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Circular RNA hsa_circ_0096157 contributes to cisplatin resistance by proliferation, cell cycle progression, and suppressing apoptosis of non-small-cell lung carcinoma cells

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Abstract

Circular RNAs (circRNAs) play a major role in cancer development and chemotherapy resistance. This study aimed to characterize circRNA profiles associated with Cisplatin (diamminedichloroplatinum, DDP) resistance of non-small-cell lung carcinoma (NSCLC) cells. The half-maximal inhibitory concentration (IC50) of A549 and A549/DDP cells was determined using CCK-8 assay. Further, circRNA profiles and differentially expressed genes in A549 and A549/DDP cells were characterized by deep sequencing and cell proliferation was measured using MTS assay. Cell cycle progression was analyzed using flow cytometry. Apoptosis experiment was performed by TUNEL assay and flow cytometry. Cell migration and invasion were assessed using the Transwell system. Finally, signalling protein levels related to cell cycle progression and migration were measured by western blot. CCK-8 assay showed that A549/DDP cells obtained strong DDP resistance. Further deep sequencing results showed that 689 circRNAs and 87 circRNAs were significantly upregulated and downregulated in A549/DDP cells compared to A549 cells, respectively. Moreover, the circRNA hsa_circ_0096157 with the highest expression level in A549/DPP cells was further analyzed for its potential mechanism of DDP resistance in A549/DDP. With or without DDP treatment, hsa_circ_0096157 knockdown inhibited proliferation, migration, invasion and cell cycle progression but promoted apoptosis of A549/DDP cells. In addition, the western blot results also showed that hsa_circ_0096157 knockdown in A549/DDP cells increased P21 and E-cadherin but decreased CDK4, Cyclin D1, Bcl-2, N-cadherin, and Vimentin protein expression levels, indicating that cell cycle progression might be inhibited by increased P21 protein level to inhibit the expression of CDK4-cyclin D1 complex and decreased Bcl-2 protein level; and migration and invasion were suppressed by the increased E-cadherin and decreased N-cadherin and Vimentin expression levels. In contrast, hsa_circ_0096157 overexpression in A549 cells caused the opposite cellular and molecular alterations. DDP resistance in NSCLC cells was associated with significant circRNA profile alterations. Moreover, increased hsa_circ_0096157 expression contributed to DDP resistance in NSCLC cells by promoting cell proliferation, migration, invasion and cell cycle progression and inhibiting apoptosis.

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Funding

This work was supported by the National Natural Science Foundation of China (Nos. 81760419 and 81760743) and Beijing Medical and Health Public Welfare Fund Medical Science Research Fund (No. B20151DS).

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  1. Huasong Lu and Xun Xie are Co-first author.

  2. Huasong Lu and Xun Xie contributed equally to this work.

Authors and Affiliations

  1. Pulmonary and Critical Care Medicine Ward, The First Affiliated Hospital of Guangxi Medical University, No. 6, Shuangyong Road, Qingxiu District, Nanning, 530021, Guangxi, People’s Republic of China

    Huasong Lu, Xun Xie, Ke Wang, Quanfang Chen, Shuangqi Cai, Dongmei Liu, Jin Luo & Jinliang Kong

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Contributions

This study concept and design, and the manuscript revision were performed by JL and JK. The experiments performance, data analysis and the manuscript draft were performed by HL and XX. The study design, study implementation and manuscript revision were performed by KW, QC, SC and DL. All authors read and approved the final manuscript.

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Correspondence to Jin Luo or Jinliang Kong.

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11010_2020_3860_MOESM1_ESM.tif

Supplemental Figure 1. The interaction network between differentially expressed circRNAs and the cell cycle and apoptosis processes. The network representing the interaction between differentially expressed circRNAs in A549/DDP cells and key components of the cell cycle progression and apoptosis processes were established using the Cytoscape software (TIF 7006 kb)

11010_2020_3860_MOESM2_ESM.xls

Supplemental Table 1. Detailed information of circRNAs identified in A549 and A549/DDP cells by next-generation RNA sequencing. (XLS 73 kb)

11010_2020_3860_MOESM3_ESM.xls

Supplemental Table 2. Differentially expressed genes between A549 and A549/DDP cells. Totally 859 genes were significantly up-regulated and 1038 genes were down-regulated in A549/DDP cells in comparison with the A549 cells. Differentially expressed genes were defined by an FDR <= 0.001 and log2Ratio >= 1. (XLS 195 kb)

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Lu, H., Xie, X., Wang, K. et al. Circular RNA hsa_circ_0096157 contributes to cisplatin resistance by proliferation, cell cycle progression, and suppressing apoptosis of non-small-cell lung carcinoma cells. Mol Cell Biochem 475, 63–77 (2020). https://doi.org/10.1007/s11010-020-03860-1

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