hsa_circ_0077837 Alleviated the Malignancy of Non-Small Cell Lung Cancer by Regulating the miR-1178-3p/APITD1 Axis

Abstract

Objective: circRNAs were a group of the most promising molecular biomarkers for clinical prognosis and diagnosis of non-small cell lung cancer (NSCLC). It was a pity that academic circle still struggled to figure out how circRNAs acted on NSCLC. This article aimed to study the function and mechanism of hsa_circ_0077837 in NSCLC progression.

Methods: Cell viability was measured via CCK-8, while apoptosis was evaluated with flow cytometry. The transwell assay and scratch test were used to detect invasion and migration, respectively. The dual-luciferase reporter gene assay verified the regulatory effect of miR-1178-3p on hsa_circ_0077837 and miR-1178-3p on apoptosis-inducing, TAF9-like domain 1 (APITD1). The TUNEL assay and immunohistochemistry were used to assess cells apoptosis and proliferation in lung tumor tissues in mice.

Results: Hsa_circ_0077837 and APITD1 expression were suppressed in NSCLC tissues and cells, and miR-1178-3p level was promoted. High amount of hsa_circ_0077837 intensely prevented cell proliferation, migration, and invasion, promoted cell apoptosis in vitro, and delayed tumor growth in mice. Further analysis indicated that hsa_circ_0077837 acted as a miR-1178-3p sponge to stabilize APITD1, the target of miR-1178-3p. Mechanistically, we discovered that hsa_circ_0077837 could prevent proliferation, viability, migration, and invasion of NSCLC cells through stimulating the miR-1178-3p/APITD1 pathway.

Conclusion: Collectively, our findings validated that hsa_circ_0077837 served as a miR-1178-3p sponge by targeting APITD1 that alleviated NSCLC progression.

Figure 1

Hsa_circ_0077837 was significantly downregulated in NSCLC tissues and cell lines. (a) qRT-PCR detected hsa_circ_0077837 expression in 110 NSCLC tumor tissues compared with 110 adjacent tissues. The data were presented as mean ± SEM, ^∗∗∗P < 0.001, adjacent group vs. tumor group. (b) qRT-PCR detected hsa_circ_0077837 expression in human bronchial epithelial cell line BEAS-2B and NSCLC cell lines NCI-H1359, A549, H1650, H1975, and HCC827. The data were presented as mean ± SEM, ^∗P < 0.05,  ^∗∗∗P < 0.001, NCI-H1359, A549, H1650, H1975, or HCC827 group vs. BEAS-2B group.

Figure 2

Hsa_circ_0077837 inhibited NSCLC cell viability, migration, and invasion and enhanced apoptosis. (a) The expression of hsa_circ_0077837 was significantly elevated or decreased in H1650 cells by transfection with over-hsa_circ_0077837 or si-hsa_circ_0077837, respectively; GAPDH acted as a control. (b) CCK-8 assay detected H1650 cell viability after transfection. (c, d) Transwell and would healing assays detected H1650 cell invasive and migratory abilities in over-hsa_circ_0077837 and si-hsa_circ_0077837 groups. (e) Flow cytometry detected the apoptosis rate. The data were presented as mean ± SEM, ^∗∗P < 0.01,  ^∗∗∗P < 0.001, over-hsa_circ_0077837 group vs. control group; ^#P < 0.05,  ^##P < 0.01,  ^###P < 0.001si-hsa_circ_0077837 group vs. control group.

Figure 3

Hsa_circ_0077837 served as a miR-1178-3p sponge. (a) The potential binding site between hsa_circ_0077837 and miR-1178-3p was predicted by using CircInteractome. (b) Relative expression of miR-1178-3p was detected by qRT-PCR in 110 NSCLC tissues. U6 acts as a control. The data were presented as mean ± SEM, ^∗∗∗P < 0.001, adjacent vs. tumor group. (c) Luciferase reporter assays detected the regulatory effect of miR-1178-3p to hsa_circ_0077837. The data were presented as mean ± SEM, ^∗∗P < 0.01, miR-1178-3p mimics group vs. miR-1178-3p NC group.

Figure 4

APITD1 was a downstream molecule of miR-1178-3p. (a) TargetScan predicted the potential binding site between miR-1178-3p and APITD1. (b) Dual-luciferase reporter assays detected the regulation of miR-1178-3p on APITD1, and the data were presented as mean ± SEM, ^∗∗P < 0.01, miR-1178-3p mimics group vs. miR-1178-3p NC group. (c) miR-1178-3p expression was upregulated in 110 NSCLC tissues in comparison with 110 adjacent tissues, and U6 acted as a control, ^∗∗∗P < 0.001, tumor group vs. adjacent group. (d) APITD1 level affected by miR-1178-3p was detected by western blot, and the data were presented as mean ± SEM, ^∗∗∗P < 0.001, miR-1178-3p mimics group vs. NC group; ^###P < 0.001, miR-1178-3p inhibitor group vs. NC group.

Figure 5

Hsa_circ_0077837 inhibited NSCLC cell growth by modulating the miR-1178-3p/APITD1 pathway. (a–d) The viability, migration, invasion, and apoptosis of H1650 cells were detected via CCK-8, transwell, would healing, and flow cytometry affected by miR-1178-3p or APITD1, respectively. ^∗P < 0.05,  ^∗∗P < 0.01,  ^∗∗∗P < 0.001, mimics group vs. NC group; ^#P < 0.05,  ^##P < 0.01,  ^###P < 0.001, si-APITD1 group vs. NC group. (e–h) Rescue assays detected the rescue function of miR-1178-3p mimics or si-APITD1 to hsa_circ_0077837 elevation on viability, migratory, invasive, and apoptotic capabilities. ^∗∗P < 0.01,  ^∗∗∗P < 0.001, over-circ group vs. control group; ^#P < 0.05,  ^###P < 0.001, over-circ + mimics group vs. over-circ group; ^&P < 0.05, ^&&&P < 0.001, over-circ group + si-APITD1 group vs. over-circ group.

Figure 6

Hsa_circ_0077837 overexpression inhibited tumor growth in vivo. (a) Relative hsa_circ_0077837 level was detected by qRT-PCR, and GAPDH act as a control. (b) The tumor volume and weight were lower in over-hsa_circ_0077837 group while higher in si-hsa_circ_0077837 group. (c) Ki-67 immunohistochemistry detected proliferation in over-hsa_circ_0077837 or si-hsa_circ_0077837 group. (d) qRT-PCR analysis detected miR-1178-3p expression in the over-hsa_circ_0077837 or si-hsa_circ_0077837 group. (e) APITD1 expression was detected by western blot. (f) TUNEL staining of the tumors. The data were presented as mean ± SEM, ^∗∗P < 0.01,  ^∗∗∗P < 0.001, over-hsa_circ_0077837 group vs. control group; ^##P < 0.01,  ^###P < 0.001, si-hsa_circ_0077837 group vs. control group.