Long Non-coding RNA LINC02474 Affects Metastasis and Apoptosis of Colorectal Cancer by Inhibiting the Expression of GZMB

Background: Colorectal cancer (CRC) is one of the most frequently diagnosed malignancies. Metastasis is the main event that impedes the therapeutic effect on CRC, and its underlying mechanisms remain largely unclear. LINC02474 is a novel long noncoding RNA (lncRNA) associated with metastasis of CRC, while little is known about how LINC02474 regulates these malignant characteristics. Methods: Expressions of LINC02474 and granzyme B (GZMB) were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) or Western blotting analysis. Cell metastasis was detected by transwell assay and metastatic nude mouse model, and apoptosis was determined by Western blotting analysis and ?ow cytometry. Besides, the interaction between LINC02474 and GZMB was detected by dual-luciferase reporter assays. Results: The expression of LINC02474 was signi?cantly up-regulated in CRC tissues. Moreover, depletion of LINC02474 damaged the metastatic abilities of CRC cells in vivo and in vitro while boosting apoptosis. Besides, up-regulation of LINC02474 could promote migration and invasion, while apoptosis was inhibited in CRC cells. Besides, down-regulation of LINC02474 promoted the expression of GZMB, and interference of GZMB could increase the metastatic abilities of CRC cells while reducing apoptosis. Furthermore, LINC02474 was related to the transcriptional repression of GZMB in CRC cells determined by the dual-luciferase reporter assay. Conclusions: The ?ndings revealed that a novel lncRNA, LINC02474, as an oncogene, could promote metastasis, but limit apoptosis partly by impeding GZMB expression in CRC. Besides, LINC02474 had the potential to be used as a biomarker in the prognosis of CRC. Overall design: there are 6 samples totally which have been grouped into 2 groups including three duplicates for each group. For group named "DLD-1-356", LINC02474 is knocked down stably, and for group named "DLD-1-NC", it is the control group.

研究背景：结直肠癌（Colorectal cancer, CRC）是最常见的确诊恶性肿瘤之一。转移是阻碍结直肠癌治疗效果的核心事件，其潜在机制尚未完全阐明。LINC02474是一种与结直肠癌转移相关的新型长链非编码RNA（long noncoding RNA, lncRNA），但目前对其调控结直肠癌细胞恶性表型的具体机制仍知之甚少。
实验方法：采用实时荧光定量聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）或蛋白质免疫印迹（Western blotting）分析检测LINC02474与颗粒酶B（granzyme B, GZMB）的表达水平。通过Transwell实验及转移性裸鼠模型检测细胞转移能力，采用Western blotting分析与流式细胞术检测细胞凋亡情况。此外，通过双荧光素酶报告基因实验（dual-luciferase reporter assays）检测LINC02474与GZMB的相互作用。
实验结果：LINC02474在结直肠癌组织中的表达显著上调。敲低LINC02474可在体内外削弱结直肠癌细胞的转移能力，并促进细胞凋亡；反之，过表达LINC02474可增强结直肠癌细胞的迁移与侵袭能力，同时抑制细胞凋亡。下调LINC02474可促进GZMB的表达，而干扰GZMB则可增强结直肠癌细胞的转移能力并降低细胞凋亡水平。双荧光素酶报告基因实验证实，LINC02474可通过转录抑制调控结直肠癌细胞中GZMB的表达。
研究结论：本研究结果表明，新型长链非编码RNA LINC02474作为致癌基因，可通过抑制结直肠癌细胞中GZMB的表达，促进肿瘤转移并抑制细胞凋亡。此外，LINC02474有望作为结直肠癌预后评估的生物标志物。
整体实验设计：本研究共纳入6份样本，分为2组，每组设置3个生物学重复。其中“DLD-1-356”组为稳定敲低LINC02474的实验组，“DLD-1-NC”组为阴性对照组。