Effect Of MiRNA-495 On Osteosarcoma Biological Behavior By Targeting HMGN5

Background: Osteosarcoma is a highly malignant and aggressive bone tumor that occurs mainly in children and adolescents between 10 and 20 years of age.Although some genes(Ezrin,NOTCH,RUNX2,FAS et al)were discovered to be involved in the osteosarcoma development,there is till lack of comprehensive understanding for the osteosarcoma molecular etiology to improve the patients' survival rate.High mobility group N(HMGN)proteins are a family of ubiquitous nuclear proteins which modify the structure of chromatin to attain a conformation that facilitates and enhances transcription,histone modifications,replication and repair.HMGN5(high-mobility group nucleosome-binding domain 5),also named NSBP1(Nucleosomal Binding Protein 1),a new member of the HMGN protein family,is reported to bind to the nucleosomes via nucleosomal binding domain(NBD),unfold chromatin,and modulate gene transcription.More and more evidence indicates that HMGN5 involved in various cancers,including osteosarcoma,bladder cancer,lung cancer,meningioma.Previous studies indicate that HMGN5 expression level was associated with pathologic staging and TNM staging.Knockdown of HMGN5 induced cell cycle arrest,inhibited invasion and increased sensitivity to doxorubicin–induced cell apoptosis in U2-OS and Sa O2 cells.Thus,HMGN5 should be a potential treatment target for osteosarcoma.Micro RNA(mi RNA),an abundant group of endogenous non-coding single strand RNAs of 18-25 nucleotides,participate in the regulation of a range of biological processes by regulating the expression of genes at post-transcriptional level.It has been suggested that micro RNAs(mi RNAs)may act as critical regulators in tumorigenesis.Micro RNA-495(mi R-495)has been suggested as a cancer-associated mi RNA in various cancers,such as pancreatic cancer,ovarian cancer,bladder cancer,renal cancer et al.mi R-495 play a role as tumor suppressor in various tumor progression,mi R-495 was determined to inhibit gastric cancer cell migration and invasion via targeting high mobility group AT-Hook 2(HMGA2).Also,mi R-495 can downregulate FOXC1 expression to suppress cell growth and migration in endometrial cancer.However,for the osteosarcoma,the role of mi R-495 in osteosarcoma and whether HMGN5 expression is regulated by specific mi RNAs in osteosarcoma is unknown.Aims: The goal of this study was(1)to determine whether mi R-495 is involved in osteosarcoma pathogenesis and how it affect osteosarcoma biological behavior;(2)to determine whether mi R-495 is target on high-mobility group nucleosome-binding domain 5(HMGN5);(3)to investigate the potential molecular mechanism of mi R-495 in regulating osteosarcoma pathogenesis.Methods: Fifteen paired human osteosarcoma tissues and adjacent normal tissues(located >3 cm from the tumor)were obtained from the Department of Orthopedics at The Second Hospital of Jilin University.Human osteosarcoma cell lines(143B,Sa OS-2,U2 OS,and MG63),normal osteoblastic cell line h FOB,and 293 T cells were purchased from the American Type Culture Collection(ATCC,Manassas,VA,USA).The mi R-495 mimics,negative control(mi R-NC),pc DNA3.1/HMGN5 overexpressing vector were transfected into cells.(1)RT-q PCR was utilized to analysis the mi R-495 in osteosarcoma tissue and cells.(2)Cells proliferation was detected by MTT assay;(3)Colony-forming capacity was determined by a 10-day culturing with 0.3% noble agar and 1% crystal violet stain before detecting;(4)for the cell cycle distribution of osteosarcoma cells,cells were treated by propidium iodide and RNase A and examined by flow cytometry;(5)cell apoptosis was determined by an Annexin V/PI double staining kit as well as caspase-3 activity assay;(6)cell invasion ability was detected by transwell assay;(7)to confirm the interaction between mi R-495 and HMGN5 3'-UTR,dual-luciferase assay was performed by using pmir GLO dual-luciferase containing either the wild-type 3'-UTR of HMGN5 or the mutant 3'-UTR of HMGN5;(8)to evaluate the HMGN5 downstream protein expression in transfected and normal osteosarcoma cells with Western blot;(9)when the pc DNA3.1/HMGN5 vector transfection was determined,these transfected cells' biological behavior(proliferation,colony formation,invasion and apoptosis)were evaluated again.Results: We found that mi R-495 was significantly downregulated in osteosarcoma tissues and cell lines,as detected by real-time quantitative polymerase chain reaction(RT-q PCR).Overexpression of mi R-495 inhibited osteosarcoma cell proliferation in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,colony formation and cell cycle assays.The overexpression of mi R-495 induced osteosarcoma cell apoptosis.Moreover,mi R-495 overexpression also inhibited osteosarcoma cell invasion.Bioinformatics and luciferase reporter assays demonstrated that mi R-495 targeted the 3'-untranslated region of high-mobility group nucleosome-binding domain 5(HMGN5),a potential oncogene in various cancers.Overexpression of mi R-495 inhibited the expression of HMGN5,cyclin B1,Bcl-2,and matrix metalloproteinase 9.In addition,restoration of HMGN5 protein expression could abrogate the mi R-495-induced anti-tumor effects.Conclusion: After a comprehensive interpretation of the forehead results,our study indicates that:(1)mi R-495 is a tumor suppressor for osteosarcoma,which suppresses proliferation and invasion of osteosarcoma cells,as well as induces apoptosis of osteosarcoma cells.(2)mi R-495 combines with HMGN5 m RNA directly and downregulates its expression in osteosarcoma cells.(3)mi R-495-HMGN5 molecular pathway play an important role in osteosarcoma biological behavior regulation.Taken together,our study provides a potential molecular target for osteosarcoma in the future translating research.