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LncRNA CALML3-AS1 modulated by m6A modification induces BTNL9 methylation to drive non-small-cell lung cancer

Heng Zhang1,2,3, Shao-Qiang Wang4,5, Jie-Bo Zhu6

  • 1Department of Thoracic Surgery, Xiangya Hospital, Central South University, Changsha, 410008, Hunan Province, P. R. China. 404346@csu.edu.cn.

Cancer Gene Therapy
|October 26, 2023
PubMed

Insights

Long non-coding RNA CALML3-AS1 promotes non-small cell lung cancer (NSCLC) progression. It enhances tumor growth and metastasis by epigenetically silencing BTNL9, modulated by m6A modification.

Area of Science:

  • Molecular Oncology
  • Epigenetics
  • Non-small Cell Lung Cancer (NSCLC) Research

Background:

  • Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related mortality.
  • The long non-coding RNA CALML3 antisense RNA 1 (CALML3-AS1) has been implicated in carcinogenesis, but its precise role and mechanisms in NSCLC require further elucidation.

Purpose of the Study:

  • To investigate the functional role of CALML3-AS1 in NSCLC progression.
  • To elucidate the underlying molecular mechanisms by which CALML3-AS1 influences NSCLC pathogenesis, including its interaction with epigenetic regulators and target genes.

Main Methods:

  • Quantitative real-time PCR (qRT-PCR) and Western blot for gene expression analysis.
  • Fluorescence in situ hybridization (FISH) for subcellular localization.
  • Cellular assays (CCK-8, colony formation, EdU, wound healing, Transwell) and in vivo models (xenograft, liver metastasis) to assess malignant behaviors.
  • RNA immunoprecipitation (RIP), RNA pull-down, and chromatin immunoprecipitation (ChIP) assays to investigate molecular interactions.
  • Methylation-specific PCR (MSP) to detect methylation levels.

Main Results:

  • CALML3-AS1 was significantly upregulated, while butyrophilin-like 9 (BTNL9) was downregulated in NSCLC tissues and cells.
  • Depletion of CALML3-AS1 suppressed NSCLC cell proliferation, migration, invasion, tumor growth, and liver metastasis.
  • CALML3-AS1 stability is regulated by N6-methyladenosine (m6A) modification, with AlkB homolog 5 (ALKBH5) enhancing its stability and YTH domain-containing 2 (YTHDC2) destabilizing it.
  • CALML3-AS1 epigenetically repressed BTNL9 expression by recruiting Zeste homolog 2 (EZH2).
  • Restoration of BTNL9 expression partially rescued the antitumor effects of CALML3-AS1 knockdown.

Conclusions:

  • CALML3-AS1 acts as a crucial oncogenic lncRNA in NSCLC, promoting malignant phenotypes and metastasis.
  • The ALKBH5/m6A/YTHDC2 axis modulates CALML3-AS1 stability, influencing its oncogenic function.
  • CALML3-AS1 drives NSCLC progression by epigenetically inhibiting BTNL9, highlighting a potential therapeutic target.

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