Circular RNA 0006349 Augments Glycolysis and Malignance of Non-small Cell Lung Cancer Cells Through the microRNA-98/MKP1 Axis

Abstract

Background: The involvement of dysregulated circular RNAs (circRNAs) in human diseases has been increasingly recognized. In this study, we focused on the function of a newly screened circRNA, circ_0006349, in the progression of non-small-cell lung cancer (NSCLC) and the molecules of action. Methods: The NSCLC circRNA dataset GSE101684, microRNA (miRNA) dataset GSE29250, and mRNA dataset GSE51852 obtained from the GEO database were used to identify the differentially expressed genes in NSCLC samples. Tumor and normal tissues were collected from 59 patients with NSCLC. The expression of circ_0006349, miR-98, and MAP kinase phosphatase 1 (MKP1) in collected tissue samples and in acquired cells was determined. The binding relationships between miR-98 and circ_0006349/MKP1 were predicted and validated. Altered expression of circ_0006349, miR-98, and MKP1 was introduced in NSCLC cells to examine their roles in cell growth, apoptosis, and glycolysis. Results: Circ_0006349 and MKP1 were upregulated, and miR-98 was poorly expressed in the collected tumor tissues and the acquired NSCLC cell lines. Circ_0006349 was identified as a sponge for miR-98 to elevate MKP1 expression. Silencing of circ_0006349 suppressed proliferation and increased apoptosis of Calu-3 and H1299 cells, and it reduced glycolysis, glucose uptake, and the production of lactate in cells. Upon circ_0006349 knockdown, further downregulation of miR-98 or upregulation of MKP1 restored the malignant behaviors of cells. Conclusion: This research demonstrated that circ_0006349 derepressed MKP1 expression by absorbing miR-98, which augmented the proliferation and glycolysis of NSCLC cells and promoted cancer development.

Keywords: Circ_0006349; MKP1; NSCLC; glycolysis; miR-98; proliferation.

FIGURE 1

Circ_0006349 is highly expressed and indicates dismal prognosis in patients with non-small-cell lung cancer (NSCLC). (A) A volcano map for circRNAs screened out from the NSCLC GSE101684 dataset; (B) a heatmap for top 50 differentially expressed circRNAs in the GSE101684 dataset; (C) circ_0006349 expression in tumor and normal tissues from 59 patients with NSCLC determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR); (D–G) relevance between circ_0006349 expression with the lymph node metastasis, tumor stage, tumor differentiation and tumor size in patients; (H) circ_0006349 expression NSCLC cell lines (A549, H1299, Calu-3, H520, H1650, and H1730) and in BEAS-2B cells detected by RT-qPCR. Differences were compared by paired t test (C), unpaired t test (D–G), or one-way ANOVA (H). All data were presented as the mean ± SD from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; n.s, no significance.

FIGURE 2

Silencing of circ_0006349 suppresses the malignant behaviors of NSCLC cells. (A) transfection efficacy of si-circ_0006349-#1 and si-circ_0006349-#2 in H1299 and Calu-3 cells determined by RT-qPCR; (B) DNA replication activity of H1299 and Calu-3 cells measured by EdU labeling assay; (C) apoptosis of H1299 and Calu-3 cells detected by the flow cytometry; (D,E) ECAR in H1299 and Calu-3 cells; (F,G) glucose uptake (F) and lactate production (G) in H1299 and Calu-3 cells; (H) colony formation ability of H1299 and Calu-3 cells examined by the colony formation assay; (I) apoptotic bodies in H1299 and Calu-3 cells examined by the TUNEL assay. Differences were compared by two-way ANOVA (A–C,F–I). All data were presented as the mean ± SD from three independent experiments. **p < 0.01.

FIGURE 3

Circ_0006349 sub-localizes in cytoplasm and binds to miR-98. (A,B) subcellular localization of circ_0006349 in Calu-3 and H1299 cells determined by nuclear-cytoplasmic RNA separation (A) and FISH (B) assays; (C) a Venn diagram for the intersection of screened out differentially expressed miRNAs from the GSE29250 dataset and the predicted target miRNAs of circ_0006349; (D) putative binding site between circ_0006349 and miR-98; (E,F) binding relationship between miR-98 and circ_0006349 validated through dual luciferase reporter gene and RIP assays. Differences were compared by two-way ANOVA (E,F). All data were presented as the mean ± SD from three independent experiments. **p < 0.01.

FIGURE 4

miR-98 is poorly expressed in NSCLC and presents a negative correlation with circ_0006349. (A) miR-98 expression in tumor and normal tissues from 59 patients with NSCLC determined by RT-qPCR; (B) a reverse correlation between miR-98 and circ_0006349 expression in tumor tissues analyzed by Pearson’s correlation test; (C–F) relevance between miR-98 expression with the lymph node metastasis, tumor stage, tumor differentiation and tumor size in patients; (G) miR-98 expression in NSCLC cell lines (A549, H1299, Calu-3, H520, H1650, and H1730) and BEAS-2B cells detected by RT-qPCR. Differences were compared by paired t test (C), unpaired t test (C–F), one-way (G) or two-way ANOVA (H). All data were presented as the mean ± SD from three independent experiments. **p < 0.01; ***p < 0.001; n.s, no significance.

FIGURE 5

miR-98 inhibitor promotes the malignant behaviors of NSCLC cells. (A) Transfection efficacy of miR-98 in H1299 and Calu-3 cells determined by RT-qPCR; (B) DNA replication activity of H1299 and Calu-3 cells measured by EdU labeling assay; (C) apoptosis of H1299 and Calu-3 cells detected by the flow cytometry; (D) ECAR in H1299 and Calu-3 cells; (E,F) glucose uptake (E) and lactate production (F) in H1299 and Calu-3 cells; (G) colony formation ability of H1299 and Calu-3 cells examined by the colony formation assay; (H) apoptotic bodies in H1299 and Calu-3 cells examined by the TUNEL assay. Differences were compared by two-way ANOVA (A–C,E–H). All data were presented as the mean ± SD from three independent experiments. **p < 0.01.

FIGURE 6

miR-98 directly binds to MKP1 mRNA. (A) Volcano plots for the differentially expressed mRNAs in the GSE51852 dataset; (B) a heatmap for to 50 differentially expressed mRNAs in the GSE51852 dataset; (C) a Venn diagram for the intersections of target mRNAs of miR-98 and the differentially expressed mRNAs in NSCLC; (D) mRNA expression of MKP1 in tissue and normal tissues in 59 NSCLC patients determined by RT-qPCR; (E) a positive correlation between MKP1 expression and circ_0006349 in tumor tissues according to the Pearson’s correlation test; (F) a negative correlation between MKP1 expression and miR-98 in tumor tissues according to the Pearson’s correlation test; (G,H) mRNA (G) and protein (H) expression of MKP1 in BEAS-2B and NSCLC cells determined by RT-qPCR and western blot analysis, respectively; (I,J) mRNA (I) and protein (J) expression of MKP1 in Calu-3 and H1299 cells after si-circ_0006349 or miR-98 inhibitor transfection determined by RT-qPCR and western blot analysis, respectively; (K) sequences of the MKP1-WT and MKP1-MTL vectors for luciferase assay; (L,M) binding relationship between miR-98 and MKP1 mRNA validated through dual luciferase reporter gene (L) and anti-Ago2-RIP (M) assays. Differences were compared by paired t test (D), one-way (G,H) or two-way ANOVA (I,J,L,M). All data were presented as the mean ± SD from three independent experiments. **p < 0.01; ***p < 0.001.

FIGURE 7

Overexpression of MKP1 promotes the malignant behaviors of NSCLC cells. (A,B) transfection efficacy of oe-MKP1 in H1299 and Calu-3 cells validated by RT-qPCR and western blot analysis, respectively; (C) DNA replication activity of H1299 and Calu-3 cells measured by EdU labeling assay; (D) apoptosis of H1299 and Calu-3 cells detected by the flow cytometry; (E) ECAR in H1299 and Calu-3 cells; (F,G) glucose uptake (F) and lactate production (G) in H1299 and Calu-3 cells; (H) colony formation ability of H1299 and Calu-3 cells examined by the colony formation assay; (I) apoptotic bodies in H1299 and Calu-3 cells examined by the TUNEL assay. Differences were compared by two-way ANOVA (A–D,F–I). All data were presented as the mean ± SD from three independent experiments. **p < 0.01.