Circ_0028826 Promotes Growth and Metastasis of NSCLC via Acting as a Sponge of miR-758-3p to Derepress IDH2 Expression

Abstract

Background: Non-small cell lung cancer (NSCLC) is one of the cancers with the highest mortality and morbidity in the world. Circular RNAs (circRNAs) are newly identified players in carcinogenesis and development of various cancers. This study is aimed at exploring the functional effects and mechanism of circ_0028826 in the development of NSCLC.

Methods: Real-time quantitative PCR (RT-qPCR) was used to detect the expression levels of circ_0028826, IDH2 mRNA, and miR-758-3p. IDH2, Bcl2, Bax, and E-cadherin protein levels were detected using a western blot. Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, wound healing, and transwell assays were used to assess the capacities of proliferation, apoptosis, migration, and invasion. Interaction between miR-758-3p and circ_0028826 or IDH2 was validated using a dual-luciferase reporter assay. The role of circ_0028826 in vivo was checked based on a xenograft tumor model.

Results: Circ_0028826 was elevated in NSCLC, and its absence inhibited NSCLC cell proliferation, migration, invasion, and induced apoptosis. In terms of mechanism, circ_0028826 increased IDH2 expression by targeting miR-758-3p. In addition, circ_0028826 knockdown also regulated IDH2 by targeting miR-758-3p to inhibit tumor growth in vivo.

Conclusion: Circ_0028826 promoted the development of NSCLC via regulation of the miR-758-3p/IDH2 axis, providing a new strategy for NSCLC treatment.

Keywords: IDH2; NSCLC; circRNA; circ_0028826; miR‐758‐3p.

FIGURE 1

The expression level of circ_0028826 in NSCLC tissues and cells. (A) Circ_0028826 structure diagram. (B, C) The relative expression level of circ_0028826 was analyzed by RT‐qPCR assay in NSCLC tissues and cells, along with in control groups. (D, E) Circ_0028826 was mainly located in the cytoplasm. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2

The functional roles of circ_0028826 on cell proliferation, apoptosis, migration, and invasion in NSCLC. (A) The expression level of circ_0028826 in A549 and NCl‐H1299 cells transfected with sh‐circ_0028826 and sh‐NC. (B, C) The cell proliferation was measured by CCK‐8 and EdU assay. (D) Flow cytometry analysis was used to assess apoptosis of transfected A549 and NCl‐H1299 cells. (E, F) The cell migration and invasion were measured by wound healing and transwell assays. (G) Western blot analysis was used to examine the expression of the apoptosis markers (Bax and Bcl2) and the epithelial marker E‐cadherin. **p < 0.01, ***p < 0.001.

FIGURE 3

miR‐758‐3p was a direct target of circ_0028826. (A) CircInteractome, starBase, and circBank analyses were carried out to predict the miRNAs that could bind to circ_0028826. (B) Detecting the expression of the seven predicted miRNA in A549 and NCl‐H1299 cells with circ_0028826 knockdown. (C) The complementary sequences between miR‐758‐3p and circ_0028826 were shown. (D) The expression level of miR‐758‐3p was detected by RT‐qPCR after A549 and NCl‐H1299 cells were transfected with miR‐758‐3p or miR‐NC. (E, F) Dual‐luciferase reporter and RIP assays were performed to confirm the association between miR‐758‐3p and circ_0028826. (G, H) The relative expression level of miR‐758‐3p was measured by RT‐qPCR assay in NSCLC tissues and cells. (I) The correlation relationship between miR‐758‐3p and circ_0028826 was analyzed by Pearson's correlation analysis. ***p < 0.001.

FIGURE 4

Circ_0028826/miR‐758‐3p axis regulated proliferation, apoptosis, migration, and invasion of NSCLC cells. (A) The expression level of miR‐758‐3p was detected by RT‐qPCR after A549 and NCl‐H1299 cells were transfected with in‐miR‐758‐3p or in‐miR‐NC. (B–I) A549 and NCl‐H1299 cells were transfected with sh‐NC, sh‐circ_0028826, sh‐circ_0028826+in‐miR‐NC, or sh‐circ_0028826+in‐miR‐758‐3p. (B) RT‐qPCR was used to examine miR‐758‐3p level in transfected NSCLC cells. (C–E) CCK‐8 and EdU were conducted to assess cell proliferation in transfected A549 and NCl‐H1299 cells. (F) The cell apoptosis was quantified by flow cytometry assay. (G, H) The cell migration and invasion were measured by wound healing and transwell assays. (I) Western blot analysis was performed to estimate Bcl2, Bax, and E‐cadherin levels. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5

miR‐758‐3p targeted IDH2 in NSCLC. (A) miR‐758‐3p had the binding regions in 3′UTR of IDH2 mRNA. (B, C) Dual‐luciferase reporter was performed in A549 and NCl‐H1299 cells. (D–H) The expression levels of IDH2 were determined by RT‐qPCR, IHC, and western blot analysis in NSCLC tissues and cells. (I, J) The correlation relationships between IDH2 and miR‐758‐3p or circ_0028826 were analyzed by Pearson's correlation analysis. **p < 0.01, ***p < 0.001.

FIGURE 6

miR‐758‐3p/IDH2 axis regulated proliferation, apoptosis, migration, and invasion of NSCLC cells. (A) The expression level of miR‐758‐3p was detected by RT‐qPCR in A549 and NCl‐H1299 cells transfected with pcDNA or IDH2. (B–J) A549 and NCl‐H1299 cells were transfected with miR‐NC, miR‐758‐3p, miR‐758‐3p+pcDNA, or miR‐758‐3p+IDH2. (B, C) RT‐qPCR and western blot were used to examine IDH2 protein level in transfected A549 and NCl‐H1299 cells. (D–F) CCK‐8 and EdU were conducted to assess cell proliferation in transfected A549 and NCl‐H1299. (G) The cell apoptosis was quantified by flow cytometry assay. (H, I) The cell migration and invasion were measured by wound healing and transwell assays. (J) Western blot analysis was performed to estimate Bcl2, Bax, and E‐cadherin protein levels. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 7

Suppression of circ_0028826 repressed tumor growth in vivo. (A) Tumors were collected and photographed when the mice were killed. (B) After inoculation, tumor volume was recorded once a week. (C) Tumor weight was measured when the mice were killed. (D) The expression of circ_0028826, miR‐758‐3p, and IDH2 mRNA in these excised tumor tissues was measured by RT‐qPCR. (E) The expression of IDH2 at the protein level in these tissues was detected by western blot. (F) IHC analysis was performed to measure the protein expression of IDH2 and Ki67 in tumors from different groups. ***p < 0.001.