Overexpression Of Long Non-Coding RNA NNT-AS1 Correlates With Tumor Progression And Poor Prognosis In Osteosarcoma

ObjectiveThe expression level of LncRNA NNT-AS1 was detected in osteosarcoma tissues and cell lines,and the relationship between LncRNA NNT-AS1 and clinicopathological features was analyzed.Furthermore,the role of LncRNA NNT-AS1 in the development of osteosarcoma and molecular mechanisms were investigated in vitro and in vivo OS-732 nude mouse tumor-bearing model,respectively.Materials and MethodsThe expression of LncRNA NNT-AS1 in 47 osteosarcoma tissues was first examined,and its expression levels in immortalized osteoblasts and osteosarcoma cell lines were subsequently examined.In vitro studies,CCK-8 assay and colony formation assay were used to detect the proliferative capacity after knockdown of LncRNA NNT-AS1.Subsequent analysis of apoptosis and cell cycle distribution of osteosarcoma cells were performed by flow cytometry after knockdown of LncRNA NNT-AS1.Transwell assay was used to analyze the invasive ability of the cells after knockdown of LncRNA NNT-AS1 and the expression of key proteins related to proliferation and metastasis were determined.OS-732 cells,which were down-regulated by endogenous LncRNA NNT-AS1,were injected subcutaneously into the flank of nude mice.The tumor size and body weight of nude mice were measured periodically.After 5 weeks,the nude mice were sacrificed,and the grafted tumor tissues were taken for subsequent experiments to detect the expression of LncRNA NNT-AS1 and C-Myc(proliferation index)in the tumor tissues.Results1.LncRNA NNT-AS1 is significantly upregulated in osteosarcoma tissues and cellsReal-time quantitative PCR was used to detect and analyze the expression of LncRNA NNT-AS1 in osteosarcoma and normal.bone tissues.Upregulation of LncRNA NNT-AS1 was positive correlated with Enneking stage staging.Subsequently,it was found that LncRNA NNT-AS1 was also up-regulated in osteosarcoma cell lines,suggesting that LncRNA NNT-AS1 may be involved in the occurrence and development of osteosarcoma as a proto-oncogene.2.In vitro experiments to explore the biological function of LncRNA NNT-AS1Cell proliferation was assessed by CCK-8 assay and colony formation assay after knockdown of endogenous expression of LncRNA NNT-AS1.Flow cytometry was used to detect cell apoptosis and cell cycle distribution after LncRNA NNT-AS1 silencing.The results showed that knockdown of LncRNA NNT-AS1 significantly reduced cell proliferation,promoted apoptosis and arrested cell cycle.3.In vitro experiments to investigate the invasive ability of LncRNA NNT-AS1 on osteosarcoma cellsTranswell chamber assay was performed to detect cell invasive ability after LncRNA NNT-AS1 silencing.In addition,Western blot was used to detect the alteration of proliferation and apoptosis-related genes.The results indicated that knockdown of LncRNA NNT-AS1 could reduce the invasive ability of osteosarcoma cells,accompanied with changes in proliferation and apoptosis protein levels.4.Knockdown of LncRNA NNT-AS1 could reduce tumorigenicity in miceTo further investigate the effect of LncRNA NNT-AS1 in vivo,we constructed a nude mouse model of osteosarcoma OS-732 cells and divided into two groups:1.control transfection group;2.Knockdown of LncRNA NNT-AS 1 group.The tumor growth curve showed that knockdown of LncRNA NNT-AS1 significantly reduced tumor growth volume.In addition,immunohistochemical staining analysis was performed to detect the expression of C-Myc in tumor tissues and found that the proliferation of tumor tissues in the LncRNA NNT-AS1 interference group(C-Myc staining)was significantly lower than that in the control transfection group.These results suggest that LncRNA NNT-AS1 plays an important role in the development and progression of osteosarcoma.Conclusions1.LncRNA NNT-AS1 is significantly up-regulated in osteosarcoma tissues and plays the role of proto-oncogenes;2.Knockdown of LncRNA NNT-AS1 could effectively inhibit osteosarcoma cell proliferation by affecting cell apoptosis and cell cycle;3.In vivo experiments showed that knockdown of LncRNA NNT-AS1 inhibited tumorigenic ability.