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

Cancer Gene Therapy volume 30, pages 1649–1662 (2023)Cite this article

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Abstract

Non-small cell lung cancer (NSCLC) is a common and lethal malignancy. The carcinogenic roles of lncRNA CALML3 antisense RNA 1 (CALML3-AS1) have been documented. However, the function and potential mechanisms of CALML3-AS1 in the progression of NSCLC need to be further explored. The molecule expression was assessed by qRT-PCR and Western blot. The subcellular localization of CALML3-AS1 was observed by fluorescence in situ hybridization (FISH). The malignant behaviors of NSCLC cells were evaluated by CCK-8, colony formation, EdU, wound healing and transwell assays. In vivo xenograft tumor and liver metastatic models were established. The molecular mechanisms were investigated by RIP, RNA pull-down and ChIP assays. The methylation level was detected by MSP. Herein, we found that CALML3-AS1 was upregulated, while butyrophilin-like 9 (BTNL9) was downregulated in NSCLC. Functionally, CALML3-AS1 depletion repressed NSCLC cell malignant phenotypes, in vivo tumor growth, and liver metastasis. Mechanistically, AlkB homolog 5 (ALKBH5) enhanced CALML3-AS1 stability via N6-methyladenosine (m6A) demethylation, whereas m6A reader YTH domain-containing 2 (YTHDC2) destabilized CALML3-AS1. Moreover, CALML3-AS1 inhibited BTNL9 transcription and expression through the recruitment of Zeste homolog 2 (EZH2). Rescue experiments demonstrated that BTNL9 downregulation counteracted sh-CALML3-AS1-mediated antitumor effects on NSCLC. Taken together, CALML3-AS1 modulated by ALKBH5 and YTHDC2 in an m6A modification dependent manner drives NSCLC progression via epigenetically repressing BTNL9.

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Fig. 1: Upregulation of CALML3-AS1 in NSCLC indicated a poor prognosis.
Fig. 2: CALML3-AS1 knockdown inhibited NSCLC cell growth and metastasis.
Fig. 3: ALKBH5 increased CALML3-AS1 stability via inhibiting m6A modification.
Fig. 4: CALML3-AS1 could be destabilized by YTHDC2 in an m6A dependent manner.
Fig. 5: CALML3-AS1 epigenetically inhibited BTNL9 expression by interaction with EZH2.
Fig. 6: BTNL9 overexpression restrained the malignant phenotypes of NSCLC cells.
Fig. 7: CALML3-AS1 promoted the malignant phenotypes of NSCLC cells via regulating BTNL9.
Fig. 8: Knockdown of CALML3-AS1 delayed NSCLC growth and metastasis in vivo.

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All data generated or analysed during this study are included in this published article.

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Acknowledgements

We would like to give our sincere gratitude to the reviewers for their constructive comments.

Funding

This work was supported by Hunan Provincial Natural Science Foundation of China (No.2023JJ30970), Changsha Municipal Natural Science Foundation (No.kq2208396) and National Multidisciplinary Cooperative Diagnosis and Treatment Capacity Building Project for Major Diseases (No. z027002).

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Authors and Affiliations

  1. Department of Thoracic Surgery, Xiangya Hospital, Central South University, Changsha, 410008, Hunan Province, P. R. China

    Heng Zhang, Jie-Bo Zhu, Hang Lin, Lin-Feng Li, Yuan-Da Cheng, Chao-Jun Duan & Chun-Fang Zhang

  2. Xiangya Lung Cancer Center, Xiangya Hospital, Central South University, Changsha, 410008, Hunan Province, P. R. China

    Heng Zhang & Chun-Fang Zhang

  3. National Clinical Research Center for Geriatric Disorders (Xiangya Hospital), Changsha, 410008, Hunan Province, P. R. China

    Heng Zhang & Chun-Fang Zhang

  4. Department of Thoracic Surgery, Weifang People’s Hospital, Weifang Medical University, Weifang, 261041, Shandong Province, P.R. China

    Shao-Qiang Wang

  5. Department of Scientific Research Management, Weifang People’s Hospital, Weifang Medical University, Weifang, 261041, Shandong Province, P.R. China

    Shao-Qiang Wang

  6. Department of Thoracic Surgery, Affiliated Hospital of Jining Medical University, Jining, 272029, Shandong Province, P. R. China

    Li-Na Wang

  7. Hunan Engineering Research Center for Pulmonary Nodules Precise Diagosis & Treatment, Changsha, 410008, Hunan Province, P. R. China

    Chun-Fang Zhang

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Contributions

HZ designed this study. S-QW, J-BZ, L-NW, HL, L-FL, Y-DC, and C-JD collected the materials and performed the experiments. HZ and C-FZ analysed the data and wrote the manuscript. HZ revised the manuscript. All authors read and approved the final version of the manuscript.

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Correspondence to Heng Zhang or Chun-Fang Zhang.

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This study was approved by the ethics committee of Xiangya Hospital, Central South University. All the NSCLC patients signed their informed consent. All animal experiments were approved by the Institutional Animal Care and Use Committee of Xiangya Hospital, Central South University.

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Zhang, H., Wang, SQ., Zhu, JB. et al. LncRNA CALML3-AS1 modulated by m6A modification induces BTNL9 methylation to drive non-small-cell lung cancer progression. Cancer Gene Ther 30, 1649–1662 (2023). https://doi.org/10.1038/s41417-023-00670-7

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