https://orcid.org/0000-0001-7925-7033
Figures & data
Table 1. Clinicopathologic details of the patients
Figure 1. High APCDD1L-AS1 is discovered in OSCC and correlative to poor prognosis of OSCC patients
(A) APCDD1L-AS1 expression in OSCC tissues and normal tissues was determined by RT-qPCR. (B) The overall survival was analyzed in OSCC patients with high or low APCDD1L-AS1 expression. (C) APCDD1L-AS1 expression was examined in 5-FU-sensitive and 5-FU-resistant OSCC tissues. (D) APCDD1L-AS1 expression was tested in OSCC cell lines (SCC-4, HSC-3, TSCC1, SCC090, and HN-4) and normal human oral keratinocyte cell line (NHOK). (E) APCDD1L-AS1 expression was detected in HSC-3 and HN-4 cells and corresponding 5-FU-resistant cells (HSC-3/5-FU and HN-4/5-FU cells). *P< 0.05, **P < 0.01.
Figure 2. APCDD1L-AS1 confers resistance to 5-FU in OSCC cells
(A) RT-qPCR was performed to measure APCDD1L-AS1 level in HSC-3/5-FU and HN-4/5-FU cells transfected with sh-NC and sh-APCDD1L-AS1. (B) CCK-8 was performed to assess IC50 of 5-FU in HSC-3/5-FU and HN-4/5-FU cells transfected with sh-NC and sh-APCDD1L-AS1. (C) Colony formation assay was conducted to examine the cell viability of HSC-3/5-FU and HN-4/5-FU cells transfected with sh-NC and sh-APCDD1L-AS1. (D) Flow cytometry analysis was conducted to probe the apoptotic rate of HSC-3/5-FU and HN-4/5-FU cells transfected with sh-NC and sh-APCDD1L-AS1. **P < 0.01.
Figure 3. APCDD1L-AS1 is transferred extracellularly via exosome incorporation in 5-FU-Resistant OSCC Cells
(A) APCDD1L-AS1 expression was detected by RT-qPCR after cells were treated with 1 μg/mL RNase A or 1 μg/mL RNase A + 0.1% Triton X100 for 30 min. (B) The exosomes images secreted by HSC-3/5-FU and HN-4/5-FU cells were showed by TEM scanning. (C) The levels of CD63 and CD9 proteins were measured by Western blot in HSC-3/5-FU and HN-4/5-FU cells. (D) APCDD1L-AS1 expression in exosomes of parental and 5-FU-sensitive NSCLC cells was detected by RT-qPCR. **P < 0.01.
Figure 4. APCDD1L-AS1 inhibition attenuates 5-FU resistance mediated by exosomes
Exosomes were isolated from 5-FU-sensitive NSCLC cells, and HSC and HN-4 cells were treated with exosome or exosome+sh-APCDD1L-AS1. (A and B) APCDD1L-AS1 expression was detected by RT-qPCR in HSC and HN-4 cells. (C and D) 5-FU IC50 was detected for HSC and HN-4 cells by cell viability assay. (E and F) The proliferation of HSC and HN-4 cells was determined by CCK-8assay. (G and H) The apoptosis of HSC and HN-4 cells were assessed by flow cytometry analysis. *P< 0.05, **P < 0.01.
Figure 5. APCDD1L-AS1 serves as a sponge for miR-1224-5p in 5-FU-resistant OSCC cells
(A) The binding sites between miR-1224-5p and APCDD1L-AS1 were shown by Starbase software. (B) RT-qPCR was performed to measure miR-1224-5p level in HSC-3/5-FU and HN-4/5-FU cells transfected with NC mimics and miR-1224-5p mimics. (C and D) Dual-luciferase reporter assay was performed to validate the combination between miR-1224-5p and APCDD1L-AS1 in 5-FU-resistant OSCC cells. (E and F) RIP assay was performed to confirm the target relationship between miR-1224-5p and APCDD1L-AS1 in 5-FU-resistant OSCC cells. (G) miR-1224-5p expression in OSCC tissues and normal tissues was determined by RT-qPCR. (H) miR-1224-5p expression was examined in 5-FU-sensitive and 5-FU-resistant OSCC tissues. (I) RT-qPCR was performed to measure APCDD1L-AS1 level in HSC-3/5-FU and HN-4/5-FU cells transfected with Vector and oe-APCDD1L-AS1. (J and K) RT-qPCR was performed to detect miR-1224-5p expression in HSC-3/5-FU and HN-4/5-FU cells transfected with sh-NC, sh-APCDD1L-AS1, Vector or oe-APCDD1L-AS1. (L) MiR-1224-5p expression was detected in HSC-3/5-FU and HN-4/5-FU cells transfected with NC inhibitor or miR-1224-5p inhibitor. (M and N) RT-qPCR was performed to detect APCDD1L-AS1 expression HSC-3/5-FU and HN-4/5-FU cells transfected with NC inhibitor, miR-1224-5p inhibitor, NC mimics, or miR-1224-5p mimics. **P < 0.01, ***P < 0.001.
Figure 6. MiR-1224-5p overexpression overcomes 5-FU resistance in 5-FU-resistant OSCC cells
(A) CCK-8 revealed the IC50 value in HSC-3/5-FU and HN-4/5-FU cells transfected with NC mimics or miR-1224-5p mimics. (B) Colony formation assay were used to test cell proliferation and HSC-3/5-FU and HN-4/5-FU cells treated with NC mimics or miR-1224-5p mimics. (C) Flow cytometry analysis was applied to examine apoptosis in HSC-3/5-FU and HN-4/5-FU cells transfected with NC mimics or miR-1224-5p mimics. (D-F) CCK-8, Colony formation assay and flow cytometry were employed to detect IC50, cell proliferation and apoptosis in HSC-3/5-FU and HN-4/5-FU cells transfected with NC inhibitor or miR-1224-5p inhibitor. *P< 0.05, **P < 0.01.
Figure 7. NSD2 is a direct target of miR-1224-5p in 5-FU-resistant OSCC cells
(A) The Venn diagram showed the downstream target genes which could bind with miR-1224-5p in PITA, RNA22, miRmap, and microT databases. (B) Starbase website predicted putative-binding sites between miR-1224-5p and NSD2. (C) NSD2 expression was detected by RT-qPCR in cells transfected with miR-1224-5p mimics. (D) RIP assays were conducted to confirm miR-1224-5p and NSD2 coexisted in RNA-induced silencing complexes (RISCs). (E) Luciferase reporter assay was conducted to verify the combination between miR-1224-5p and NSD2. (F) NSD2 expression was detected in HSC-3/5-FU and HN-4/5-FU cells transfected with NC mimics, miR-1224-5p mimics, or miR-1224-5p mimics+oe-APCDD1L-AS1. **P< 0.01, ***P < 0.001.
Figure 8. APCDD1L-AS1 knockdown enhances susceptibility to 5-FU in 5-FU-resistant OSCC cells by decreasing NSD2
(A) NSD2 expression was tested in cells transfected with Vector or oe-NSD2. (B-D) CCK-8, Colony formation assay and flow cytometry were employed to detect IC50, cell proliferation and apoptosis in HSC-3/5-FU and HN-4/5-FU cells transfected with sh-NC, sh-APCDD1L-AS1, or sh-APCDD1L-AS1+ oe-NSD2. *P< 0.05, **P < 0.01.
