Cancer-released exosomal circular RNA circ_0008717 promotes cell tumorigenicity through microRNA-1287-5p/P21-activated kinase 2 (PAK2) axis in non-small cell lung cancer

Abstract

Circular RNA (circRNA) circ_0008717 has been revealed to promote cell carcinogenesis in non-small cell lung cancer (NSCLC). Exosomal circRNA packaged into exosomes has been defined as a potential diagnostic and therapeutic biomarker of cancers. However, little attention is focused on the role of circRNAs within exosomes in NSCLC. Exosomes were isolated by ultracentrifugation method and qualified by nanoparticle tracking analysis and Western blot. Levels of circ_0008717, microRNA (miR)-1287-5p, and P21-activated kinase 2 (PAK2) were detected using qRT-PCR and western blot. The interaction between miR-1287-5p and circ_0008717 or PAK2 was investigated. The phenotypes of NSCLC cells with circ_0008717 downregulation were tested. Circ_0008717 was highly expressed in NSCLC. Functionally, circ_0008717 deficiency suppressed cell malignant phenotypes in NSCLC in vitro and in nude mice. Circ_0008717 sponged miR-1287-5p to elevate PAK2, a downstream target of miR-1287-5p. Silencing of miR-1287-5p blocked the antitumor effects of circ_0008717 knockdown in NSCLC cells. Besides, miR-1287-5p repressed cell oncogenic behaviors in NSCLC by targeting PAK2. Besides that, we confirmed that circ_0008717 was incorporated into exosomes in NSCLC cells. Circ_0008717 knockdown inhibited NSCLC tumorigenesis via miR-1287-5p/PAK2 axis, and the extracellular circulating circ_0008717 was transferred through incorporation in exosomes.

Keywords: Exosomes; NSCLC; PAK2; circ_0008717; miR-1287-5p.

Graphical abstract

Figure 1.

Circ_0008717 is packaged into exosomes of NSCLC. (a) Exosomes from serum were analyzed under transmission electron microscopy; (b) Nanoparticle tracking analysis was used to measure exosome particle size and concentration; (c) Western blot for CD63 and CD81 expression in exosomes; (d) Levels of circ_0008717 expression in exosomes from serum in NSCLC and normal; N = 48 per group; (e) QRT-PCR detection of circ_0008717 expression in exosomes from bronchial epithelial cells BEAS-2B and NSCLC cells; (f–g) Circ_0008717 expression was determined by qRT-PCR in NSCLC and normal tissues, as well as BEAS-2B and NSCLC cells. (h) Circ_0008717 expression was detected by qRT-PCR in A549 and H1299 incubating with RNase A alone or in combination with Triton X-100. All experiments repeated three times. *P < 0.05 and ** P < 0.01.

Figure 2.

Circ_0008717 knockdown suppresses cell growth and mobility in NSCLC. Following assigned transfection, (a) qRT-PCR analysis of circ_0008717 expression in A549 and H1299 cells; (b–c) The cell proliferation of A549 and H1299 cells were determined by MTT (b) and colony formation assay (c); (d) Transwell assay (100 ×) was used to detect cell invasion and migration; (e) Cell cycle was analyzed by Flow cytometry. Error bars stand for the mean ± SD of three independent measurements. *P < 0.05.

Figure 3.

Circ_0008717 knockdown suppresses angiogenesis and induces apoptosis in NSCLC. A549 and H1299 cells were transfected with circ_0008717 siRNA (si-circ_0008717) and siRNA NC (si-NC); (a) Tube formation assay for HUVECs with the conditioned medium of indicated cells. (b) Flow cytometry of cell apoptosis rate. (c–d) Protein levels of Bax and Bcl-2 protein in cells were assayed by western blot. Error bars stand for the mean ± SD of three independent measurements. *P < 0.05.

Figure 4.

MiR-1287-5p is a target of circ_0008717 in NSCLC cells. (a) The binding sites of miR-1287-5p on circ_0008717. (b–g) The binding between miR-1287-5p and circ_0008717 was validated using dual-luciferase reporter, pull-down and RIP assays. (h,i) qRT-PCR analysis of miR-1287-5p expression in NSCLC tissues and normal adjacent tissues, as well as in normal BEAS-2B cells and NSCLC cells. (j) The interference efficiency of miR-1287-5p inhibitor or inhibitor NC was detected by qRT-PCR in A549 and H1299 cells. (k) qRT-PCR analysis was conducted to investigate the effects of circ_0008717 on miR-1287-5p expression in A549 and H1299 cells. Error bars stand for the mean ± SD of three independent measurements. *P < 0.05 and ** P < 0.01.

Figure 5.

Silencing of miR-1287-5p attenuates the antitumor effects mediated by si-circ_0008717 in NSCLC cells. After indicated transfection, (a–c) The proliferation analysis of cells with MTT assay and colony formation assay. (d,e) Transwell assay for cell invasion and migration. (f,g) Flow cytometry for cell cycle analysis. (h) Tube formation in HUVECs by conditioned medium from indicated cells. (i) Flow cytometry of cell apoptosis rate. (j,k) Bax and Bcl-2 protein levels in cells were tested by western blot. Error bars stand for the mean ± SD of three independent measurements. *P < 0.05 and ** P < 0.01.

Figure 6.

PAK2 is a target of miR-1287-5p in NSCLC cells. (a) The predicted binding sites of miR-1287-5p on PAK2. (b–e) Dual-luciferase reporter and RIP assays were utilized to confirm the interaction between PAK2 and miR-1287-5p in A549 and H1299 cells. (f,g) Western blot analysis of PAK2 expression in NSCLC tissues and normal adjacent tissues, as well as in normal BEAS-2B cells and NSCLC cells. (h) The transfection efficiency of pc-NC or pc-PAK2 was verified by western blot. (i) Western blot analysis was employed to investigate the impact of miR-1287-5p on PAK2 expression profile. Error bars stand for the mean ± SD of three independent measurements. *P < 0.05 and ** P < 0.01.

Figure 7.

MiR-1287-5p inhibits malignant phenotypes in NSCLC cells by targeting PAK2. A549 and H1299 cells were transfected with miR-1287-5p and/or pc-PAK2. (a–c) MTT assay and colony formation assay for the cells proliferation; (d–e) Transwell assay for A549 and H1299 cell invasion and migration abilities. (f–g) Flow cytometry for cell cycle in A549 and H1299 cells. (h) Tube formation in HUVECs by conditioned medium from indicated A549 and H1299 cells. (i) Flow cytometry of A549 and H1299 cell apoptosis rate. (j,k) Bax and Bcl-2 protein levels in A549 and H1299 cells were detected by western blot. Error bars stand for the mean ± SD of three independent measurements. *P < 0.05 and ** P < 0.01.

Figure 8.

Circ_0008717 indirectly regulates PAK2 through binding to miR-1287-5p. After indicated transfection, the PAK2 expression in A549 and H1299 cells was detected by western blot. Error bars stand for the mean ± SD of three independent measurements. *P < 0.05 and ** P < 0.01.

Figure 9.

Circ_0008717 knockdown impedes tumor growth in vivo. (a,b) Tumor size and weight of xenograft were detected. (c–e) qRT-PCR or western blot was utilized to assay the levels of circ_0008717, miR-1287-5p and PAK2 in tumors from each group. (f) Representative images of PAK2 staining in the tumors. *P < 0.05 and ** P < 0.01.