Circular RNA_0120376 regulates microRNA-148b-3 and centrosomal protein 55 to promote non-small cell lung cancer development

Abstract

Circular RNAs (circRNAs) are non-coding RNAs with covalent closed-loop structures that are vital in regulating diverse pathological processes. This work is aimed to investigate the role of circ_0120376 in non-small cell lung cancer (NSCLC). Circ_0120376, microRNA (miR)-148b-3p, and centrosomal protein 55 (CEP55) mRNA expression in NSCLC tissues and cells were determined using qRT-PCR. The influences of circ_0120376 and miR-148b-3p on the proliferation of NSCLC cell lines were analyzed by CCK-8 and colony formation assays. Apoptosis was analyzed by flow cytometry. Cell migration and invasion were analyzed using the Transwell experiment. Binding relationships between circ_0120376 and miR-148b-3p and between miR-148b-3p and CEP55 3'UTR were investigated using the dual-luciferase reporter experiment and the RIP experiment. Western blot was conducted to analyze the regulatory effect of circ_0120376 and miR-148b-3p on CEP55 expression. We found that circ_0120376 was markedly overexpressed in NSCLC, and its overexpression was positively associated with increased T stage and lymph node metastasis of the patients. Functional experiments unveiled that circ_0120376 enhanced the proliferation, migration and invasion of NSCLC cells and impeded apoptosis, while knocking down circ_0120376 remarkably suppressed the malignant features of NSCLC cells mentioned above. Circ_0120376 could adsorb miR-148b-3p to reduce miR-148b-3p expression, and circ_0120376 could increase CEP55 expression via adsorbing miR-148b-3p. In summary, circ_0120376 contributes to the malignancy of NSCLC cells through a ceRNA mechanism via regulating miR-148b-3p/CEP55 axis. Circ_0120376 is likely to be a potential diagnostic biomarker and therapeutic target for NSCLC.

Keywords: CEP55; Circ_0120376; NSCLC; miR-148b-3p.

Graphical abstract

Figure 1.

Circ_0120376 was overexpressed in NSCLC. Circ_0120376 expression in human NSCLC tissues and paracancerous tissues was examined by qRT-PCR (n = 113). (b) Circ_0120376 expression was remarkably increased in the cancer tissues of T3 + T4 patients relative to T1+ T2 patients, which was examined by qRT-PCR. (c) qRT-PCR showed that circ_0120376 expression was remarkably increased in the NSCLC tissues of patients with lymph node metastasis. (d) Circ_0120376 expression in 16HBE and 4 types of NSCLC cell lines (A549, H596, H2087, and H520 cells) was detected by qRT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2.

Circ_0120376 enhanced the proliferation, migration and invasion of NSCLC cells, and restrained apoptosis. (a) A549 cells were transfected with circ_0120376 overexpression plasmid, and H520 cells were transfected with si-circ_0120376, and successful transfection was proved by qRT-PCR. (b, c) Cell proliferation was monitored using CCK-8 (b) and colony formation (c) assays. (d) Flow cytometry was utilized to examine the apoptosis of NSCLC cells. (e, f) Transwell experiments were adopted to examine NSCLC cell migration (e) and invasion (f). *P < 0.05, ***P < 0.001.

Figure 3.

MiR-148b-3p was the target of circ_0120376 in NSCLC. (a) StarBase database predicted a potential binding site between circ_0120376 and miR-148b-3p. (b) qRT-PCR was applied to examine miR-148b-3p expression after up-regulation or knockdown of circ_0120376 in NSCLC cells. (c) Pearson’s correlation analysis showed a negative correlation between miR-148b-3p expression and circ_0120376 expression in NSCLC tissues. (d) Dual-luciferase report assay showed that miR-148b-3p suppressed the luciferase activity of wild type CEP55 reporter, while it had no remarkable effect on the mutant reporter. (e) RIP experiments showed that miR-148b-3 directly interacted with circ_0120376. **P < 0.01, ***P < 0.001.

Figure 4.

miR-148b-3p restrained the proliferation, migration and invasion of NSCLC cells and promoted apoptosis. (a) MiR-148b-3p expression in NSCLC tissues and paracancerous tissues was analyzed by qRT-PCR. (b) MiR-148b-3p expression in HBE cell line and NSCLC cell lines was analyzed by qRT-PCR. (c) A549 cells were transfected with miR-148b-3p inhibitors, and H520 cells were transfected with miR-148b-3p mimics, and qRT-PCR was utilized to detect the transfection efficacy. (d-h) colony formation assay (d), CCK-8 test (e), Transwell assay (f, g) and flow cytometry (h) were used to detect the effect of miR-148b-3p on the malignant biological behaviors of NSCLC cells. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5.

CEP55 was a direct target of miR-148b-3p in NSCLC cells. (a) The dataset GSE29249 was analyzed using GEO2R, and the results showed that CEP55 was dysregulated in NSCLC. (b) CEP55 mRNA expression in NSCLC tissues and paracancerous tissues was analyzed by qRT-PCR. (c) CEP55 mRNA expression in NSCLC cell lines was analyzed by qRT-PCR. (d) StarBase database predicted a binding site between miR-148b-3p and CEP55 3'UTR. (e) Pearson’s correlation analysis suggested that miR-148b-3p was negatively correlated with CEP55 expression in NSCLC tissues. (f, g) qRT-PCR (f) and Western blot (g) showed that miR-148b-3p overexpression remarkably inhibited CEP55 expression at the mRNA and protein levels. (h) MiR-148b-3p significantly inhibited the luciferase activity of wild type CEP55 reporter but had no significant effect on that of mutant CEP55 reporter. (i) The Pearson’s correlation analysis suggested that miR-148b-3p expression was positively correlated with circ_0120376 expression in NSCLC tissues. (j, k) qRT-PCR (j) and Western blot (k) were used to detect the expression of CEP55 in NSCLC cells after circ_0120376 and miR-148b-3p were selectively regulated. *P < 0.05, **P < 0.01, ***P < 0.001.