Department of Chemistry, University of Manitoba, Room 380 Parker Building, 144 Dysart Road, Winnipeg, MB, R3T 2N2, Canada.
Department of Immunology, University of Manitoba, 750 McDermot Ave, Winnipeg, R3E 0T5, MB, Canada.
Mol Cancer. 2017 Jun 26;16(1):109. doi: 10.1186/s12943-017-0679-7.
BC200 is a long non-coding RNA expressed at high levels in the brain and elevated in a variety of tumour types. BC200 has a hypothesized role in translational regulation; however, to date the functional role of BC200 in both normal and diseased states remains poorly characterized.
Detailed BC200 expression analyses were performed in tumor cell lines, primary and non-tumorigenic cultured breast and lung cells, and a panel of normal human tissues by quantitative real-time PCR and confirmed by northern blot. Subcellular fractionation was performed to assess BC200 distribution and efficient knock-down of BC200 was established using both locked nucleic acid (LNA) GapmeRs and conventional siRNAs. Cell viability following BC200 knockdown and overexpression was assessed by MTT assay and induction of apoptosis was monitored by Annexin V/PI staining and flow cytometry. Cell cycle arrest and synchronization were performed using serum withdrawal as well as the specific inhibitors Lovastatin, Thymidine, RO3306 and Nocodazole. Synchronization was monitored by fluorescent analysis of cellular DNA content by flow cytometry RESULTS: BC200 expression was substantially upregulated in brain and elevated expression was also observed in testes, small intestine and ovary. Expression in cultured tumour cells was dramatically higher than corresponding normal tissue; however, expression in cultured primary cells was similar to that in immortalized and cancer cell lines. BC200 knockdown resulted in a dramatic loss of viability through growth arrest and induction of apoptosis that could be partially rescued by overexpression of wild-type BC200 but not an siRNA-resistant sequence mutant. A substantial decrease in BC200 expression was observed upon cell confluence or serum deprivation, as well as drug induced cell cycle arrest in G1 or G2 but not S- or M-phases. Upon release from cell cycle arrest, BC200 expression was recovered as cells entered S-phase, but did not follow a periodic expression pattern during synchronized progression through the cell cycle. This elevated expression was critical for the survival of proliferating cancerous and non-cancerous cells, but is dispensable upon senescence or cell cycle arrest.
BC200 expression is elevated in proliferating cultured cells regardless of origin. In primary cells, expression is dramatically reduced upon cell cycle arrest by confluence, serum deprivation or chemical inhibition. The lethality of BC200 knockdown is restricted to actively proliferating cells, making it a promising therapeutic target for a broad spectrum of cancers.
BC200 是一种在大脑中高水平表达的长非编码 RNA，在多种肿瘤类型中升高。BC200 在翻译调节中具有假设作用；然而，迄今为止，BC200 在正常和疾病状态下的功能作用仍未得到充分描述。
通过定量实时 PCR 对肿瘤细胞系、原代和非肿瘤培养的乳腺和肺细胞以及一系列正常人体组织进行详细的 BC200 表达分析，并通过 northern blot 进行验证。进行亚细胞分级分离以评估 BC200 分布，并使用锁核酸 (LNA) GapmeRs 和常规 siRNAs 成功建立了 BC200 的有效敲低。通过 MTT 测定评估 BC200 敲低和过表达后的细胞活力，并通过 Annexin V/PI 染色和流式细胞术监测凋亡的诱导。通过血清撤出以及特定抑制剂洛伐他汀、胸苷、RO3306 和诺考达唑来进行细胞周期阻滞和同步。通过流式细胞术荧光分析细胞 DNA 含量来监测同步化。
BC200 的表达在大脑中大量上调，在睾丸中也观察到升高的表达；在培养的肿瘤细胞中的表达明显高于相应的正常组织；然而，在原代培养细胞中的表达与永生化和癌细胞系相似。BC200 敲低导致通过生长停滞和诱导凋亡导致活力显著丧失，野生型 BC200 的过表达可部分挽救，但不能挽救 siRNA 抗性序列突变体。在细胞汇合或血清剥夺时观察到 BC200 表达大量减少，以及药物诱导的 G1 或 G2 期细胞周期阻滞，但不是 S 或 M 期。从细胞周期阻滞释放后，随着细胞进入 S 期，BC200 表达得到恢复，但在同步通过细胞周期时没有遵循周期性表达模式。这种升高的表达对于增殖性癌性和非癌性细胞的存活至关重要，但在衰老或细胞周期阻滞时是可有可无的。
BC200 的表达在起源无关的增殖培养细胞中升高。在原代细胞中，通过细胞汇合、血清剥夺或化学抑制使细胞周期停滞时，表达显著降低。BC200 敲低的致死性仅限于活跃增殖的细胞，使其成为广泛癌症的有前途的治疗靶点。
