Abstract：Objective: To observe the expression of circular RNA (circRNA) BIRC6 (circBIRC6) in non-small cell lung cancer (NSCLC) and to explore its role and molecular mechanism in tumor cell immune escape. Methods: Human NSCLC cell lines (H1975, HCC827, H1650, H1299, and A549) and human normal lung epithelial cell lines (BEAS-2B) were cultured in vitro. The expressions of circBIRC6, miR-944, cluster of differentiation 44 (CD44) and programmed cell death ligand 1 (PD-L1) were detected by qRT-PCR. A549 cells were collected and separated into normal control (NC) group, si-NC group, si-circBIRC6 group, si-circBIRC6+anti-NC group, and si-circBIRC6+anti-miR-944 group. 48h after transfection, qRT-PCR and Western blot were applied to detect the mRNA and protein expression levels of circBIRC6, miR-944, CD44 and PD-L1 in cells; MTT assay was applied to detect cell proliferation activity; Transwell chambers were used to detect cell migration and invasion ability; A549 cells were co-cultured with CD8+ T cells; Trypan blue staining was applied to determine CD8+T cell viability in co-culture systems; flow cytometry was applied to detect CD8+T cell apoptosis in co-culture systems; ELISA was applied to detect the levels of soluble PD-L1 (sPD-L1), interferon-γ (INF-γ), tumor necrosis factor alpha (TNF-α), granzyme B, and perforin in culture supernatants of co-cultured cells; the role of knockdown of circBIRC6 in NSCLC immune escape was validated. Dual-luciferase reporter (DLRA), RNA immunoprecipitation (RIP) and RNA pull-down experiments were applied to validate the regulatory mechanisms of circBIRC6, miR-944 and CD44 in A549 cells. Results: CircBIRC6, CD44 and PD-L1 were highly expressed in NSCLC, while miR-944 was lowly expressed in NSCLC. Knockdown of circBIRC6 (0. 36±0. 05 vs 1. 01±0. 12) could up-regulate miR-944 (3. 45±0. 38 vs 0. 99±0. 10), inhibit the expression of CD44 mRNA (0. 39±0. 05 vs 1. 00±0. 10), CD44 protein (0. 25±0. 05 vs 0. 64±0. 07)and PD-L1 mRNA (0. 31±0. 04 vs 1. 00±0. 13), PD-L1 protein (0. 17±0. 03 vs 0. 52±0. 06), and greatly reduce the proliferation, migration and invasion abilities of A549 cells (all P<0. 05). In the co-culture system, knockdown of circBIRC6 in A549 cells could activate CD8+T cells [(68. 35±7. 20) % vs (35. 82±4. 14) %], inhibit the apoptosis of CD8+T cells [(23. 85±3. 76) % vs (56. 01±6. 22) %], reduce the level of sPD-L1 in the supernatant, increase the levels of IFN-γ, TNF-α, granzyme B and perforin (all P<0. 05). Down-regulation of miR-944 increased the expressions of CD44 and PD-L1, and attenuated the effects of circBIRC6 knockdown on the proliferation, migration and invasion of A549 cells and activation of CD8+ T cells (all P<0. 05). DLRA, RIP and RNA pull-down experiments confirmed that circBIRC6 could target and bind to miR-944 in A549 cells, and CD44 was the target of miR-944. Conclusion: Knockdown of circBIRC6 in A549 cells can activate CD8+ T cells in the tumor microenvironment by down-regulating the expression and secretion of PD-L1, thereby inhibiting the immune escape of NSCLC. The molecular mechanism may be related to the regulatory effect of miR-944/CD44 axis on PD-L1.
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2022, Vol. 28 
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