Department of Oncology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui Province, China.
Cell Death Dis. 2019 Mar 15;10(4):252. doi: 10.1038/s41419-019-1479-3.
Diffuse large B cell lymphoma (DLBCL) is a common and fatal hematological malignancy. Long noncoding RNAs (lncRNAs) have emerged as crucial biomarkers and regulators in many cancers. Novel lncRNA biomarker in DLBCL needs to be investigated badly, as well as its function and molecular mechanism. To further explore, microarray analysis was performed to identify the differentially expressed lncRNAs in DLBCL tissues. To investigate the biological functions of SMAD5-AS1, we performed gain- and loss-of-function experiments in vitro and in vivo. Furthermore, bioinformatics analysis, dual-luciferase reporter assays, Argonaute 2-RNA immunoprecipitation (AGO2-RIP), RNA pull-down assay, quantitative PCR arrays, western blot assay, TOPFlash/FOPFlash reporter assay, and rescue experiments were conducted to explore the underlying mechanisms of competitive endogenous RNAs (ceRNAs). We found that SMAD5-AS1 was down-regulated in DLBCL tissues and cell lines. Functionally, SMAD5-AS1 downregulation promoted cell proliferation in vitro and in vivo, whereas SMAD5-AS1 overexpression could lead to the opposite effects in vitro and in vivo. Bioinformatics analysis and luciferase assays revealed that miR-135b-5p was a direct target of SMAD5-AS1, which was validated by dual-luciferase reporter assays, AGO2-RIP, RNA pull-down assay, and rescue experiments. Also, dual-luciferase reporter assays and rescue experiments demonstrated that miR-135b-5p targeted the adenomatous polyposis coli (APC) gene directly. SMAD5-AS1/miR-135b-5p inhibits the cell proliferation via inactivating the classic Wnt/β-catenin pathway in the form of APC dependency. Our results indicated that SMAD5-AS1 inhibits DLBCL proliferation by sponging miR-135b-5p to up-regulate APC expression and inactivate classic Wnt/β-catenin pathway, suggesting that SMAD5-AS1 may act as a potential biomarker and therapeutic target for DLBCL.
弥漫性大 B 细胞淋巴瘤（DLBCL）是一种常见且致命的血液恶性肿瘤。长链非编码 RNA（lncRNA）已成为许多癌症中重要的生物标志物和调控因子。急需研究新型 DLBCL lncRNA 标志物及其功能和分子机制。为了进一步探索，我们进行了微阵列分析，以鉴定 DLBCL 组织中差异表达的 lncRNA。为了研究 SMAD5-AS1 的生物学功能，我们在体外和体内进行了增益和缺失功能实验。此外，还进行了生物信息学分析、双荧光素酶报告基因实验、Argonaute 2-RNA 免疫沉淀（AGO2-RIP）、RNA 下拉实验、定量 PCR 阵列、western blot 实验、TOPFlash/FOPFlash 报告基因实验和挽救实验，以探讨竞争性内源 RNA（ceRNA）的潜在机制。我们发现 SMAD5-AS1 在 DLBCL 组织和细胞系中下调。功能上，SMAD5-AS1 的下调促进了体外和体内的细胞增殖，而 SMAD5-AS1 的过表达则导致了相反的效果。生物信息学分析和荧光素酶报告基因实验表明，miR-135b-5p 是 SMAD5-AS1 的直接靶标，这通过双荧光素酶报告基因实验、AGO2-RIP、RNA 下拉实验和挽救实验得到了验证。此外，双荧光素酶报告基因实验和挽救实验表明，miR-135b-5p 直接靶向腺瘤性息肉病基因（APC）。SMAD5-AS1/miR-135b-5p 通过 APC 依赖性方式抑制经典 Wnt/β-catenin 通路的激活，抑制细胞增殖。我们的研究结果表明，SMAD5-AS1 通过海绵吸附 miR-135b-5p 抑制 DLBCL 增殖，上调 APC 表达并失活经典 Wnt/β-catenin 通路，提示 SMAD5-AS1 可能作为 DLBCL 的潜在生物标志物和治疗靶点。
