circZNF707 promoted glycolysis and tumor progression through miR-668-3p-PFKM axis in NSCLC

Abstract

Background: Circular RNA (circRNA) plays an important regulatory role in the development of human malignancies, but the potential mechanisms of circRNA in non-small cell lung cancer (NSCLC) remain largely unknown.

Methods: Microarray analysis was used to test for circRNAs differing in expression between NSCLC tumors and healthy adjacent tissues. Using qRT-PCR, the expression of circZNF707 was determined. Through a number of loss-of-function and gain-of-function investigations, the biological behavior of NSCLC cells was evaluated. Finally, tests using Western blotting, RIP, qRT-PCR, and luciferase reporter gene detection and rescue assays revealed the potential mechanism of circZNF707.

Results: Increased expression of circZNF707 was found in NSCLC tissues. Functionally, circZNF707 enhances proliferation, migration, invasion, and glycolysis of NSCLC cells. Mechanistically, circZNF707 can upregulate PFKM by acting as a sponge for miR-668-3p, thus contributing to the progression of NSCLC.

Conclusions: Through the circZNF707/miR-668-3p/PFKM axis, upregulation of circZNF707 promotes tumor development. CircZNF707 may provide new insights into the treatment and diagnosis of NSCLC.

Keywords: CircRNA; Glycolysis; MiR-668-3p; NSCLC; PFKM.

Fig. 1

Expression and characteristics of circZNF707 in NSCLC. A Differentially expressed circRNA in GSE101586 dataset. B Differentially expressed circRNA in GSE101684 dataset. C Heatmap of differentially expressed circRNA in GSE101586 and GSE101684 datasets. D, E Expression levels of circZNF707 detected by qRT-PCR in NSCLC and adjacent tissues. F Expression levels of circZNF707 detected by qRT-PCR in normal human lung epithelial cells, bronchial epithelial cells, and NSCLC cell lines. G circZNF707 formed by reverse splicing of exons 2 and 3 of the ZNF707 gene. H Expression of circZNF707 and ZNF707 mRNA detected by qRT-PCR after RNase R treatment in A549 cells. I Expression levels of circZNF707 and ZNF707 mRNA detected by qRT-PCR after resveratrol D treatment in A549 cells. J Expression levels of circZNF707 detected by qRT-PCR in the cytoplasm and nucleus of A549 cells. K FISH staining confirmed the expression of circZNF707 in the cytoplasm. Scale bar, 100 μm. **P < 0.01, ***P < 0.001

Fig. 2

circZNF707 promotes cell proliferation in vitro. A Transfection of siRNA in A549 cells and qRT-PCR detection of circZNF707 expression levels. B Transfection of circZNF707 overexpression vector in NCI-H1299 cells and qRT-PCR detection of circZNF707 expression levels. C–E CCK-8 and EDU assays to assess the proliferation ability of NSCLC cells after circZNF707 overexpression or knockdown. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

circZNF707 enhances cell migration and invasion ability in vitro. A–D Wound healing and Transwell assays to evaluate the migration and invasion ability of NSCLC cells after circZNF707 overexpression or knockdown. Scale bar, 100 μm. E, F Measurement of lactate and glucose uptake in NSCLC cells after circZNF707 overexpression or knockdown. G Analysis of extracellular acidification rate (ECAR) using a Seahorse analyzer after circZNF707 overexpression or knockdown. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

circZNF707 functions as a sponge for miR-668-3p. A Prediction of potential miRNAs binding to circZNF707 by miRanda, Circinteractome, and RNAhybrid databases. B qRT-PCR detection of the expression levels of miR-326, miR-330, and miR-668-3p in NCI-H1299 cells transfected with circZNF707 overexpression vector. C qRT-PCR detection of the expression levels of miR-326, miR-330, and miR-668-3p in A549 cells transfected with siRNA. D Protein expression levels of AGO2 and ACTIN detected by Western Blot experiment. E, F Co-immunoprecipitation of AGO2 group and IgG group, with qRT-PCR detection of the expression levels of miR-668-3p and circZNF707. G Prediction of the binding site between miR-668-3p and circZNF707 by Circinteractome database. H Luciferase assay to detect the binding between miR-668-3p and circZNF707. I qRT-PCR detection of the expression levels of miR-668-3p in NSCLC and adjacent tissues. J Pearson correlation analysis of the expression correlation between miR-668-3p and circZNF707 in NSCLC tissues. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

circZNF707's oncogenic effect in NSCLC can be reversed by miR-668-3p. After transfection with PCD5 + mimics-NC, PCD5 + miR-668-3pmimics, and PCD5-circZNF707 + miR-668-3pmimics in NCI-H1299 cells, A, B CCK-8 and EDU experiments were conducted to assess cell proliferation ability. C, D Wound healing and Transwell experiments were performed to evaluate the migration and invasion capability of NSCLC cells. Scale bar, 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

PFKM is a downstream target gene of miR-668-3p. A The expression levels of downstream genes were measured by transcriptome sequencing after transfection of miR-668-3p mimic and miRNA-NC in NCI-H1299 cells. B Target genes potentially binding to miR-668-3p predicted by PITA, miRmap, microT, and TargetScan databases. C The expression level of PFKM was detected by qRT-PCR after transfection of miR-668-3p mimic and circZNF707 overexpression vector in NCI-H1299 cells. D The expression level of PFKM was detected by qRT-PCR and Western Blot after transfection of miR-668-3p inhibitor and circZNF707 siRNA in A549 cells. E Predicted binding sites of miR-668-3p with PFKM according to TargetScan database. F Luciferase assay to detect the binding sites between miR-668-3p and PFKM. G Expression levels of PFKM in NSCLC and adjacent tissue detected by Western Blot. H Expression levels of PFKM in human bronchial epithelial cells and NSCLC cell lines detected by qRT-PCR. I Expression levels of PFKM in NSCLC and adjacent tissue detected by qRT-PCR. J Pearson correlation analysis of the expression correlation between PFKM and circZNF707 in NSCLC tissues. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

PFKM reverses the anticancer effect of miR-668-3p in NSCLC. In A549 cells, transfection of si-NC + inhibitor NC, si-PFKM + inhibitor NC, and si-PFKM + miR-668-3p inhibitor was performed A, B Cell proliferation ability was detected by CCK-8 and EDU assays. C, D Wound healing and Transwell assays were conducted to measure cell migration and invasion in NSCLC cells. Scale bar, 100 μm. E, F Measurement of lactate and glucose uptake in NSCLC cells. G Analysis of extracellular acidification rate (ECAR) using a Seahorse analyzer. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 8

circZNF707/miR-668-3p/PFKM axis promotes glycolysis in NSCLC cells. Western blot assay of PFKM, HK1, and LDHA proteins in NSCLC cells transfected with si-circZNF707 (A), PCD5-circZNF707 (B), PCD5-circZNF707 + miR-668-3p mimics (C), OE-PFKM + miR-668-3p mimics (D), and si-circZNF707 + miR-668-3p inhibitor (E)

Fig. 9

Knockdown of circZNF707 inhibits the progression of NSCLC in vivo. A Representative images of subcutaneous tumor tissues in the sh-circZNF707 group and sh-NC group. B, C Tumor volume and weight of subcutaneous tumor tissues. D Western blot analysis of PFKM expression levels in subcutaneous tumor tissues of each group. E qRT-PCR analysis of circZNF707, miR-668-3p, and PFKM expression levels in subcutaneous tumor tissues of each group. F Immunohistochemical staining of PFKM, CD31, and Ki67 expression levels in subcutaneous tumor tissues of each group. Scale bar, 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001