagy and epithelial mesenchymal transformation have not been reported.This project studied the effect of miR-506 and SPHK1 on autophagy and epithelium mesenchymal transformation of osteosarcoma,further to explain the mechanism of osteosarcoma,providing a new target and theoretical basis for the treatment of osteosarcoma.Methods:(1)The following human osteosarcoma cells: MG63,143 B,MNNG/HOS,SaOS-2,U-2OS cells were cultured in vitro.The expression of hsa-miR-506-3p and SPHK1 were detected by Real-time PCR.The expression of SPHK1 was detected by Western blot.(2)Synthetize hsa-miR-506-3p mimics and its NC control(3)According to the results,two cells with low expression of hsa-miR-506-3p and high expression of SPHK1 were selected from the above cell lines to transfect hsa-miR-506-3p mimics and its NC control.(4)After 24 hours of transfection,the expression of hsa-miR-506-3p and SPHK1 were detected by Real-time PCR.(5)After 48 h transfection,the expression of SPHK1 was detected by Western blot 2.Target Detection of SPHK1 by hsa-miR-506-3p Double luciferase method was used to detect the targeting conditions of has-miR-506-3p and SPHK1 in tool cells.3.Effects of hsa-miR-506-3p/SPHK1 on epithelial mesenchymal transformation and autophagy of human osteosarcoma cells(1)The overexpression plasmid of SPHK1 was constructed.(2)SPHK1 overexpression plasmid and empty vector were co-transfected with hsa-miR-506-3p mimics or NC mimics into human osteosarcoma cells.(3)After 48 hours of transfection,phase contrast microscopy was used to observe the morphological changes of the cells in each group.(4)After 24 hours of transfection,Transwell assay was used to detect the 24 h invasion ability of the cells in each group.(5)After 48 hours of transfection,the expression of E-cadherin was observed by immunofluorescence assay in each group.(6)After 48 hours of transfection,the expression of E-cadherin Vimentind and Fibronectin were detected by Western blot in each group.(7)After 48 hours of transfection,Western blot was used to detect the expression of LC3II/I.4.Effects of hsa-miR-506-3p/SPHK1 on epithelial mesenchymal transformation of human osteosarcoma cells through autophagy(1)Hsa-miR-506-3p mimics and NC controls were transfected into SaOS-2 cells.The cells were treated with autophagy accelerator 0.5 μ M Rapamycin for 24 h at 24 h after transfection.Transwell assay was used to detect the 24 h invasion ability of the cells in each group.The expression of E-cadherin,Vimentind and Fibronectin were detected by Western blot in each group.Similarly,the expression of LC3II/I was detected by Western blot in each group.(2)SPHK1 mimics and NC controls were transfected into human osteosarcoma cell line 143 B and SaOS-2.The cells were treated with autophagy inhibitor 10 mM 3-methyladenine for 6 h at 48 h after transfection.The expression of E-cadherin,Vimentind and Fibronectin were detected by Western blot in each group.Result: The hsa-miR-506-3p was expressed at a low level in 143 B and SaOS-2 cell lines,and SPHK1 was expressed at a high level in 143 B and SaOS-2 cell lines.When hsa-miR-506-3p mimics/NC controls was transfected into 143 B and SaOS-2,the detected expression of hsa-miR-506-3p mRNA was significantly increased while the MRNA and protein levels of SPHK1 were decreased significantly in hsa-miR-506-3p mimics group.Double luciferase assay was used to determine that SPHK1 was a direct target of hsa-miR-506-3p.Transwell invasion assay showed that the overexpression of hsa-miR-506-3p inhibited the ability of cell invasion,while the overexpression of SPHK1 promoted the ability of cell invasion.Overexpression of hsa-miR-506-3p partially attenuated the role of SPHK1 in promoting the invasion of OS cells.The expression and location of E-cadherin were detected by immunofluorescence staining.It was found that hsa-miR-506-3p increased the expression of E-cadherin by inhibiting SPHK1 in 143 B and SaOS-2 cells,and E-cadherin was mainly expressed in the cytoplasm of osteosarcoma cells,which was confirmed by Western blotting at the protein level.When hsa-miR-506-3p mimics was transfected into 143 B and SaOS-2 cells,the level of E-cadherin was up-regulated and the ratio of SPHK1,Vimentind,Fibronectin and LC3II/I were down-regulated.When SPHK1 mimics was transfected into 143 B and SaOS-2 cells,the ratio of LC3II/I increased.From the above results,hsa-miR-506-3p can inhibit the autophagy of 143 B and SaOS-2 cells,and SPHK1 can promote the autophagy of 143 B and SaOS-2 cells.MiR-506-3p mimics and NC mimics were transfected into SaOS-2 cells,and miR-506-3p mimics group was treated with 0.5 μ M rapamycin for 24 h.The invasion ability of each group was detected by transwell invasion assay.The expression of EMT markers(E-cadherin,Vimentind,Fibronectin)and autophagy related gene LC3II/I were detected by western blotting.Compared with miR-506-3p mimics group,the invasion ability was increased,the protein expression of epithelial marker(E-cadherin)was down-regulated,the protein expression of mesenchymal markers(Vimentind and Fibronectin)were up-regulated,and the expression of autophagy related gene LC3II/I was increased in rapamycin+miR-506-3p mimics group.SPHK1 mimics and NC mimics were transfected into 143 B and SaOS-2 cells,and the cells were treated with autophagy inhibitor 10 mM 3-methyladenine for 6 h.Then,the expression of EMT markers(E-cadherin,Vimentind,Fibronectin)were detected by western blotting.Compared with SPHK1 mimics group,the protein expression of epithelial marker(E-cadherin)was up-regulated,and the protein expression of mesenchymal markers(Vimentind and Fibronectin)were down-regulated in 3-methyladenine+SPHK1 mimics group.Conclusion: Hsa-miR-506-3p targets SPHK1 and affects EMT of human OS cells through autophagy.