Establishment And Ananlysis Of MG-63 Osteosarcoma Cell Sublines And The Effects Of MiR-130b In Proliferation And Apoptosis Of MG-63 Osteosarcoma Cell Sublines

Osteosarcoma(OS),one of the most common primary bone malignancies,is the eighth leading cancer with an incidence of 4.4cases per million population per year,which occurs most commonly in the metaphyseal regions of long bones.Although the 5-year survival rate of OS patients has been greatly improved from only 20%to nearly 75%after the introduction of combination chemotherapy over the last three decades,a significant proportion of them have a poor response to chemotherapy and are still at high risk of relapse or metastasis even after curative resection of the primary site and intensive chemotherapy.Research study on osteosarcoma metastasis mechanism has long been the focus in orthopedics and relevant disciplines.The osteosarcoma MG-63 cell is a human osteosarcoma cell line with a high metastatic potential.Currently,we have not discovered any reports on studying the metastatic mechanism using the human osteosarcomaMG-63 cell subline with high or without metastasis.MicroRNAs(miRNAs)are a class of small and noncoding RNAs.miRNAs regulate various biological processes including cell maintenance,proliferation,differentiation,apoptosis and development.Increasing amounts of evidence shows that aberrant expression of many miRNAs is associated with human diseases,including cancer.In fact,it has been widely reported that dysregulated expression of miRNAs occurs in various tumors,and some function as tumor suppressors or oncogenes which depend on the role of mRNA targets.miR-130b has been reported to act as an oncogene,and is significantly up-regulated in various types of malignant tumors.We recently discovered that miR-130b is up-regulated in OS cells by analysis of a published micro-array-based high-throughput assessment.We found that the naked cuticle homolog 2(NKD2)is a potential target of miR-130b.Recently,NKD2 has been reported to inhibit tumor growth and metastasis in OS by suppressing Wnt signaling.Therefore,we hypothesize that miR-130b might play a role in OS development and progression by regulation the expression of NKD2.Part 1 Establishment and analysis of osteosarcoma cell sublines with different metastatic characteristicsObjectiveTo establish human osteosarcoma MG-63 cell sublines with different metastatic characteristics and provide good experimental models for mechanism study of osteosarcoma metastasis.Method:Six cell sublines were screened and established by using the in vitro cloning technology.In vitro invasion experiments,cellular electrophoretic mobility determinations,cellular proliferation rate determinations,and soft agar clone formation assays were used to compare,analyze,and identify the metastatic characteristics of various cell sublines.ResultThe invasion capacity,cellular electrophoretic mobility,cell proliferation,and soft agar clone formation capacity of A2,A3,and A16 sublines were higher than those of A1,A6,and A20 sublines.There was no significant difference in various values determined among A2,A3,and A16,and among A1,A6,and A20(P>0.05),but there were significant differences in various values determined between the former three and the latter three(P<0.05).ConclusionMG-63 cell sublines with different metastatic characteristics can be established by combining several technologies and can contribute to further research on the mechanism of osteosarcoma metastasis.Part 2 miR-130b targets NKD2 and regulates the Wnt signaling to promote proliferation and inhibit apoptosis in osteosarcoma cellsObjectiveTo analyze that miR-130b might target NKD2 and regulate the Wnt signaling to promote OS growth and offer new clues for OS development and progression,and novel potential therapeutic targets for OS.MethodQuantitative Real-Time PCR(qRT-PCR).Western blot assays,MTT and flow cytometry assays were used to compare,analyze,and identify that the effects of miR-130b in osteosarcoma cell sublines.Luciferase reporter assay was used to identify thatNKD2 is a direct target of miR-130b.ResultUp-regulation of miR-130b and down-regulation of NKD2 mRNA and protein exist in OS cell lines.miR-130b inhibitors inhibit proliferation and promote apoptosis in OS cells.NKD2 is a direct target of miR-130b,and miR-130b regulated proliferation and apoptosis of OS cells by targeting NKD2.miR-130b and NKD2 regulate OS cell proliferation and apoptosis by inhibiting the Wnt signaling.ConclusionmiR-130b targets NKD2 and regulates the Wnt signaling to promote proliferation and inhibit apoptosis of OS cells.