Exosomal Circ-MEMO1 Promotes the Progression and Aerobic Glycolysis of Non-small Cell Lung Cancer Through Targeting MiR-101-3p/KRAS Axis

Abstract

Circular RNA mediator of cell motility 1 (circ-MEMO1) was identified as an oncogene in non-small cell lung cancer (NSCLC). Nevertheless, the working mechanism behind circ-MEMO1-mediated progression of NSCLC is barely known. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to detect the expression of circ-MEMO1, microRNA-101-3p (miR-101-3p), and KRAS proto-oncogene, GTPase (KRAS). Cell proliferation and aerobic glycolysis were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and glycolysis detection kits. Flow cytometry was used to evaluate cell cycle progression and apoptosis of NSCLC cells. Western blot assay was used to measure the protein expression of hexokinase 2 (HK2), lactate dehydrogenase A (LDHA), KRAS, CD9, CD81, tumor susceptibility 101 (TSG101), and Golgi matrix protein 130 kDa (GM130). The target relationship between miR-101-3p and circ-MEMO1 or KRAS was predicted by StarBase software and confirmed by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay, and RNA-pull down assay. In vivo tumor growth assay was conducted to assess the effect of circ-MEMO1 in vivo. Exosomes were isolated using the ExoQuick precipitation kit. Circ-MEMO1 was up-regulated in NSCLC, and high expression of circ-MEMO1 predicted poor prognosis in NSCLC patients. Circ-MEMO1 accelerated the proliferation, cell cycle progression, and glycolytic metabolism and inhibited the apoptosis of NSCLC cells. Circ-MEMO1 negatively regulated the expression of miR-101-3p through direct interaction, and si-circ-MEMO1-induced biological effects were attenuated by the introduction of anti-miR-101-3p. MiR-101-3p directly interacted with the 3' untranslated region (3' UTR) of KRAS messenger RNA (mRNA), and KRAS level was regulated by circ-MEMO1/miR-101-3p axis. Circ-MEMO1 silencing suppressed the NSCLC tumor growth in vivo. ROC curve analysis revealed that high expression of serum exosomal circ-MEMO1 (exo-circ-MEMO1) might be a valuable diagnostic marker for NSCLC. Circ-MEMO1 facilitated the progression and glycolysis of NSCLC through regulating miR-101-3p/KRAS axis.

Keywords: KRAS; NSCLC; circ-MEMO1; exosome; glycolysis; miR-101-3p.

FIGURE 1

High expression of circ-MEMO1 is associated with poor prognosis of NSCLC patients. (A) The relative expression of circ-MEMO1 in NSCLC tumor tissues (n = 52) and adjacent normal tissues (n = 52) was detected by qRT-PCR, and the ratio of circ-MEMO1 expression in tumor tissues and normal tissues was analyzed. (B–F) NSCLC patients were divided into two groups according to the age, gender, tumor size, clinical stage, or lymph node metastasis, and the expression of circ-MEMO1 in tumor tissues of each group was analyzed. (G) NSCLC patients were split into circ-MEMO1 high expression group (n = 26) and low expression group (n = 26) according to the median of circ-MEMO1 level, and the overall survival curve was generated by Kaplan–Meier plot and analyzed by log-rank test. (H) Circ-MEMO1 level was examined in human bronchial epithelial cell line (HBE) and NSCLC cell lines (H1650, PC9, H1299, and A549) by qRT-PCR. *P < 0.05.

FIGURE 2

Circ-MEMO1 stably distributes in cytoplasmic fraction of NSCLC cells. (A) The schematic diagram revealed the formation of circ-MEMO1 from MEMO1 gene. Back-splicing occurs in the end of exon 3 and exon 5 to allow these three exons to form the closed loop structure. (B,C) Actinomycin D was used to inhibit transcription to test the stability of circ-MEMO1, and GAPDH served as the internal reference. qRT-PCR was used to detect the expression of circ-MEMO1 and GAPDH. (D,E) RNase was used to test the circular structure of circ-MEMO1, and qRT-PCR was conducted to detect the expression of circ-MEMO1 and GAPDH in A549 and H1299 cells with or without RNase treatment. (F,G) qRT-PCR was performed to measure the expression of circ-MEMO1 in cytoplasmic fraction and nuclear fraction of NSCLC cells. *P < 0.05.

FIGURE 3

Circ-MEMO1 silencing restrains cell proliferation and cell cycle progression and triggers cell apoptosis in NSCLC cells. (A–I) A549 and H1299 cells were transfected with si-NC or si-circ-MEMO1. (A) Circ-MEMO1 level was detected in transfected NSCLC cells by qRT-PCR. (B,C) MTT assay was utilized to analyze the proliferation of NSCLC cells. (D–G) Flow cytometry was used to analyze the percentage of NSCLC cells in G0/G1, S, or G2/M phase with the silencing of circ-MEMO1 or not. (H,I) The percentage of apoptotic NSCLC cells in the early stage and late stage was analyzed by flow cytometry. *P < 0.05.

FIGURE 4

Circ-MEMO1 knockdown hampers the glycolysis of NSCLC cells. (A–F) A549 and H1299 cells were transfected with si-circ-MEMO1 or si-NC, respectively. (A,B) The glucose uptake and lactate production were measured by Glucose Assay Kit and Lactate Assay Kit. (C–F) The levels of glycolysis-related proteins (HK2 and LDHA) were examined in transfected NSCLC cells by Western blot assay, and the protein quantification was performed using Image J software. *P < 0.05.

FIGURE 5

Circ-MEMO1 directly interacts with miR-101-3p in NSCLC cells. (A) The binding sequence between circ-MEMO1 and miR-101-3p was predicted by StarBase software, and the binding sites in circ-MEMO1 (GUACUGU) were mutated by CAUGACA to perform dual-luciferase reporter assay. (B,C) Dual-luciferase reporter assay was conducted to verify the direct target interaction between miR-101-3p and circ-MEMO1. NSCLC cells were transfected with the following four groups: miR-NC + circ-MEMO1-wt, miR-101-3p + circ-MEMO1-wt, miR-NC + circ-MEMO1-mut, and miR-101-3p + circ-MEMO1-mut. (D,E) RIP assay was utilized to confirm the spatial interaction between miR-101-3p and circ-MEMO1 in NSCLC cells, and IgG group was used as the control group. (F,G) The abundance of miR-101-3p was analyzed in NSCLC cells transfected with si-NC or si-circ-MEMO1 by qRT-PCR. (H,I) RNA-pull down assay was conducted to verify the interaction between miR-101-3p and circ-MEMO1 using biotinylated miR-101-3p (bio-miR-101-3p) or bio-NC in A549 and H1299 cells. (J) The ratio of miR-101-3p expression in tumor samples (n = 52) and corresponding normal samples (n = 52) was analyzed. (K) qRT-PCR was applied to detect the abundance of miR-101-3p in HBE cell line and NSCLC cell lines. *P < 0.05.

FIGURE 6

MiR-101-3p knockdown partly alleviates si-circ-MEMO1-mediated effects in NSCLC cells. (A) The knockdown efficiency of anti-miR-101-3p was assessed by qRT-PCR. (B–J) We transfected si-NC + anti-NC, si-circ-MEMO1 + anti-NC, or si-circ-MEMO1 + anti-miR-101-3p into NSCLC cells. (B,C) The proliferation ability of transfected NSCLC cells was evaluated by MTT assay. (D,E) The cell cycle of NSCLC cells was analyzed by flow cytometry. (F) The apoptosis rate of NSCLC cells was analyzed by flow cytometry. (G,H) The aerobic glycolysis of NSCLC cells was analyzed using Glucose Assay Kit and Lactate Assay Kit. (I,J) The expression of HK2 and LDHA in transfected NSCLC cells was detected by Western blot assay. *P < 0.05.

FIGURE 7

MiR-101-3p directly interacts with the 3′ UTR of KRAS in NSCLC cells. (A) The interactions of miR-101-3p-mRNAs were screened by StarBase software, and KRAS was a candidate target of miR-101-3p. The mutant binding sites with miR-101-3p in KRAS were also shown. (B,C) Dual-luciferase reporter assay was performed to test the direct interaction between miR-101-3p and KRAS in NSCLC cells. (D,E) The regulatory relationship between miR-101-3p and KRAS in NSCLC cells was analyzed through transfecting miR-101-3p, anti-miR-101-3p, and their negative controls into NSCLC cells, and then the expression of KRAS was measured via Western blot assay. (F,G) The expression of KRAS was detected in A549 and H1299 cells co-transfected with si-NC + anti-NC, si-circ-MEMO1 + anti-NC, or si-circ-MEMO1 + anti-miR-101-3p by Western blot assay. (H) The expression of KRAS mRNA in NSCLC tumor tissues and matching normal tissues was examined by qRT-PCR assay. (I) Western blot assay was used to detect the protein expression of KRAS in NSCLC tumor tissue and matching normal tissue. (J) The expression of KRAS in HBE, H1299, and A549 cell lines was detected by Western blot assay. *P < 0.05.

FIGURE 8

Circ-MEMO1 interference restrains the NSCLC tumor growth in vivo. BALB/c nude mice were divided into sh-NC group (n = 7) and sh-circ-MEMO1 group (n = 7). A549 cells stably expressing sh-NC or sh-circ-MEMO1 were subcutaneously injected into the right back of the nude mice in the corresponding group. (A) The tumor volume was measured after injection for 7 days every 3 days with the formula: length × width² × 0.5. (B) Tumor weight in sh-NC group and sh-circ-MEMO1 group was measured with an analytical balance. (C,D) The expression of circ-MEMO1 and miR-101-3p in two groups was detected by qRT-PCR. (E) Western blot assay was used to detect the protein expression of KRAS in two groups. *P < 0.05.

FIGURE 9

Exosomal circ-MEMO1 is higher in the serum from NSCLC patients compared with healthy volunteers. (A) The electron microscopy images showed the exosomes isolated from serum of NSCLC patient and healthy volunteer. Ruler length indicates 200 nm. (B) The expression of exosomal markers (CD9, CD81, and TSG101) and Golgi matrix protein (GM130) was detected by Western blot assay. (C) The expression of exosomal circ-MEMO1 in the serum from NSCLC patients (n = 30) and healthy volunteers (n = 25) was detected by qRT-PCR. (D) ROC curve was generated to evaluate the diagnostic value of exosomal circ-MEMO1 level in serum of NSCLC patients. *P < 0.05.