MicroRNA-615-5p Regulates Proliferation,invasion And Migration Of Osteosarcoma Cells By Targeting AKT1

Background: Osteosarcoma(OS)is an invasive bone cancer.Currently,osteosarcoma(OS)is treated with the standard treatment regimen based on relevant guidelines,including surgical resection,chemotherapy,targeted therapy,and radiotherapy.The 5-year survival rate of patients with osteosarcoma is about 60～70%..The 5-year survival rate of patients with locally advanced stage,metastasis or recurrence is only 20%.Regrettably,although radiotherapy,chemotherapy,targeted therapy,and other adjuvant treatments have been used in most patients,the survival time of patients with osteosarcoma has not been significantly prolonged.Traditional treatment methods are limited in the treatment of osteosarcoma.By looking for treatment targets based on abnormal changes in the genetics or epigenetics of tumor patients,it is possible to improve the treatment effect of osteosarcoma and improve the prognosis of patients.Among them,Micro RNAs(mi RNAs)have been extensively studied.Mi RNAs may play a role in promoting cancer in the occurrence and development of tumors,and may also play a role in suppressing cancer.When certain mi RNAs are pro-oncogenic genes,their overexpression will promote the development of tumors.,on the contrary,if some mi RNAs are tumor suppressor genes,their overexpression will inhibit the development of tumors.For example,mi R-135-b,mi R-150,mi R-542-5p and mi R-652 have been confirmed to be abnormally expressed in osteosarcoma cells.The expression level of mi R-615-5p is down-regulated in a variety of tumor tissues such as liver cancer,lymphoma and malignant glioma,and this abnormal expression is directly or indirectly related to tumor cell proliferation,invasion and migration.The targets of mi R-615-5p include AKT,CCND2,IGFR1/IGF2 and etc.The physiological function of AKT1 gene is to encode serine/threonine protein kinase.Studies have shown that the up-regulation of AKT1 promotes the lung metastasis of osteosarcoma,and the down-regulation of AKT1 can significantly inhibit the promotion of MAT1-mediated migration and invasion of osteosarcoma cells in vitro,inhibit tumor growth in xenograft nude mice,and reduce the number of metastatic lung tumors.Therefore,mi R-615-5p has a significant tumor suppressor effect in most tumors,and the AKT1 signaling pathway plays an important role in the occurrence and development of tumors.Its role in many tumors including osteosarcoma has been confirmed and a variety of AKT1 regulatory factors have been discovered,but the specific functions of mi R-615-5p and AKT1 in osteosarcoma and the interaction between the two remain to be further studied.Objective: To investigate the expression level of mi R-615-5p /AKT1 regulating axis in osteosarcoma and its effect on the biological function of osteosarcoma cells by molecular biological experiments.Methods: A total of 29 pairs of osteosarcoma tissues and normal tissues adjacent to the tumor were collected from June 2018 to May 2019 in our department.The osteosarcoma cells(U2OS)and human normal osteoblasts(h FOB)were purchased and cultured by the Institute of Cell Research,Chinese Academy of Sciences.(1)The q RT-PCR method was used to detect the expression level of mi R-615-5p in cancerous tissues and normal tissues adjacent to cancer,U2 OS cells and h FOB cells.(2)Divided U2 OS cells into control group,overexpression group and nonsense control group,and transfected mi R-615-5p mimic and nonsense RNA into osteosarcoma U2 OS cells with liposomes respectively.The q RT-PCR method was used to detect the expression level of mi R-615-5p in the cells and investigated the transfection efficiency(3)CCK-8 method was used to detect cell viability and proliferation of each group.(4)The scratch test detected the invasion ability of each group of cells.(5)Transwell experiment detected the in vitro migration ability of each group of cells.(6)Obtained the sequence information of mi R-615-5p and AKT1 through the bioinformatics software Targetscan7.2,investigate whether the two sequences had fragmentary complementary sequences,and analyzed their possible existence binding site.(7)Western blot method was used to detect the expression level of AKT1 protein in the three groups of cells.(8)The dual luciferase reporter experiment detected the binding relationship between mi R-615-5p and AKT1.Results :(1)The effect of osteosarcoma on the expression level of mi R-615-5P.(1)The relative expression level of mi R-615-5p in osteosarcoma tissues was significantly lower than that in adjacent normal tissues,and the difference was statistically significant(P < 0.05).(2)The relative expression level of mi R-615-5p in U2 OS was significantly lower than that of h FOB,with a statistically significant difference(P < 0.05).(2)The effect of mi R-615-5p overexpression on the biological behavior of osteosarcoma cells.(1)The relative expression level of mi R-615-5p in the overexpressed group was significantly higher than that in the control group,with statistically significant difference(P < 0.05).(2)After the overexpression of mi R-615-5p,the cell activity and proliferation ability of U2 OS cells were significantly inhibited,with statistically significant differences(P < 0.05).(3)After mi R-615-5p overexpression,the degree of scratch healing in the U2 OS cell group was far lower than that in the control group,and the difference was statistically significant(P < 0.05).(4)After the overexpression of mi R-615-5p,the number of cells passing through the compartment in the U2 OS cell group was significantly lower than that in the control group,and the difference was statistically significant(P < 0.05).(3)The relevant mechanism of mi R-615-5p affecting the biological behavior of U2 OS cells through AKT1.(1)Sequence comparison results showed that mi R-615-5p had binding sites with AKT1 3 ’-UTR.(2)After the overexpression of mi R-615-5p,the AKT1 protein expression level in U2 OS cells was significantly decreased,and the difference was statistically significant(P < 0.05).(3)Analysis of the interaction between mi R-615-5p and AKT1 expression by double luciferase reporter gene showed that,compared with the Control group,the luciferase activity of AKT1 3’-UTR WT in the cells transfected with mi R-615-5p was decreased,with statistically significant difference(P < 0.05).Conclusions :(1)mi R-615-5p was low expressed in osteosarcoma tissues and osteosarcoma cell line U2 OS.(2)Overexpression of mi R-615-5p can inhibit the proliferation,migration and invasion of U2 OS.(3)AKT1 was a direct target of mi R-615-5p,and mi R-615-5p can regulate the biological behavior of U2 OS through AKT1.(4)In this study,mi R-615-5p was found to be a potential molecular therapeutic target for osteosarcoma.