Research On Mechanism Of MiR-21 In Human Osteosarcoma Cells MG63

Background Osteosarcoma is one of the most highly malignant types of cancer in adolescents and young adults and has high mortality rate. Despite advances in surgery, radiation therapy and chemotherapy, the prognosis for patients with osteosarcoma has not significant improvement over the past several decades. The problems of metastasis, recurrence, drug resistance and the appearance of secondary malignancies are still puzzling to most doctors.Therefore, it is important for the development of more effective tumor therapies to understand how the signaling pathways were involved in proliferation, invasion and apoptosis of osteosarcoma.Micro RNAs(mi RNAs)are a class of endogenous small non-coding RNAs. They are approximately 20 to 25 nucleotides long in eukaryotic organisms that genetically regulate the expression of some potential target m RNAs at the post-transcriptional level. Mi RNAs are aberrantly expressed in numerous cancers. Many mi RNAs have been linked to different cancer-related processes. More and more researchers have discovered that mi RNAs may play a role of oncogene or tumor suppression in diverse biological processes. So more and more researches about the effects of mi RNAs in cancer-related processes is underway. Researchers have discovered that micro RNA-21(mi R-21) is one of the most common oncogenes in numerous cancers. Accumulating evidences indicate that mi R-21 is overexpressed in many cancers. However, the function of mi R-21 in human osteosarcoma has not been totally elucidated. In the present study, the effects of mi R-21 in osteosarcoma and the possible mechanism by which mi R-21 affected the proliferation, invasion and apoptosis of osteosarcoma cells were investigated.Objective Based on the research of the function of mi R–21 in human MG63 osteosarcoma cells,our study suggested that mi R-21 may play an oncogenic role in osteosarcoma and may have potential as a therapeutic target.Methods(1) To study the function of mi R-21, we transfected MG63 cells with chemically synthesized double-stranded oligonucleotides(anti-21, mi-21 and Scr) to up or down regulate the expression level of mi R-21. The expression of mi R-21 was detected by real-time quantitative PCR analysis. The blank(MG63 cells transfect nothing)was used as control.(2) The effects of mi R-21 were assessed by cell viability, cell cycle analysis, invasion assay and apoptosis assay through up-regulating or inhibiting the expression of mi R-21.(3) We also examined the relationship between mi R-21 and the resistance of cisplatin in osteosarcoma cells through up or down-regulateing the mi R-21 level.(4)To identify how mi R-21 regulated the expression of PDCD4 in osteosarcoma cells MG63, we designed a mi RNA screening assay using a mi RNA target database of mammalian cells. We performed bioinformatics search for putative mi RNAs predicted to target PDCD4 coding or non-coding sequences using Pic Tar(http://pictar.mdc-berlin.de/), mi Randa(http://www.microrna.org) and Target Scan(http://www. targetscan.org/).(5)To further confirm the relationship between mi R-21 and PDCD4 expression in osteosarcoma cells. We also detected the m RNA level of PDCD4 by real-time quantitative PCR and the protein level of PDCD4 by western blotting.(6)We also repeated the luciferase assay to validate PDCD4 as a target of mi R-21 in osteosarcoma cells MG63. Moreover, we examined PDCD4 expression by immunohistochemical staining.Results(1) 24 h after transfection, the expression of mi R-21 was evaluated by quantitative real-time PCR analysis. The results demonstrated that cells transfected with anti-21 were significantly suppressed the expression level of mi R-21 compared with blank group. MG63 cells transfected with mi-21 expressed significantly higher level of mi R-21 compared with blank group(P<0.05).(2) MTT assay was performed following the procedure as described in Methods every 24 h. And then proliferation curve was depicted. The results demonstrated that up-regulation of mi R-21 significantly enhanced proliferation compared with control. In contrast, inhibition of mi R-21 significantly suppressed cell proliferation compared with control. Cell cycle distribution was measured by flow cytometry. Results showed that through up-regulation of mi R-21 had the significant difference in G0/G1 phase and S phase compared with blank group, respectively. In contrast, through down-regulation of mi R-21, cells had the significant difference in G0/Gphase and S phase. There are no significant statistical difference between Scr and blank group in G0/G1 and S phase. No significant statistical was observed in the percentage of cells in the G2/M phase among the five groups. These data demonstrated that mi R-21 may induce the proliferation in MG63 cells. Cell apoptosis assay showed that up-regulation of mi R-21 significantly decreased spontaneous apoptosis compared with control. In contrast, inhibition of mi R-21 significantly promoted spontaneous apoptosis compared with control. Invasion assay demonstrated that cells transfected with anti-21 exhibited significant declines in invasion capacities compared with blank group.While cells transfected with mi-21 significantly increased invasion capacities compared with blank group. However, there is no significant difference between Scr and blank group. The results strongly indicated that mi R-21 was an important participant in promoting invasive potential of osteosarcoma in vitro.(3) Up or down-regulation of the mi R-21 level can affect the resistance of cisplatin in osteosarcoma cells.(4)Bioinformatic search for putative mi RNAs predicted to target PDCD4 coding or non-coding sequences showed that the 3′-UTR of human PDCD4 gene harbored putative mi R-21 target sites.(5) Western blotting demonstrated that the endogenous PDCD4 protein level in mi-21 group cells exhibited remarkable decrease compared with blank. Real time PCR demonstrated that the endogenous PDCD4-m RNA level in mi-21 group cells was also significantly decreased. Meanwhile, down-regulation of mi R-21 showed the contrary results.(6) Luciferase assay demonstrated that the luciferase activity of the p MIR-PDCD4-3’-UTR construct was significantly inhibited after the introduction of mi R-21. Immunohistochemical staining showed the weak PDCD4 protein staining and significantly proliferation in mi-21 group. The anti-21 group showed the reverse results.Conclusion(1) MG63 cells transfected with mi R-21 mimics or mi R-21 inhibitors effectively up or down-regulated mi R-21 expression.(2) Mi R-21 mimics significantly enhanced proliferation,invasion and reduced cell apoptosis compared with control. While mi R-21 inhibitors inhibited cell growth,invasion and induced cell apoptosis compared with control. The results demonstrated that mi R-21 probably served as an oncogene in osteosarcoma cells.(3) Up or down-regulation of the mi R-21 level can affect the resistance of cisplatin in osteosarcoma cells.(4) PDCD4 protein levels not only correlated inversely with mi R-21 levels in a number of tumor cell lines, but also PDCD4 m RNA was a direct functional target of mi R-21 in osteosarcoma cells. So mi R-21 may function as an oncogenes gene partly through down-regulation of PDCD4 in osteosarcoma.