Circular RNA hsa_circ_0000317 inhibits non-small cell lung cancer progression through regulating microRNA-494-3p/phosphatase and tensin homolog deleted on chromosome 10 axis

Abstract

Background: Circular RNA (circRNA), a group of non-coding RNA, is pivotal in the progression of various cancers, including Non-Small Cell Lung Cancer (NSCLC). Some circRNAs have been reported to be implicated in the progression of NSCLC, however, the function and molecular mechanism of hsa_circ_0000317 (circ_0000317) in NSCLC have not been fully understood.

Methods: The significantly differentially expressed circRNA in NSCLC tissues, circ_0000317, was screened out by microarray. Circ_0000317, microRNA(miR)-494-3p and Phosphatase and Tensin Homolog Deleted on Chromosome 10 (PTEN) expressions in NSCLC tissues were respectively probed by quantitative real-time polymerase chain reaction and western blot assay. MTT and Transwell assays were adopted to examine the growth, migration, and invasion of NSCLC cells. Bioinformatics, luciferase reporter gene assay, RNA immunoprecipitation, and RNA pull-down assay were conducted to probe the relationships among circ_0000317, miR-494-3p, and PTEN.

Results: Circ_0000317 expression level was reduced in NSCLC tissues and cell lines. Circ_0000317 expression in NSCLC patients was associated with TNM stage and lymphatic metastasis. Circ_0000317 overexpression restrained the proliferation, migration, and invasion of NSCLC cells, but co-transfection of miR-494-3p mimics partially reversed this effect. In addition, circ_0000317, was identified as a competitive endogenous RNA, which could sponge miR-494-3p to increase PTEN expression and activate PI3K/AKT pathway.

Conclusion: Circ_0000317, inhibits NSCLC progression via modulating miR-494-3p/PTEN/PI3K/AKT pathway.

Keywords: Circ_0000317; Non-small cell lung cancer cells; PTEN; miR-494-3p.

Fig. 1

Circ_0000317 is downregulated in NSCLC tissues. (A) Volcano plot showed the differentially expressed circRNAs of lung adenocarcinoma tissues and matched non-tumor tissues in GSE101684. The cut-off criteria of values were as follows: ∣Log2(fold change)∣ > 1 and p < 0.05. The red points represented significantly up-regulated circRNAs, and the green points represented significantly down-regulated differentially expressed circRNAs, and the black points represented circRNAs with no statistically significant difference. (B) Heat map showed that 24 circRNAs were significantly differentially expressed in lung adenocarcinoma tissues. (C) The expression levels of circ_0000317 in 67 paired of NSCLC tissues (red points) and adjacent normal tissues (black points) were examined by qRT-PCR. (D) The expression levels of circ_0000317 in NSCLC tissues with lower TNM stage (black points, I‒II, n = 27) and higher stage (red points, III‒IV, n = 40) were examined by qRT-PCR. (E) The expression levels of circ_0000317 in NSCLC tissues with (red points, Positive, n = 32) and without (black points, Negative, n = 35) distant metastasis were analyzed by qRT-PCR. (F) The levels of circ_0000317 in NSCLC cell lines (A549, H460, PC9, H1299 and SPC-A1) and the immortalized Bronchial Epithelium Cell line (BEAS-2B) were examined by qRT-PCR. **p < 0.01, and ***p < 0.001.

Fig. 2

Knocking down circ_0000317 promotes the proliferation, migration, and invasion of NSCLC cells. (A) Si-NC, si-circ_0000317#1, si-circ_0000317#2 and si-circ_0000317#3 were transfected into A549 and PC9 cells, respectively, and the expression of circ_0000317 was detected by qRT-PCR. (B) MTT assay was used to detect the proliferation of A549 and PC9 cells transfected with si-circ_0000317#1 or si-circ_0000317#2. (C-D). Transwell assay was used to detect the migration and invasion of A549 and PC9 cells transfected with si-circ_0000317#1 or si-circ_0000317#2. si-NC, siRNA Negative Control; si-circ, siRNA targeting circ_0000317; ns, not statistically significant. *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 3

MiR-494-3p is the downstream target of circ_0000317. (A) FISH assays were performed to determine the localization of circ_0000317 in A549 and PC9 cells. (B) According to the binding site between circ_0000317 and miR-494-3p, circ_0000317-WT and circ_0000317-MUT luciferase reporter gene vectors were constructed. And miR-494-3p mimics or miR-control were co-transfected into HEK293T cells with circ_0000317-WT or circ_0000317-MUT. Then the luciferase activity of cells in each group was measured. (C-D) RIP assay (C) and RNA pull-down assay (D) were performed to verify the interaction of circ_0000317 and miR-494-3p. (E) Pearson's correlation analysis was employed to analyze the correlation between circ_0000317 expression and miR-494-3p expression in NSCLC tissues. (F) The expression of miR-494-3p in A549 and PC9 cells transfected with si-circ_0000317#1 and si-circ_0000317#2 was detected by qRT-PCR. Circ-WT, circ_0000317-WT; circ-MUT, circ_0000317-MUT; miR-con, miR-control; miR-494-3p, miR-494-3p mimic; ns, not statistically significant. ***p < 0.001.

Fig. 4

PTEN is the direct target of miR-494-3p. (A) According to the binding sites between PTEN 3′UTR and miR-494-3p, PTEN-WT, PTEN-MUT1, PTEN-MUT2 and PTEN-MUT1&2 luciferase reporter gene vectors were constructed. (B) MiR-494-3p mimic or miR-control were cotransfected into HEK293T cells with PTEN-WT or PTEN-MUT, respectively, and the luciferase activity of each group was determined. (C) Pearson's correlation analysis was employed to analyze the correlation between PTEN expression and miR-494-3p expression or circ_0000317 expression in NSCLC tissues. (D) The miR-494-3p mimic and miR-control were transfected into A549 and PC9 cells, and the expression of miR-494-3p was detected by qRT-PCR. (E) Western blot assay was used to detect the expression of PTEN in A549 and PC9 cells after overexpression of miR-494-3p. miR-con, miR-control; miR-494-3p, miR-494-3p mimic; ns, not statistically significant. *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 5

Circ_0000317 inhibits the progression of NSCLC through miR-494-3p/PTEN axis. PC9 and A549 cells were transfected with control vector, pcDNA3.1-circ_0000317, pcDNA3.1-circ_0000317 + miR-control, or pcDNA3.1-circ_0000317 + miR-494-3p mimics, respectively. (A) The expression of circ_0000317 in PC9 and A549 cells after transfection was detected by qRT-PCR. (B) The expression level of miR-494-3p in PC9 and A549 cells after transfection was detected by qRT-PCR. (C) Western blot assay was adopted to detect the protein expressions of PTEN, p-AKT, and p-PI3K in PC9 and A549 cells after transfection. (D) MTT assay was used to detect the proliferation of PC9 and A549 cells after transfection. (E‒F) Transwell assay was used to detect migration and invasion of PC9 and A549 cells after transfection. NC, Negative Control; circ, pcDNA3.1-circ_0000317; miR-con, miR-control; miR-494-3p, miR-494-3p mimic; ns, not statistically significant. * p < 0.05, **p < 0.01, and *** p < 0.001.