Abstract
Background Circular RNAs (circRNAs) are reported as competing endogenous RNAs (ceRNAs) and play key roles in non-small-cell lung cancer (NSCLC) progression. Thus, this study was aimed at clarifying underlying molecular mechanisms of circHUWE1 in NSCLC. Methods The quantitative real-time polymerase chain reaction (RT-qPCR) and western blot analyses were used for examining circHUWE1, microRNA-34a-5p (miR-34a-5p), and tumor necrosis factor alpha-induced protein 8 (TNFAIP8). IC50 of cisplatin (DDP) in A549/DDP and H1299/DDP cells and cell viability were analyzed by the Cell Counting Kit 8 (CCK-8) assay. Colony forming assay was performed to assess colony forming ability. Cell apoptosis and cell cycle distribution were determined by flow cytometry. Migrated and invaded cell numbers were examined by transwell assay. The association among circHUWE1, miR-34a-5p, and TNFAIP8 was analyzed by dual-luciferase reporter and RNA immunoprecipitation assays. A xenograft experiment was applied to clarify the functional role of circHUWE1 in vivo. Results circHUWE1 was upregulated in NSCLC tissues and cells, especially in DDP-resistant groups. circHUWE1 downregulation inhibited DDP resistance, proliferation, migration, and invasion while it induced apoptosis and cell cycle arrest of DDP-resistant NSCLC cells, which was overturned by silencing of miR-34a-5p. TNFAIP8 was a functional gene of miR-34a-5p, and the suppressive effects of miR-34a-5p overexpression on DDP-resistant NSCLC progression were dependent on the suppression of TNFAIP8. circHUWE1 inhibition also delayed tumor growth of DDP-resistant NSCLC cells. Conclusion circHUWE1 functioned as a promoter in DDP-resistant NSCLC by interaction with miR-34a-5p-TNFAIP8 networks, providing novel insight into DDP-resistant NSCLC diagnosis and treatment.
Highlights
Non-small-cell lung cancer (NSCLC) is a most common pulmonary subtype and is a major reason of tumor-caused death all over the world [1, 2], accounting 80% proportion of primary lung cancer [3]
0 Proliferating Cell Nuclear Antigen (PCNA) c-caspase 3 matrix metalloproteinases 13 (MMP 13) shRNA control (sh-NC) sh-circHUWE1 (q) cells. (a–q) A549/DDP and H1299/DDP cells were transfected with sh-circHUWE1 or sh-NC. (a) The expression of circHUWE1 was assessed by RT-qPCR. (b–e) IC50 of DDP in non-smallcell lung cancer (NSCLC) cells was analyzed by the Cell Counting Kit 8 (CCK-8) assay. (f) Colony forming assay was performed in A549/DDP and H1299/DDP cells. (g, h) The proliferation ability of A549/DDP and H1299/DDP cells was measured by the CCK-8 assay. (i, j) Cell apoptosis and cell cycle distribution were determined by flow cytometry. (k–n) Migrated and invaded cell number were analyzed by transwell assay. (o–q) The protein levels of PCNA, c-caspase 3, and MMP 13 were assessed by western blot assay. ∗P < 0:05
We found that circHUWE1 was upregulated in NSCLC tissues and cells relative to controls; importantly, circHUWE1 level was higher in DDP-resistant tissues and cells than DDP-sensitive tissues and cells, suggesting that circHUWE1 was related to DDP resistance (Figures 1(c) and 1(d))
Summary
Non-small-cell lung cancer (NSCLC) is a most common pulmonary subtype and is a major reason of tumor-caused death all over the world [1, 2], accounting 80% proportion of primary lung cancer [3]. As well-documented, noncoding RNAs including circular RNAs (circRNAs) and microRNAs (miRNAs) are one of the pharmacological resistance mechanisms in tumor cells [8, 9]. CircHUWE1 was upregulated in NSCLC tissues and cells, especially in DDP-resistant groups. CircHUWE1 downregulation inhibited DDP resistance, proliferation, migration, and invasion while it induced apoptosis and cell cycle arrest of DDP-resistant NSCLC cells, which was overturned by silencing of miR-34a-5p. TNFAIP8 was a functional gene of miR-34a-5p, and the suppressive effects of miR34a-5p overexpression on DDP-resistant NSCLC progression were dependent on the suppression of TNFAIP8. CircHUWE1 inhibition delayed tumor growth of DDP-resistant NSCLC cells. CircHUWE1 functioned as a promoter in DDP-resistant NSCLC by interaction with miR-34a-5p-TNFAIP8 networks, providing novel insight into DDP-resistant NSCLC diagnosis and treatment Conclusion. circHUWE1 functioned as a promoter in DDP-resistant NSCLC by interaction with miR-34a-5p-TNFAIP8 networks, providing novel insight into DDP-resistant NSCLC diagnosis and treatment
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