circRACGAP1 promotes non-small cell lung cancer proliferation by regulating miR-144-5p/CDKL1 signaling pathway

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Abstract

Circular RNAs (circRNAs) are involved in the regulation of many pathophysiological processes as non-coding RNAs. This study focuses on the role of circRACGAP1 in the development of non-small cell lung cancer (NSCLC). Expression patterns of circRACGAP1 and miR-144-5p in NSCLC tissues and cell lines were quantified by qRT-PCR analysis. Then, the function of circRACGAP1 on cell proliferation and tumorigenesis were confirmed in vitro and in vivo using CCK-8 assay, colony formation, EdU incorporation, and xenograft technique. The regulation of circRACGAP1 on Gefitinib resistance of NSCLC cells was evaluated by flow cytometry. The regulatory network of circRACGAP1/miR-144-5p/CDKL1 was verified by luciferase reporter assay and RNA pull-down. Western blotting analysis was performed to assess the biomarkers of cell cycle and apoptosis-associated proteins. CircRACGAP1 was highly expressed and miR-144-5p was inhibited both in NSCLC tissues and cell lines, suggesting their negative correlation in NSCLC. Knockdown of circRACGAP1 suppressed cell proliferation via arresting the cell cycle. miR-144-5p was identified as a downstream target to reverse circRACGAP1-mediated cell proliferation. miR-144-5p directly targeted the 3′-UTR of CDKL1 to regulate cell cycle of NSCLC cells. circRACGAP1 knockdown dramatically inhibited the tumor growth and enhanced the sensitivity of NSCLC to Gefitinib in vitro and in vivo. In summary, our study revealed a novel machinery of circRACGAP1/miR-144-5p/CDKL1 for the NSCLC tumorigenesis and development, providing potential diagnostic and therapeutic targets for NSCLC.

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Fig. 1: Expression of circRACGAP1 and miR-144-5p in NSCLC tissues and cell lines.
Fig. 2: Knockdown of circRACGAP1 inhibits the NSCLC cell proliferation.
Fig. 3: circRACGAP1 acts as a sponge of miR-144-5p in NSCLC cells.
Fig. 4: miR-144-5p reverses NSCLC cells proliferation induced by circRACGAP1.
Fig. 5: miR-144-5p targeted CDKL1 in NSCLC cells.
Fig. 6: circRACGAP1 promoted the NSCLC cell proliferation through miR-144-5p/CDKL1.
Fig. 7: circRACGAP1 promoted the NSCLC cell proliferation and drug resistance in vitro.
Fig. 8: circRACGAP1 promoted NSCLC tumor growth and enhanced drug resistance.

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Guarantor of integrity of the entire study: PL; Study concepts: C-HH, PL; Study design: PL, C-HH; Definition of intellectual content: ML, HX; Literature research: ML, HX, FW, H-XZ; Clinical studies: ML, HX, Z-KX; Experimental studies: ML, HX, BL, Z-KX; Data acquisition: ML, HX, BL, Z-KX; Data analysis: ML, HX, BL, Z-KX; Statistical analysis: FW, H-XZ; Manuscript preparation: HX, FW, Z-KX; Manuscript editing: ML, PL, H-XZ; Manuscript review: PL.

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Correspondence to Ping Liu.

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This study was approved by the Ethics Committee of the Second Xiangya Hospital of Central South University.

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Lu, M., Xiong, H., Xia, ZK. et al. circRACGAP1 promotes non-small cell lung cancer proliferation by regulating miR-144-5p/CDKL1 signaling pathway. Cancer Gene Ther (2020). https://doi.org/10.1038/s41417-020-00209-0

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