Background. Long noncoding RNAs (lncRNAs) have critical regulatory functions in biological and pathological activities during osteosarcoma progression. It is important to elucidate the expression pattern and reveal the underlying mechanisms of the newly identified lncRNAs. Methods. Herein, we screened the differentially expressed lncRNAs in osteosarcoma tumors and cell lines using lncRNA microarray. The candidate lncRNA was further verified by qRT-PCR, and the association of gene expression with clinicopathological features was evaluated by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The targeting miRNA was identified using starBase analysis, and the competing endogenous RNA (ceRNA) network was established by STRING. Overexpression and silence of RNA were detected by qRT-PCR. Osteosarcoma cell proliferation was measured with CCK-8 assay, and the migration and invasion were evaluated with Transwell assay. Colony formation assay was observed. Flow cytometry evaluated the cell cycle. Western blot was performed to detect the mitotic markers and apoptosis-related proteins. A nude mouse tumor formation experiment was used to evaluate osteosarcoma progression in vivo. Cooverexpressing miR-34b-3p with RAD51 reversed the miR-34b-3p-induced changes in proliferation, the cell cycle, the expression of H2A.X, and that of apoptosis-related proteins. Results. HCG9 was identified as osteosarcoma-associated lncRNA. Osteosarcoma tissues and cell lines expressed higher levels of HCG9 as compared to normal tissues and osteoblasts, and high expression of HCG9 was further proved to be related to metastasis and the grade of osteosarcoma in clinical cases. Knockdown of HCG9 inhibited the proliferation, migration, and invasion of osteosarcoma cells. miR-34b-3p was identified as the target of HCG9, and RAD51 acted as a potential target of miR-34b-3p. Cooverexpressing miR-34b-3p with HCG9 partially suppressed the HCG9-stimulated proliferation, migration, and invasion of osteosarcoma cells in vitro and delayed the tumor progression in vivo. Conclusion. We discovered that lncRNA HCG9 promoted the proliferation of osteosarcoma cells via suppressing miR-34b-3p. Our study provides novel biomarkers and potential therapeutic targets for osteosarcoma treatment.

中文翻译：

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长链非编码 RNA HCG9 作为 miR-34b-3p 的 ceRNA 通过 RAD51 促进骨肉瘤进展

背景。长链非编码 RNA (lncRNA) 在骨肉瘤进展过程中在生物和病理活动中具有关键的调节功能。阐明表达模式并揭示新发现的 lncRNA 的潜在机制非常重要。方法. 在此，我们使用 lncRNA 微阵列筛选了骨肉瘤肿瘤和细胞系中差异表达的 lncRNA。通过qRT-PCR进一步验证候选lncRNA，并通过京都基因和基因组百科全书（KEGG）通路分析评估基因表达与临床病理学特征的关联。使用starBase分析鉴定靶向miRNA，并通过STRING建立竞争性内源性RNA（ceRNA）网络。通过qRT-PCR检测RNA的过表达和沉默。用CCK-8法测定骨肉瘤细胞增殖，用Transwell法测定迁移和侵袭。观察到集落形成测定。流式细胞术评估细胞周期。进行蛋白质印迹以检测有丝分裂标志物和凋亡相关蛋白。在体内。将 miR-34b-3p 与 RAD51 共过表达可逆转 miR-34b-3p 诱导的增殖、细胞周期、H2A.X 表达和凋亡相关蛋白的变化。结果. HCG9被鉴定为骨肉瘤相关的lncRNA。与正常组织和成骨细胞相比，骨肉瘤组织和细胞系表达更高水平的HCG9，临床病例进一步证明HCG9的高表达与骨肉瘤的转移和分级有关。敲除 HCG9 可抑制骨肉瘤细胞的增殖、迁移和侵袭。miR-34b-3p 被确定为 HCG9 的靶标，RAD51 充当 miR-34b-3p 的潜在靶标。与 HCG9 共过表达 miR-34b-3p 在体外部分抑制了 HCG9 刺激的骨肉瘤细胞的增殖、迁移和侵袭，并在体内延缓了肿瘤进展。结论. 我们发现lncRNA HCG9通过抑制miR-34b-3p促进骨肉瘤细胞的增殖。我们的研究为骨肉瘤的治疗提供了新的生物标志物和潜在的治疗靶点。