The Effect Of MiR-34a On Osteosarcoma Proliferation,Migration,Programmed Death,Adherence And Its Molecular Mechanism

[objective]Osteosarcoma is the most prevalent primary malignant bone cancer which happens generally in young people affecting rapidly growing bones and with an annual estimated incidence of about 4 million worldwide.During the past ten years,surgery combined with chemotherapy for osteosarcoma treatment by inhibiting cell growth or invasion,has been explored.Still,despite outstanding treatment,the progress of metastasis and chemotherapy resistance stays main problems accounting for the failure of treatment.The overall survival rate for osteosarcomas is only 50%to 80%and local relapse happens in about 10%of patients Therefore,there is a marked requirement to develop effective therapeutic treatments that inhibit metastasis,the major reason of death in osteosarcoma.Finding out the underlying molecular mechanisms that drive the progression and metastasis of osteosarcoma would help the advance of better therapeutic strategies to improve the patient prognosis and management,and it could also help to identify novel molecular prognostic factors and therapeutic targets.Reports have explored the genes related to the metastasis of osteosarcoma,and miRNAs have grown into a novel exploration hotspot.MicroRNAs are small non-coding RNAs,constituting about 22 nucleotides,which classically participating in post-transcriptional regulation of gene.Growing evidences have demonstrated that miRNAs have vital functions in biological and pathological processes,for example,cell proliferation,apoptosis,stress response,and development.Moreover,frequent dysregulations of miRNAs have been detected in many types of cancer,including ovarian,liver,bladder,and colorectal cancer.Especially,miR-34a is a recognized tumor inhibitor that controls cell proliferation,invasion,and other courses in various kinds of tumors.Yet,the functions of miR-34a in OS have not been fully illustrated.Thus,it is important to demonstrate the activity and potential mechanisms of miR-34a in OS.miR-34a has many target genes that have numerous functions in shaping the cellular function in OS,such as p53,Eag1,Wnt and Notch.Mitogen-activated protein kinases(MAPKs)constitute an extremely conserved clan of kinases that transmit signals from outside to the intracellular molecules has been implicated in proliferation and differentiation.The largest groups of enzymes that mediate the dephosphorylation of MAPK are the protein tyrosine phosphatase superfamily,termed the dual-specificity protein phosphatases(DUSPs).DUSP1 was firstly replicated as a gene induced by growth factor involved in the G0/G1 phase transition and apoptosis.miR-101 has been found targeting DUSP1 to affect the secretion of TGF-β in sorafenib.miR-940 inhibition retains DUSP1 expression and attenuates JNK-mediated apoptosis in lung cancer cells.However,the regulation of DUSP1 by miR-34a in OS remains unknown.Our study intends to study the roles of miR-34a and DUSP1 in proliferation,apoptosis and adhesion of OS cells and the involved molecular mechanisms.This may help develop potential diagnostic and therapeutic targets for OS.[Methods]By applying pre-miR-34a,anti-miR-34a and negative control in MG63 and U-20S cells,adjusting the expression of miR-34a,we studied the role of miR-34a in osteosarcoma cells.Cell proliferation,cell cycle,cell migration and programmed death were respectively detected by MTT,colony formation,tranwell assay and flow cytometry,and the expression of DUSP1 regulated by miR-34a and molecular mechanism of miR-34a on osteosarcoma was detected by luciferase reporter assay,QPCR and western blot.[Results]MiR-34a reduced the proliferation of MG63 cells through prompting cell cycle arrest at G0/G1 phase,cell inducing apoptosisby by 6 fold,and suppressed cell adhesion and cell migration abilityby 40%.Whereas anti-miR-34a increases U-20S cell proliferation by preventing cell apoptosis,and promotes cell adherence.Dual-specificity phosphatase 1(DUSP1)was identified as the target gene of miR-34a in osteosarcoma cells and confirmed that DUSP1 enhanced the proliferation through inhibiting cell cycle arrest at G0/G1 phase and apoptosis,and inhibits the decreased cell adhesion induced by miR-34a.However,inhibition of DUSP1 resulted in substantially decreased proliferation and adhesion,and cell cycle arrest in G0/G1 phase and cell apoptosis,which was consistent with what observed of miR-34a in U-20S cells.[conclusion]MiR-34a plays an important role in the pathogenesis of osteosarcoma,inhibiting the development of cancer by inhibiting DUSP1.MiR-34a may work as a novel tumor-suppressor in osteosarcoma pathogenesis through inhibition of DUSP1,suppressing osteosarcoma cell proliferation,migration,blocking cell cycle and arresting at G0/G1,inhibiting adherence and promoting apoptosis.