Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Jan 2;26(12):919-933.
doi: 10.3779/j.issn.1009-3419.2023.102.45.

[Study on the Role and Mechanism of METTL3 Mediating the Up-regulation of m6A Modified Long Non-coding RNA THAP7-AS1 in Promoting the Occurrence of Lung Cancer]

[Article in Chinese]
Yu Zhang  1 , Yanhong Wang  1 , Mei Liu  2
Affiliations

[Study on the Role and Mechanism of METTL3 Mediating the Up-regulation of m6A Modified Long Non-coding RNA THAP7-AS1 in Promoting the Occurrence of Lung Cancer]

[Article in Chinese]
Yu Zhang et al. Zhongguo Fei Ai Za Zhi. .

Abstract
in English, Chinese

Background: Lung cancer is a major threat to human health. The molecular mechanisms related to the occurrence and development of lung cancer are complex and poorly known. Exploring molecular markers related to the development of lung cancer is helpful to improve the effect of early diagnosis and treatment. Long non-coding RNA (lncRNA) THAP7-AS1 is known to be highly expressed in gastric cancer, but has been less studied in other cancers. The aim of the study is to explore the role and mechanism of methyltransferase-like 3 (METTL3) mediated up-regulation of N6-methyladenosine (m6A) modified lncRNA THAP7-AS1 expression in promoting the development of lung cancer.

Methods: Samples of 120 lung cancer and corresponding paracancerous tissues were collected. LncRNA microarrays were used to analyze differentially expressed lncRNAs. THAP7-AS1 levels were detected in lung cancer, adjacent normal tissues and lung cancer cell lines by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The diagnostic value of THAP7-AS1 in lung cancer and the relationship between THAP7-AS1 expression and survival rate and clinicopathological parameters were analyzed. Bioinformatics analysis, methylated RNA immunoprecipitation (meRIP), RNA pull-down and RNA-immunoprecipitation (RIP) assay were used to investigate the molecular regulation mechanism of THAP7-AS1. Cell proliferation, migration, invasion and tumorigenesis of SPC-A-1 and NCI-H1299 cells were determined by MTS, colony-formation, scratch, Transwell and xenotransplantation in vivo, respectively. Expression levels of phosphoinositide 3-kinase/protein kenase B (PI3K/AKT) signal pathway related protein were detected by Western blot.

Results: Expression levels of THAP7-AS1 were higher in lung cancer tissues and cell lines (P<0.05). THAP7-AS1 has certain diagnostic value in lung cancer [area under the curve (AUC)=0.737], and its expression associated with overall survival rate, tumor size, tumor-node-metastasis (TNM) stage and lymph node metastasis (P<0.05). METTL3-mediated m6A modification enhanced THAP7-AS1 expression. The cell proliferation, migration, invasion and the volume and mass of transplanted tumor were all higher in the THAP7-AS1 group compared with the NC group and sh-NC group of SPC-A-1 and NCI-H1299 cells, while the cell proliferation, migration and invasion were lower in the sh-THAP7-AS1 group (P<0.05). THAP7-AS1 binds specifically to Cullin 4B (CUL4B). The cell proliferation, migration, invasion, and expression levels of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), phosphoinositide-3 kinase, catalytic subunit delta (PIK3CD), phospho-phosphatidylinositol 3-kinase (p-PI3K), phospho-protein kinase B (p-AKT) and phospho-mammalian target of rapamycin (p-mTOR) were higher in the THAP7-AS1 group compared with the Vector group of SPC-A-1 and NCI-H1299 cells (P<0.05).

Conclusions: LncRNA THAP7-AS1 is stably expressed through m6A modification mediated by METTL3, and combines with CUL4B to activate PI3K/AKT signal pathway, which promotes the occurrence and development of lung cancer.

【中文题目：METTL3介导m6A修饰长链非编码RNA THAP7-AS1表达上调促进肺癌发生的作用及机制研究】 【中文摘要：背景与目的 肺癌是对人类健康的一大威胁，有关肺癌发生发展的分子机制复杂且知之尚少，探索与肺癌发展相关的分子标志物有利于提高早期诊断和治疗的效果。长链非编码RNA（long non-coding RNA , lncRNA）THAP7-AS1已知在胃癌中高表达，但在其他癌症中研究较少。本研究旨在探究甲基转移酶样3（methyltransferase-like 3, METTL3）介导N6-甲基腺苷（N6-methyladenosine, m6A）修饰lncRNA THAP7-AS1表达上调促进肺癌发生的作用及机制。方法 收集120例肺癌与对应癌旁组织样本，lncRNA微阵列分析差异表达的lncRNA，实时荧光定量聚合酶链式反应（real-time quantitative polymerase chain reaction, qRT-PCR）检测肺癌、癌旁组织、肺癌细胞系THAP7-AS1表达，分析THAP7-AS1对肺癌的诊断价值以及其表达水平与肺癌患者生存率、临床病理特征的关系。通过生物信息学分析、甲基化RNA免疫共沉淀（methylated RNA immunoprecipitation, meRIP）、RNA pull-down实验、RIP实验探究THAP7-AS1的分子调节机制；通过MTS、克隆形成、划痕、Transwell、体内异种移植实验测定各组SPC-A-1、NCI-H1299细胞增殖、迁移、侵袭、成瘤能力，Western blot检测磷脂酰肌醇-3激酶/蛋白激酶B（phosphoinositide 3-kinase/protein kenase B, PI3K/AKT）信号通路蛋白表达。结果 肺癌组织、细胞系THAP7-AS1表达升高（P<0.05），对肺癌具有一定的诊断价值［曲线下面积（area under the curve, AUC）=0.737］，其表达水平与患者总生存率、肿瘤大小、肿瘤原发灶-淋巴结-转移（tumor-node-metastasis, TNM）分期、淋巴结转移相关（P<0.05）。METTL3介导的m6A修饰能够增强THAP7-AS1表达。与SPC-A-1、NCI-H1299细胞NC组、sh-NC组相比，THAP7-AS1组增殖、迁移、侵袭能力提高（P<0.05），移植瘤体积、质量增大（P<0.05），sh-THAP7-AS1组增殖、迁移、侵袭能力下降（P<0.05）。THAP7-AS1与Cullin蛋白4B（Cullin 4B, CUL4B）存在特异结合。与SPC-A-1、NCI-H1299细胞Vector组相比，THAP7-AS1组增殖、迁移、侵袭能力、磷脂酰肌醇-4,5-二磷酸3-激酶催化亚基α（phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha, PI3KCA）、磷脂酰肌醇-3激酶催化亚基δ（phosphoinositide-3 kinase-catalytic subunit delta, PI3KCD）、磷酸化磷脂酰肌醇3-激酶（phospho-phosphatidylinositol 3-kinase, p-PI3K）、磷酸蛋白激酶B（phospho-protein kinase B, p-AKT）、磷酸哺乳动物雷帕霉素靶蛋白（phospho-mammalian target of rapamycin, p-mTOR）表达水平升高（P<0.05）。结论 LncRNA THAP7-AS1通过METTL3介导的m6A修饰稳定表达，与CUL4B结合激活PI3K/AKT信号通路，促进肺癌发生发展。 】 【中文关键词：甲基转移酶样3；N6-甲基腺苷；长链非编码RNA THAP7-AS1；肺肿瘤；增殖】.

Keywords: Long non-coding RNA THAP7-AS1; Lung neoplasms; Methyltransferase-like 3; N6-methyladenosine; Proliferation.

PubMed Disclaimer

Figures

Fig 1
图1. THAP7-AS1在肺癌中表达上调。A：10例肺癌与2例癌旁组织lncRNA表达谱聚类热图；B：120对肺癌与癌旁组织qRT-PCR检测THAP7-AS1、LOC100133669表达水平；C：肺癌细胞系qRT-PCR检测THAP7-AS1表达水平。*P<0.05。
Fig 2
图2. THAP7-AS1在SPC-A-1、NCI-H1299细胞中的定位（×400）。A：FISH实验染色结果；B：THAP7-AS1在SPC-A-1、NCI-H1299细胞的胞核和胞浆中的相对表达水平。
Fig 3
图3. THAP7-AS1与肺癌诊断、预后及临床病理特征的关系。A：ROC曲线评价THAP7-AS1对肺癌的诊断价值；B：Kaplan-Meier Plotter数据库不同THAP7-AS1水平患者生存分析结果。
Fig 4
图4. METTL3介导m6A修饰增强THAP7-AS1表达。A：m6A修饰的THAP7-AS1位点3、5在SPC-A-1、NCI-H1299细胞中富集；B：qRT-PCR检测过表达或下调METTL3对SPC-A-1、NCI-H1299细胞THAP7-AS1表达水平变化；C：qRT-PCR检测120对肺癌与癌旁组织THAP7-AS1表达情况；D：肺癌组织THAP7-AS1与METTL3表达水平的相关性分析。*P<0.05。
Fig 5
图5. THAP7-AS1促进肺癌细胞增殖。A：构建SPC-A-1、NCI-H1299 THAP7-AS1过表达与沉默稳定表达细胞株，MTS检测细胞增殖率；B：EdU实验检测细胞DNA复制活性（×200）；C：克隆形成实验检测细胞克隆形成能力。*：与NC组相比，P<0.05；#：与sh-NC组相比，P<0.05。
Fig 6
图6. THAP7-AS1促进肺癌细胞迁移和侵袭。A：构建SPC-A-1、NCI-H1299 THAP7-AS1过表达与沉默稳定表达细胞株，划痕实验检测细胞迁移能力（×100）；B：Transwell实验检测细胞侵袭能力（×200）。*：与NC组相比，P<0.05；#：与sh-NC组相比，P<0.05。
Fig 7
图7. THAP7-AS1增强肺癌细胞成瘤能力。A：构建稳定过表达THAP7-AS1的SPC-A-1、NCI-H1299细胞株，体内异种移植实验肿瘤图片；B：移植瘤体积检测结果；C：移植瘤质量检测结果。*P<0.05。
Fig 8
图8. THAP7-AS1特异性结合蛋白筛选验证。A：THAP7-AS1蛋白质复合体银染实验结果；B：蛋白质组学Western blot验证结果；C：RIP检测CUL4B与THAP7-AS1结合情况。
Fig 9
图9. THAP7-AS1结合CUL4B促进肺癌细胞增殖、迁移、侵袭。A：构建SPC-A-1、NCI-H1299 THAP7-AS1过表达与沉默CUL4B稳定表达细胞株，MTS检测细胞增殖率；B：划痕实验检测细胞迁移能力（×100）；C：Transwell实验检测细胞侵袭能力（×200）。*P<0.05。
Fig 10
图10. THAP7-AS1结合CUL4B启动PI3K/AKT信号通路。SPC-A-1（A）、NCI-H1299（B）THAP7-AS1过表达与沉默CUL4B稳定表达细胞株，Western blot检测PI3K/AKT信号通路相关蛋白表达情况。*P<0.05。
Fig 11
图11. METTL3介导m6A修饰lncRNA THAP7-AS1表达上调促进肺癌发生的具体作用机制
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Cao W, Chen HD, Yu YW, et al. . . Changing profiles of cancer burden worldwide and in China: a secondary analysis of the global cancer statistics 2020. Chin Med J (Engl), 2021, 134(7): 783-791. doi: 10.1097/CM9.0000000000001474 - DOI - PMC - PubMed
    1. Dama E, Colangelo T, Fina E, et al. . . Biomarkers and lung cancer early detection: state of the art. Cancers (Basel), 2021, 13(15): 3919-3939. doi: 10.3390/cancers13153919 - DOI - PMC - PubMed
    1. Yang CY, Lin YT, Lin LJ, et al. . . Stage shift improves lung cancer survival: real-world evidence. J Thorac Oncol, 2023, 18(1): 47-56. doi: 10.1016/j.jtho.2022.09.005 - DOI - PubMed
    1. Rahman MM, Hossain MT, Reza MS, et al. . . Identification of potential long non-coding RNA candidates that contribute to triple-negative breast cancer in humans through computational approach. Int J Mol Sci, 2021, 22(22): 12359-12372. doi: 10.3390/ijms222212359 - DOI - PMC - PubMed
    1. Luo L, Zhen Y, Peng D, et al. . . The role of N6-methyladenosine-modified non-coding RNAs in the pathological process of human cancer. Cell Death Discov, 2022, 8(1): 325-339. doi: 10.1038/s41420-022-01113-2 - DOI - PMC - PubMed

Publication types

MeSH terms