https://orcid.org/0000-0002-6564-5657
Figures & data
Figure 1. LINC01605 is upregulated in NPC cells.
(a) RT-qPCR analysis was done for detecting LINC01605 expression in NPC cells and NP69 cells. One-way ANOVA was applied for statistical analysis. (b) LINC01605 level in HNSC tumour tissues or normal tissues was obtained on GEPIA. Student’s t-test was applied for statistical analysis. (c-d) FISH and subcellular fractionation assays were done to determine the cellular position of LINC01605. Student’s t-test was applied for statistical analysis in subcellular fractionation assay. *P < 0.05, **P < 0.01.
Figure 2. LINC01605 silence inhibits cell proliferation and enhances cell apoptosis in NPC. (a) Efficiency of sh-LINC01605#1/2 in CNE-1 and 5–8 F cells was evaluated by RT-qPCR. (b–d) The changes in cell viability and proliferation under the influence of sh-LINC01605 transfection were evaluated by CCK-8, EdU and colony formation assays. (e–f) Apoptosis of sh-LINC01605-transfected CNE-1 and 5–8 F cells was measured by JC-1 and TUNEL assays. One-way ANOVA was applied for statistical analysis. **P < 0.01.
Figure 3. LINC01605 regulates Ikbkb expression to promote nuclear translocation of p65 and thereby activates the NF-κB pathway.
(a) The specific pathway involved in the LINC01605 modulation mechanism in NPC cells was preliminarily screened out via luciferase reporter assay by measuring the changes in the activity of several signalling pathways in HEK293T and CNE-1 cells under the influence of LINC01605 depletion. (b) Western blot detected the impacts of LINC01605 silence and overexpression on the protein levels of p-p65, p65, IKKβ, p-IKBα (Ser36) and p-IKBβ (Ser23). (c) RT-qPCR detected the impacts of LINC01605 silence on the mRNA levels of CHUK, Ikbkb, Ikbkg and RelA. One-way ANOVA was applied for statistical analysis. (d) Immunofluorescence staining detected the impacts of LINC01605 silence on p-p65 nuclear enrichment. **P < 0.01, n.s.: no significance.
Figure 4. LINC01605 sequesters miR-942-5p to regulate Ikbkb mRNA.
(a) MiRNAs combining with LINC01605 and Ikbkb in common were predicted by DIANA and starBase websites. (b) Respective binding sites of LINC01605 and Ikbkb on miR-942-5p were predicted by DIANA and starBase websites. (c) RT-qPCR analysis was done for the measurement of miR-942-5p expression in NPC cells and NP69. One-way ANOVA was applied for statistical analysis. (d) RT-qPCR analysis was applied for the detection of miR-942-5p expression in CNE-1 and 5–8 F cells transfected with miR-942-5p mimics. Student’s t-test was utilized for statistical analysis. (e) Luciferase activities were assessed in CNE-1 and 5–8 F cells co-transfected with miR-942-5p mimics and LINC01605-WT/MUT or miR-942-5p mimics and Ikbkb-WT/MUT. Two-way ANOVA was utilized for statistical analysis. (f) Luciferase activities of Ikbkb-WT/MUT were assessed in CNE-1 and 5–8 F cells co-transfected with miR-942-5p mimics or miR-942-5p mimics+pcDNA/LINC01605. Two-way ANOVA was utilized for statistical analysis. (g) In RIP assay, enrichment of LINC01605, miR-942-5p and Ikbkb in Ago2 was assessed via RT-qPCR. Student’s t-test was taken for statistical analysis. (h) Level of Ikbkb mRNA as well as that of IKKβ protein was analysed in CNE-1 and 5–8 F cells transfected with sh-LINC01605#1 or sh-LINC01605#1+ miR-942-5p inhibitor. One-way ANOVA was taken for statistical analysis. *P < 0.05, **P < 0.01, n.s.: no significance.
Figure 5. LINC01605 regulates USP3 to stabilize IKKβ protein.
(a) CNE-1 and 5–8 F cells with LINC01605 knockdown were treated with CHX and IKKβ protein levels were analysed via Western blot. (b) Western blot analysis was done for the detection of IKKβ protein levels in CNE-1 and 5–8 F cells with or without LINC01605 knockdown and MG132 treatment. (c) LINC01605-silenced CNE-1 and 5–8 F cell lysates were immunoprecipitated with IKKβ antibody and then immunoblotted for ubiquitin and IKKβ.
Figure 6. LINC01605 recruits IGF2BP2 to stabilize USP3 mRNA.
(a) Luciferase activity of the pGL3-USP3 promoter was detected in CNE-1 and 5–8 F cells transfected with sh-LINC01605#1. (b) The stability of USP3 mRNA was measured after actinomycin D treatment in LINC01605-silenced CNE-1 and 5–8 F cells. (c) RNA pull-down assay, followed by Western blot, detected the combination between LINC01605 and IGF2BP2. (d) RIP assay detected the bond of LINC01605 and IGF2BP2. (e) RNA pull-down assay followed by Western blot detected the combination between USP3 and IGF2BP2. (f) RIP assay detected the impacts of LINC01605 silence on combination between USP3 and IGF2BP2. (g) The stability of USP3 mRNA was measured after treatment of Actinomycin D in IGF2BP2-silenced CNE-1 and 5–8 F cells. Student’s t-test was used for statistical analysis. **P < 0.01, n.s.: no significance.
Figure 7. Overexpression of p65 enhances LINC01605 expression.
(a) ChIP assay detected the combination between the LINC01605 promoter and p65. (b) Luciferase activity of the pGL3-LINC01605 promoter was assessed in CNE-1 and 5–8 F cells treated with the p65 inhibitor or transfected with the p65 overexpression vector. (c) Expression level of LINC01605 was analysed in CNE-1 and 5–8 F cells treated with p65 inhibitor or transfected with p65 overexpression vector using RT-qPCR. Student’s t-test was utilized for statistical analysis. **P < 0.01.
Supplemental material