Department of Colorectal Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China.
Department of Plastic Surgery, Central South University Third Xiangya Hospital, Changsha 410013, Hunan, China.
Int J Biol Sci. 2022 Apr 24;18(7):3048-3065. doi: 10.7150/ijbs.70630. eCollection 2022.
Long noncoding RNAs (lncRNAs) regulate multiple biological effects in cancers. Recently, RNA methylation has been found to modify not only coding RNAs but also some noncoding RNAs. How RNA methylation affects lncRNAs to affect colorectal cancer (CRC) progression remains elusive. The expression of LINC01559 was explored through RNA sequencing, quantitative real-time PCR (qRT-PCR) and hybridization (ISH). The preliminary exploration of its function was performed using Western blotting (WB) and immunohistochemistry (IHC). Functional experiments and were conducted to explore the biological functions of LINC01559 in CRC. The LINC01559/miR-106-5p/PTEN axis was verified through fluorescence hybridization (FISH), luciferase assays, and rescue experiments. RIP-sequencing, m6A RNA immunoprecipitation (MeRIP) assays and bioinformatic analysis were conducted to determine the upstream mechanism of LINC01559. The results showed that LINC01559 was downregulated in CRC compared with normal controls. Lower expression of LINC01559 in CRC patients predicted a poor prognosis. In addition, PTEN was found to be positively correlated with LINC01559, and miR-106b-5p could be the link between LINC01559 and PTEN. Then, silencing LINC01559 restored the malignant phenotype of CRC cells, while cotransfection of miR-106b-5p inhibitor neutralized this effect. Mechanistically, we found abundant m6A modification sites on LINC01559. Then, we uncovered these sites as potential targets of METTL3 through experiments . The results revealed a negative functional regulation of the LINC01559/miR-106b-5p/PTEN axis in CRC progression and explored a new mechanism of METTL3-mediated m6A modification on LINC01559. These results elucidate a novel potential therapeutic target for CRC treatment.
长链非编码 RNA（lncRNA）在癌症中调节多种生物学效应。最近，人们发现 RNA 甲基化不仅可以修饰编码 RNA，还可以修饰一些非编码 RNA。RNA 甲基化如何影响 lncRNA 从而影响结直肠癌（CRC）的进展仍然难以捉摸。通过 RNA 测序、定量实时 PCR（qRT-PCR）和杂交（ISH）探索 LINC01559 的表达。通过 Western blot（WB）和免疫组织化学（IHC）初步探索其功能。进行功能实验以探索 LINC01559 在 CRC 中的生物学功能。通过荧光杂交（FISH）、荧光素酶测定和挽救实验验证 LINC01559/miR-106-5p/PTEN 轴。进行 RIP-seq、m6A RNA 免疫沉淀（MeRIP）测定和生物信息学分析以确定 LINC01559 的上游机制。结果表明，与正常对照相比，CRC 中 LINC01559 的表达下调。CRC 患者中 LINC01559 的低表达预示着预后不良。此外，发现 PTEN 与 LINC01559 呈正相关，miR-106b-5p 可以作为 LINC01559 和 PTEN 之间的联系。然后，沉默 LINC01559 恢复了 CRC 细胞的恶性表型，而共转染 miR-106b-5p 抑制剂则中和了这种作用。从机制上讲，我们在 LINC01559 上发现了丰富的 m6A 修饰位点。然后，我们通过实验发现这些位点可能是 METTL3 的潜在靶标。结果揭示了 LINC01559/miR-106b-5p/PTEN 轴在 CRC 进展中的负功能调节，并探索了 METTL3 介导的 LINC01559 上 m6A 修饰的新机制。这些结果阐明了 CRC 治疗的新潜在治疗靶点。
