| Osteosarcoma is a mesenchymal tumor and more common occurs in children and adolescents.Osteosarcoma occurs frequently in areas where epiphysis is active,such as femur,humerus and tibia.It can be observed in tissue structure that osteosarcoma is a malignant mesenchymal tumor formed by abnormal immature bone matrix that produced by spindle cells.Tumor cells of osteosarcoma are unique that they can produce immature bone-like tissue,therefore osteosarcoma is also referred to as primary bone sarcoma.80% of patients with osteosarcoma have metastasized or micrometastasized at the time of diagnosis,so most patients undergo surgery and multiple drug chemotherapy.With increasing understanding of pathogenesis,development and prognosis of osteosarcoma at molecular level in recent years,molecular targeted therapy is likely to be the most potential treatment for osteosarcoma.Therefore,there is an urgent need to develop new treatment strategies and innovative methods to further improve survival rate of osteosarcoma patients.Micro RNAs（mi RNAs）play an important role in regulating the development and progression of tumors.Mi RNAs are single-stranded,non-coding RNAs that are not translated into proteins by themselves,but they recognize specific target genes and regulates expression levels of target genes through post-transcriptional modifications.Mi R-708-5p is expressed in a variety of tumor-related diseases,mi R-708-5p is also involved in neurodegenerative diseases,cardiovascular diseases and immune responses,suggesting that mi R-708-5p may involve in tumor immune response.At present,there is no research report on how mi R-708-5p regulates the molecular mechanism of osteosarcoma cells.Up-regulated gene 4/Up-regulator of cell proliferation（URG4/URGCP）was first discovered in the X antigen encoded by hepatitis B virus（HBx Ag）,and later was found in cancers such as gastric cancer and liver cancer.However,the biological mechanism of URG4/URGCP in carcinogenesis remains unclear.In studies of bladder cancer,URG4/URGCP can activate the NF-κB pathway and promote the translocation of P65 to nucleus.In this study,mi R-708-5p was found to be lower expressed in osteosarcoma by comparing expression levels of mi R-708-5p in osteosarcoma and corresponding paracancerous tissues.At the same time,it was speculated that URG4/URGCP would also be a valuable prognostic marker for patients with osteosarcoma.Therefore,we designed a series of experiments,finally proved that mi R-708-5p inhibited the activation of NF-κB by regulating expression of URG4/URGCP,and also inhibited proliferation,invasion and migration of osteosarcoma cell lines（SAOS-2）.These findings provide an experimental basis for diagnosis and treatment of osteosarcoma and search for new molecular targets.Objective: To investigate whether mi R-708-5p played a role in proliferation,invasion and migration of osteosarcoma cells and to clarify the molecular mechanism involved in its regulatory effects on these biological processes.Methods: Firstly,we collected specimens of osteosarcoma and adjacent normal tissues from 60 patients with osteosarcoma in the first affiliated Hospital of Anhui Medical University and the first affiliated Hospital of the University of Science and Technology of China.The expression of mi R-708-5p in 60 cases of osteosarcoma specimens was detected by RT-PCR,and the expression of mi R-708-5p in the corresponding adjacent tissues of osteosarcoma tissues was also detected to study effects of mi R-708-5p on the biological function of osteosarcoma cells.The osteosarcoma cell line SAOS-2 and osteoblast OB-3 were simultaneously selected,and the expression of mi R-708-5p in tumor cell line and normal cells was detected by real-time fluorescent quantitative PCR.Then,the mi R-708-5p mimetic,negative control and control group were transiently transfected into SAOS-2 cells using liposome2000 to achieve up-regulation of mi R-708-5p expression,transfection efficiency was detected by real-time fluorescent quantitative PCR.Then,CCK-8 and flow cytometry were used to detect proliferation and apoptosis of each group.Finally,invasion and metastasis ability of three groups were detected by scratch test and cell invasion test to demonstrate whether mi R-708-5p affected the biological behavior of osteosarcoma cells by regulating target gene URG4/URGCP.Thirdly,the bioinformatics software Targetscan 7.2 was applied to（http://www.targetscan.org/vert71/）predict that mi R-708-5p may act on target gene URG4/URGCP.Mi R-708-5p mimics,NC and URG4/URGCP wild-type or mutant URG4/URGCP 3 by lipo2000 ’UTR binding was co-transfected into SAOS-2 cells respectively,the expression of wild-type and mutant URG4/URGCP binding to targeted targets was verified by dual luciferase assay.At last,the protein and m RNA expression of URG4/URGCP and NF-κB were detected by Western blot and RT-PCR.The URG4/URGCP overexpression vector（p-c DNA-URGCP）was synthesized and transfected into SAOS-2 cells by lipo2000.The NC group was also used as a control group.The expression of URG4/URGCP and NF-κB in each group of cells was detected by RT-PCR and Western blot.Results: 1.The expression of micro RNAs-708-5p in tumor tissues of 60 patients with osteosarcoma was significantly lower than that in adjacent tissues（P<0.05）;the expression of micro RNAs-708-5p in osteosarcoma cell lines SAOS-2 was also lower than that in normal osteoblasts cell line OB-3,which showed that the expression of micro RNAs-708-5p in vitro was similar to that in tumor tissues.2.Mimic was transfected into osteosarcoma cell SAOS-2 by PCR.The results showed that the expression of m RNA in mi R-708-5p and the apoptosis of SAOS-2 cells were significantly promoted,and the proliferation of SAOS-2 cells was inhibited,but there was no significant difference between the control group and the negative control group.The invasion and migration experiment showed that the migration and invasion ability of the mi R-708-5p transfected group was stronger than that of the control group.However,there was no significant difference between control group and NC group.3.Bioinformatics showed that mi R-708-5p might bind to URGCP and inhibit its expression.Luciferase reporter gene assay showed that after co-transfection with URGCP 3’UTR wild-type,the expression of mi R-708-5p luciferase activity was lower than that in NC group,but there was no significant difference in luciferase activity between the two groups.These results indicated that the expression of m RNA and protein of URGCP and NF-κB decreased in the mi R-708-5p mimic transfection group,but the m RNA and protein levels of NF-κB increased significantly after p-c DNA-URGCP transfection,but was lower than that in NC and control groups.Conclusion: The expression level of mi R-708-5p was closely related to the incidence of human osteosarcoma,and the low expression of mi R-708-5p in tumor tissues may play a role in inhibiting the development of osteosarcoma.In addition,we also found that mi R-708-5p promoted the apoptosis of osteosarcoma cell line SAOS-2 by targeting URGCP,which may provide a new therapeutic approach for osteosarcoma. |
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