Fig 2: Immunostaining of NETO2 protein in human CRC and adjacent normal tissues. a Representative immunohistochemical expression patterns of NETO2 in cancerous and adjacent normal mucosa specimens were shown. (Magnification, upper panel, ×100; lower panel, ×400) Right panel: human CRC cell line HCT116 cells were used as a negative control and HCT15 cells were used as a positive control for immunostaining of NETO2 protein. (Magnification × 100) (b) Percentage of cases with different staining intensity of NETO2 in the tumor or adjacent normal tissues in the study cohort. (p < 0.001)

Fig 3: NETO2 prompts the proliferation, migration and invasion of NPC cells but inhibits cell apoptosis(A) The expression of 5 mRNAs was examined in C17 and C666-1 cells with or without miR-876-5p augmentation. (B) Binding sites between miR-876-5p and NETO2 were predicted. (C) The enrichment of NETO2 in Bio-NC, Bio-miR-876-5p-Wt/Mut was quantified. (D) The binding affinity between miR-876-5p and NETO2 was appraised based on the luciferase activity of NETO2-Wt/Mut in C17 and C666-1 cells transfected with miR-876-5p mimics. (E) The enrichment of NETO2, TTN-AS1, and miR-876-5p in RISCs was examined. (F) TTN-AS1 expression was assessed in C17 and C666-1 cells transfected with pcDNA3.1/TTN-AS1. (G) NETO2 expression was detected in C17 and C666-1 cells respectively co-transfected with NC mimics, miR-876-5p mimics, or miR-876-5p mimics + pcDNA3.1/TTN-AS1. (H) NETO2 expression was evaluated in C17 and C666-1 cells with NETO2 knockdown. (I–N) The effects of NETO2 depletion on cell proliferative, migratory, and invasive capabilities as well as the apoptotic ability were examined in C17 and C666-1 cells successfully transfected with sh-NC or sh-NETO2#1/2 Scale bar = 200µm, Scale bar = 200µm, Scale bar = 100µm. Data obtained from three or more than three independent experimental results were shown as mean±SD. **p < 0.01; n.s., not significant.

Fig 4: Kaplan-Meier survival analysis for CRC patients. a Kaplan-Meier curves for disease-specific survival of all CRC patients in the study cohort according to NEOT2 expression status. b Kaplan-Meier curves for disease-specific survival of all CRC patients according to TNM stage. c-d Kaplan-Meier curves for disease-specific survival of patients with early stage tumors (c) or advanced stage tumors (d) according to NETO2 expression status. The p-value was determined using the log-rank test. The absolute number of patients at risk in each subgroup is listed below

Fig 5: NETO2 function is affected by miR-193a-5p and SNHG17. (A) The proliferative ability of A549 and H1299 cells was assessed following transfection with the indicated groups. *P<0.05, **P<0.01 vs. si-NC; &P<0.05, &&P<0.01 vs. si-NETO2. (B) Western blot analysis was performed to detect the protein expression levels of the epithelial-to-mesenchymal markers following transfection with the indicated groups. *P<0.05, **P<0.01, ***P<0.001 vs. si-NC; &P<0.05, &&P<0.01 vs. si-NETO2. (C) Correlation analysis between NETO2 and miR-193a-5p expression. (D) Correlation analysis between NETO2 and SNHG17 expression. (E and F) The effects of miR-193a-5p and SNHG17 on NETO2 expression. **P<0.01, ***P<0.001 vs. si-NC; &&P<0.01, &&&P<0.001 vs. si-NETO2. miR, microRNA; si, small interfering; NC, negative control; OD, optical density.