CircRNA circ_0006677 Inhibits the Progression and Glycolysis in Non-Small-Cell Lung Cancer by Sponging miR-578 and Regulating SOCS2 Expression

Abstract

Objective: Circular RNAs (circRNAs) have been demonstrated in playing an important role in the physiological and pathological processes (such as cancer). This paper aims to clarify the role of Circ_0006677 in non-small-cell lung cancer (NSCLC) progression. Methods: Using clinical data and in vitro cell line models, we revealed the tumor-suppressive role of circ_0006677 in lung cancer. Using the online bioinformatics tool, we predicted the target of circ_0006677 and further validated its regulatory mechanisms responsible for its tumor suppressor function in NSCLC. Results: Circ_0006677 expression was reduced in NSCLC tissues of patients and lung cancer cells in comparison to adjacent normal tissues. Lower expression of circ_0006677 was significantly associated with poorer patient survival. Overexpression of circ_0006677 significantly inhibited the ability of NSCLC cell proliferation, migration, invasion, and glycolysis. Mechanically, circ_0006677 could inhibit NSCLC progression and glycolysis by regulating the expression of the signal transducer inhibitor SOSC2 through sponging microRNA-578 (miR-578). Conclusion: Circ_0006677 prevents the progression of NSCLC via modulating the miR-578/SOSC2 axis.

Keywords: SOSC2; circ_0006677; lung cancer; microRNA-578; non–small-cell lung cancer.

FIGURE 1

Circ_0006677 is reduced in NSCLC tissues and cells. (A) Analysis of the differentially expressed circRNAs in NSCLC tissues and adjacent normal tissues (GSE112214). Three pairs of tumor and respective control tissues were analyzed. (B) The expression of circ_0006677 in 88 pairs of NSCLC tissues and adjacent normal tissues was detected using qRT-PCR assays. (C) The Kaplan–Meier survival analysis of the correlation between circ_0006677 levels with the prognosis of NSCLC patients. Eighty-eight patients were divided into circ_0006677-high and circ_0006677-low according to the median expression value of circ_0006677 in NSCLC tissues. (D) The expression of circ_0006677 in NSCLC cell lines and human bronchial epithelial cells (HBE) were detected with qRT-PCR analysis. (E) The expression of WDR78 and circ_0006677 in A549 and H1229 cells after RNase R treatment were detected by qRT-PCR analysis. (F) Analysis of the subcellular location of circ_0006677 in A549 and H1229 cells. The cytoplasm and nucleus were separated followed by detecting the circ_0006677 level using qRT-PCR assays. U6 and GAPDH were used as internal references of the nucleus and cytoplasm, respectively. ***p < 0.0001.

FIGURE 2

Overexpression of circ_0006677 inhibits the proliferation, migration, invasion, and glycolysis of NSCLC cells. (A) A549 and H1299 cells were infected with lentivirus coding circ_0006677 and overexpression of circ_0006677 in A549, and H1299 cells were validated using qRT-PCR analysis. (B) Impact of circ_0006677 expression on the proliferation of A549 and H1299 cells was investigated using CCK-8 assays at indicated time points. (C) Impact of circ_0006677 expression on colony formation capacity of A549 and H1299 cells. (D). Impact of circ_0006677 expression on cell migration. (E) Impact of circ_0006677 expression on cell invasion ability. (F) Western blot assay of cell migration/invasion–related proteins in A549 and H1229 cells transfected with a control vector or circ_0006677 vector. Circ: circ_0006677. Lower: quantitative analysis of Western blot data. (G) Glucose assay was performed to evaluate the impact of circ_0006677 expression on glucose consumption. (H) The effects of circ_0006677 on lactic acid production in A549 and H1299 cells were evaluated using the lactate assay. ***p < 0.0001.

FIGURE 3

Circ_0006677 is a direct target of miR-578. (A) Venn diagram illustrates five overlapping miRNAs as predicted by circBank and CircInteractome databases. (B) qRT-PCR assays were used to detect the expression of miRNAs in A549 and H1299 cells after overexpression of circ_0006677. (C) Reporter assay was done to evaluate the binding between miR-578 and circ_0006677. (D) RNA pull-down assays were conducted to verify the binding between miR-578 and circ_000667 in A549 and H1299 cells. (E) The expression of miR-578 in NSCLC and normal tissues were compared using qRT-PCR assays. (F) Spearman correlation analysis of circ_0006677 and miR-578 in NSCLC tissues. ***p < 0.0001.

FIGURE 4

Knockdown of miR-578 expression inhibits NSCLC cell proliferation, migration, invasion, and glycolysis. (A) Comparison of miR-578 expression in NSCLC cell lines and HBE with qRT-PCR analysis. (B) qRT-PCR assays were used to examine miR-578 expression in A549 and H1299 cells transfected with miR-578 inhibitor or the respective control. (C–H) Cell proliferation assays (C), colony formation assays (D), cell migration assays (E), invasion assays (F), glucose consumption assays (G), and lactic acid assays (H) of A549 and H1299 cells transfected as indicated. ***p < 0.0001.

FIGURE 5

MiR-578 directly targets SOCS2 in NSCLC cells. (A) Reporter assays were used to evaluate the binding between miR-578 and 3′-UTR of SOCS2 mRNA in A549 and H1299 cells. (B) Western blot assays were performed to examine the protein expression of SOCS2 in A549 and H1299 cells transfected with miR-578 mimic or control mimic. (C) Western blot assays showed that miR-578 attenuated the effects of circ_0006677 on SOCS2 protein expression in A549 and H1299 cells. ***p < 0.0001.

FIGURE 6

Circ_0006677 regulates NSCLC progression by the miR-578/SOCS2 axis. (A) Western blot assays of SOCS2 expression in A549 and H1299 cells transfected as indicated. (B–G) Cell proliferation assays (B), colony formation assays (C), cell migration assays (D), invasion assays (E), glucose consumption assays (F), and lactic acid assays (G) of A549 and H1299 cells transfected as indicated. ***p < 0.0001.

FIGURE 7

Circ_0006677 inhibits NSCLC growth in vivo. (A, B) The volume (A) and weight (B) of the xenografts derived from mice inoculated with A549 cells overexpressing c_0006677 or the control cells. (C) qRT-PCR analysis was used to detect circ_0006677 expression in the xenografts derived from mice inoculated with A549 cells overexpressing c_0006677 or the control cells. (D) Left: Immunostaining of Ki-67 and SOCS2 expression in the xenografts derived from mice inoculated with A549 cells overexpressing c_0006677 or the control cells, scale bar: 25 μm. Right: quantitative analysis of immunostaining data. *p < 0.05, **p < 0.001, ***p < 0.0001.