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Abstract：
OBJECTIVE: Moesin-ezrin-radixin-like protein (Merlin) has been identified as a tumor suppressor in several types of cancers. However, the biological function of Merlin in osteosarcoma remains unclear. MicroRNAs (miRNAs) can influence cancer progression by targeting oncogenes or anti-oncogenes. In this study, we sought to evaluate the regulation of Merlin expression by miR-25-3p and the role of the miR-25-3p/Merlin axis in osteosarcoma progression, with the aim of identifying a potential therapeutic target for osteosarcoma.
METHODS: TCGA (The Cancer Genome Atlas) database was used to analyze the correlation between Merlin expression and prognosis. RT-qPCR and Western blotting analyses were performed to compare Merlin expression between normal and malignant cells. A dual-luciferase reporter assay was performed to evaluate the direct targeting of Merlin by miR-25-3p. We overexpressed miR-25-3p, or/and Merlin, in U-2 OS and 143B cells, and studied their cellular functions in vitro. MTT and colony formation assays were performed to determine the effects on cell growth. EdU and cell cycle assays were performed to analyze the effects in cell replication. We used annexin V-fluorescein isothiocyanate and propidium iodide to stain apoptotic cells, and analyzed the cells using flow cytometry. The effects on cell metastasis were studied in wound healing and transwell assays. Lastly, the underlying mechanism was determined in RT-qPCR and Western blotting experiments.
RESULTS: Low Merlin expression was linked to poor prognosis. miR-25-3p was observed to directly target Merlin and downregulate its expression. miR-25-3p promoted cell growth, migration, and invasion, and inhibited apoptosis induced by cisplatin. Moreover, the overexpression of Merlin reversed the abovementioned effects of miR-25-3p. Further, the miR-25-3p/Merlin axis was observed to play an important role in the Hippo pathway, and regulated the expression of genes such as BIRC5, CTGF, and CYR61.
CONCLUSIONS: miR-25-3p functions as an oncogenic microRNA in osteosarcoma by targeting Merlin, and may serve as a potential therapeutic target for osteosarcoma.
METHODS: TCGA (The Cancer Genome Atlas) database was used to analyze the correlation between Merlin expression and prognosis. RT-qPCR and Western blotting analyses were performed to compare Merlin expression between normal and malignant cells. A dual-luciferase reporter assay was performed to evaluate the direct targeting of Merlin by miR-25-3p. We overexpressed miR-25-3p, or/and Merlin, in U-2 OS and 143B cells, and studied their cellular functions in vitro. MTT and colony formation assays were performed to determine the effects on cell growth. EdU and cell cycle assays were performed to analyze the effects in cell replication. We used annexin V-fluorescein isothiocyanate and propidium iodide to stain apoptotic cells, and analyzed the cells using flow cytometry. The effects on cell metastasis were studied in wound healing and transwell assays. Lastly, the underlying mechanism was determined in RT-qPCR and Western blotting experiments.
RESULTS: Low Merlin expression was linked to poor prognosis. miR-25-3p was observed to directly target Merlin and downregulate its expression. miR-25-3p promoted cell growth, migration, and invasion, and inhibited apoptosis induced by cisplatin. Moreover, the overexpression of Merlin reversed the abovementioned effects of miR-25-3p. Further, the miR-25-3p/Merlin axis was observed to play an important role in the Hippo pathway, and regulated the expression of genes such as BIRC5, CTGF, and CYR61.
CONCLUSIONS: miR-25-3p functions as an oncogenic microRNA in osteosarcoma by targeting Merlin, and may serve as a potential therapeutic target for osteosarcoma.
摘要：
目的：Moesin-ezrin-radixin-like蛋白（Merlin）已被确定为几种癌症的肿瘤抑制因子。然而,Merlin在骨肉瘤中的生物学功能尚不清楚。MicroRNAs（miRNA）可以通过靶向癌基因或抗癌基因来影响癌症进展。在这项研究中,我们试图评估miR-25-3p对Merlin表达的调节以及miR-25-3p/Merlin轴在骨肉瘤进展中的作用，目的是确定骨肉瘤的潜在治疗靶点。
方法：使用TCGA（癌症基因组图谱）数据库分析Merlin表达与预后的相关性。进行RT-qPCR和Western印迹分析以比较正常和恶性细胞之间的Merlin表达。进行双荧光素酶报告基因测定以评估miR-25-3p对Merlin的直接靶向。我们过表达miR-25-3p，或/和梅林，在U-2OS和143B单元中，并在体外研究了它们的细胞功能。进行MTT和集落形成测定以确定对细胞生长的影响。进行EdU和细胞周期测定以分析对细胞复制的影响。我们使用膜联蛋白V-异硫氰酸荧光素和碘化丙啶染色凋亡细胞，并使用流式细胞仪分析细胞。在伤口愈合和transwell测定中研究了对细胞转移的影响。最后,在RT-qPCR和Western印迹实验中确定了潜在的机制.
结果：Merlin低表达与不良预后相关。观察到miR-25-3p直接靶向Merlin并下调其表达。miR-25-3p促进细胞生长，迁移,和入侵,并抑制顺铂诱导的细胞凋亡。此外，Merlin的过表达逆转了miR-25-3p的上述作用。Further,观察到miR-25-3p/Merlin轴在Hippo途径中起重要作用，并调节BIRC5，CTGF等基因的表达，CYR61
结论：miR-25-3p通过靶向Merlin在骨肉瘤中作为致癌microRNA发挥作用，并可作为骨肉瘤的潜在治疗靶点。
方法：使用TCGA（癌症基因组图谱）数据库分析Merlin表达与预后的相关性。进行RT-qPCR和Western印迹分析以比较正常和恶性细胞之间的Merlin表达。进行双荧光素酶报告基因测定以评估miR-25-3p对Merlin的直接靶向。我们过表达miR-25-3p，或/和梅林，在U-2OS和143B单元中，并在体外研究了它们的细胞功能。进行MTT和集落形成测定以确定对细胞生长的影响。进行EdU和细胞周期测定以分析对细胞复制的影响。我们使用膜联蛋白V-异硫氰酸荧光素和碘化丙啶染色凋亡细胞，并使用流式细胞仪分析细胞。在伤口愈合和transwell测定中研究了对细胞转移的影响。最后,在RT-qPCR和Western印迹实验中确定了潜在的机制.
结果：Merlin低表达与不良预后相关。观察到miR-25-3p直接靶向Merlin并下调其表达。miR-25-3p促进细胞生长，迁移,和入侵,并抑制顺铂诱导的细胞凋亡。此外，Merlin的过表达逆转了miR-25-3p的上述作用。Further,观察到miR-25-3p/Merlin轴在Hippo途径中起重要作用，并调节BIRC5，CTGF等基因的表达，CYR61
结论：miR-25-3p通过靶向Merlin在骨肉瘤中作为致癌microRNA发挥作用，并可作为骨肉瘤的潜在治疗靶点。
