Identification of TERT Delta 2-4 knockdown effect by Long read RNA seq.

All cancer cells must adopt a telomere maintenance mechanism to achieve replicative immortality. Most human cancer cells utilize the enzyme telomerase to maintain telomeres. Alternative splicing of TERT regulates the amount and function of telomerase, however many alternative splicing isoforms of TERT have unknown functions. Single molecule long read RNA/cDNA sequencing of TERT revealed 45 TERT mRNA variants including 13 known and 32 novel variants. Among the variants, TERT Delta 2-4, which lacks exons 2-4 but retains the original open reading frame, was selected for further study. Induced pluripotent stem cells and cancer cells express higher levels of TERT Delta 2-4 compared to primary human bronchial epithelial cells. Overexpression of TERT Delta 2-4 enhanced clonogenicity and resistance to cisplatin- induced apoptosis. Knockdown of endogenous TERT Delta 2-4 in Calu-6 cells reduced clonogenicity and resistance to cisplatin. Our results suggest that TERT Delta 2-4 enhances cancer cells’ resistance to intrinsic apoptosis. RNA sequencing following knockdown of Delta 2-4 TERT indicates that translation is downregulated and that mitochondrial related proteins are upregulated compared to controls. Overall, our data indicate that TERT produces many isoforms that influence the function TERT and the abundance and activity of telomerase. Calu-6 cells, which are non-small cell lung cancer (NSCLC) cells, treated with siRNA targeting TERT Delta 2-4 had RNA extracted, mRNA purified, cDNA made and RNA-Seq libraries generated. The libraries were then sequenced on an Oxford Nanopore MinIonMk1c

所有癌细胞必须获得端粒维持机制，方可实现复制永生。绝大多数人类癌细胞通过端粒酶这一核心酶类维持端粒稳态。端粒酶逆转录酶（Telomerase Reverse Transcriptase, TERT）的可变剪接可调控端粒酶的表达量与功能，但目前多数TERT可变剪接异构体的功能仍未明确。研究人员针对TERT开展单分子长读长RNA/互补DNA（complementary DNA, cDNA）测序，共鉴定出45种TERT mRNA变异体，其中13种为已报道的已知变异体，剩余32种为全新发现的变异体。在上述变异体中，缺失外显子2-4但保留完整开放阅读框的TERT Δ2-4被选为后续研究靶点。与原代人支气管上皮细胞相比，诱导多能干细胞与癌细胞中TERT Δ2-4的表达水平显著更高。过表达TERT Δ2-4可增强癌细胞的集落形成能力，并提升其对顺铂诱导细胞凋亡的抵抗性。而在Calu-6细胞中敲低内源性TERT Δ2-4后，细胞的集落形成能力与顺铂抵抗性均显著降低。本研究结果表明，TERT Δ2-4可增强癌细胞对内在凋亡通路的抵抗性。对TERT Δ2-4敲低后的细胞进行RNA测序分析显示，与对照组相比，细胞内翻译相关通路被显著下调，而线粒体相关蛋白的表达水平则出现上调。综上，本研究数据证实，TERT可产生多种功能性异构体，这些异构体可调控端粒酶的功能、丰度与活性。本研究中，研究人员对经靶向TERT Δ2-4的小干扰RNA（siRNA）处理的非小细胞肺癌（NSCLC）Calu-6细胞进行样本制备：提取总RNA、纯化mRNA、合成cDNA并构建RNA测序文库，随后使用牛津纳米孔MinIon Mk1c测序平台完成文库测序。