Fig 2: MiR‐635 directly interacts with the 3′UTR of NOVA alternative splicing regulator 2 (NOVA2) in non‐small cell lung cancer (NSCLC) cells. (a) The putative target sites between miR‐635 and NOVA2 were predicted by starBase. (b, c) Luciferase activity was detected in NSCLC cells cotransfected with WT‐NOVA2 3′UTR or MUT‐NOVA2 3′UTR and miR‐NC or miR‐635. (d, e) The enrichment of miR‐635 or NOVA2 was measured by RNA immunoprecipitation (RIP) assay in NSCLC cells. (f) The mRNA of NOVA2 was measured in NSCLC tissues and adjacent noncancer tissues. (g, h) The correlation of NOVA2 with circ_0102231 and miR‐635 was analyzed by Spearman correlation analysis in NSCLC tissues. (i, j) The protein expression of NOVA2 was detected in NSCLC tissues, normal lung tissues, BEAS‐2B cells, A549 cells, H1299 cells, and H1581 cells. (k) The transfection efficiency of NOVA2 overexpression plasmid was analyzed by western blot. (l) NOVA2 expression was measured in A549 and H1299 cells introduced with miR‐NC, miR‐635, miR‐635 + vector, or miR‐635 + NOVA2. Data are presented as mean ± SD. *p < 0.05.

Fig 3: NOVA alternative splicing regulator 2 (NOVA2) acted as the downstream target gene of circ_0102231/miR‐635. A549 and H1299 cells were transfected with si‐NC, si‐circ_0102231, si‐circ_0102231 + anti‐miR‐NC, or si‐circ_0102231 + anti‐miR‐635. (a, b) The mRNA and protein levels of NOVA2 in A549 cells were measured by quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blot. (c, d) qRT‐PCR and western blot was conducted to detect the mRNA and protein levels of NOVA2 in H1299 cells, respectively. Data are presented as mean ± SD. *p < 0.05.

Fig 4: Overexpression of NOVA alternative splicing regulator 2 (NOVA2) also led to recovery of cellular malignant phenotypes after upregulation of miR‐635. A549 and H1299 cells were transfected with miR‐NC, miR‐635, miR‐635 + vector, or miR‐635 + NOVA2. CCK8 assays (a, b), 5‐ethynyl‐2′‐deoxyuridine (EdU) assays (c) and clone formation experiments (d) were used to detect cell proliferation. (f) The angiogenic ability of HUVEC was illustrated by tube formation experiments. (g) Apoptosis of A549 and H1299 cells was detected by flow cytometry. (h, i) The protein levels of PCNA, Bax and Bcl‐2 were measured by western blot. Data are presented as mean ± SD. *p < 0.05.