The Expression And Effects Of MiR-885-5p On Osteosarcoma

Background Osteosarcoma(OS),a most frequent primary malignancy bone tumor in children and adolescents,normally occurs in the metaphysis of long bones and is characterized by a high level of relapse and poor prognosis.Despite the combination of adjuvant chemotherapy and advanced surgery,the cumulative 5-year survival of patients is still less optimistic.Micro RNAs(miRNAs)are a small class of endogenous non-coding RNAs that range in length from 18 to 25 nucleotides.The accumulating evidences indicated that miRNAs have influence on tumor microenvironment and are closely associated with the development of cancer.Objective 1.To observe the expression of miR-885-5p in OS cells and tissues.2.To discuss the correlation between the expression of miR-885-5p in OS tissues and clinicopathological features.3.To observe the biological role of miR-885-5p on OS cells.4.To explore a target gene of miR-885-5p,and illuminate the mechanism of miR-885-5p on its target gene.Methods 1.To detect the expression of miR-885-5p between OS cell lines MG63,U2 OS and normal human osteoblastic cell line h FOB1.19 by RT-q PCR.2.A total of 31 OS tissue and matched adjacent normal tissues were surgically acquired from patients in the First Affiliated Hospital of Fujian Medical University between September 2015 and August 2017.The expression of miR-885-5p was detected by RT-q PCR to compare the differences of OS tissue and matched adjacent normal tissue.3.To reveal the correlation between the expression of miR-885-5p in OS tissues and clinicopathological features through the analysis of patients data,including age,gender,tumor size,anatomic location,clinical stage and metastasis.4.Recombinant lentivirus containing miR-885-5p mimic,miR-885-5p inhibitor and negative control were synthesized to transfect into MG63 cells,and we tried to establish stable cell lines with miR-885-5p-up/down groups.The cell experimental groups were as follows:(1)Blank group: MG63 cells without any treatment.(2)NC group: transfected miR-885-5p negative control.(3)miR-885-5p-up group: transfected miR-885-5p mimic.(4)miR-885-5p-down group: transfected miR-885-5p inhibitor.5.MTT assay and colony formation assay were used to observe the effect of miR-885-5p on OS cells proliferation.6.Transwell assay was used to observe the effect of miR-885-5p on OS cells migration.7.We predicted the target genes of miR-885-5p through analysis of biological data on Target Scan,miRanda and miRBase Targets.Based on bioinformatics algorithms,constructed reporter vectors of both wild-type and mutant-type containing 3’-UTR of CDC73 and miR-885-5p or the miRNA negative control were co-transfected into MG63 cells.The Dual-Luciferase assay was used to confirm that CDC73 is a target gene of miR-885-5p.8.Western blot was used to detect the expression of CDC73.Results 1.Compared with normal human osteoblastic cell line,miR-885-5p was significantly upregulated in OS cells.2.Compared with matched adjacent normal tissues,miR-885-5p was significantly upregulated in the human OS tissues.3.Upregulation of miR-885-5p is associated with advanced clinicopathological features of OS,including tumor size and clinical stage.4.After transfection and selection of cells,the results revealed that miR-885-5p expression levels were significantly higher in miR-885-5p-up group and lower in miR-885-5p-down group compared with blank and NC groups,respectively.5.In the MTT assay,the absorbance of MG63 cells at OD570 in miR-885-5p-up group was remarkably increased and exhibited a significant climb in proliferation curve compared with blank group and NC group while the cells in miR-885-5p-down group presented a opposite trend.6.The results of Transwell assay showed that the number of migrated cells in the miR-885-5p-up group significantly increased compared with blank group and NC group while cells in miR-885-5p-down were significantly suppressed.7.The Dual-Luciferase assay results revealed that CDC73 is a target gene of miR-885-5p.8.Western Blot assay results suggested that the expression of CDC73 was significantly inhibited in miR-885-5p-up group compared with blank group and NC group,while the expression presented a opposite result.Conclusions 1.The expression of miR-885-5p was significantly upregulated in OS tissues,which associated with the development of OS.2.miR-885-5p promotes OS cells proliferation and migration.3.miR-885-5p exerts a biological role by negatively regulating CDC73 expression.