Silencing LINC00663 inhibits inflammation and angiogenesis through downregulation of NR2F1 via EBF1 in bladder cancer

Figures & data

Table 1. The clinical data of patients with bladder cancer.

Pathological features	Cases
Gender
Male	25
Female	5
Age
≤60	14
>60	16
TNM stage
I	10
II	8
III	8
IV	4
Lymph node metastasis
no	20
yes	10

TNM, tumour-node-metastasis.

Figure 1. NR2F1 expressed at a high level in BC tissues.

(A) bioinformatics prediction was used to analyse the DEGs in the occurrence and development of BC. (B) univariate Cox analysis on survival data was used to analyse the genes related to BC prognosis. (C) multivariate Cox analysis was used to analyse the genes related to BC prognosis. (D) the relationship between NR2F1 and BC prognosis was analysed using a pan-cancer dataset downloaded from the UCSC database and a prognostic dataset from the TCGA database in a previous study. (E) the expression of NR2F1 in BC tissues and normal para-cancer tissues was measured by qRT-PCR and western blot. (F) the expression of NR2F1 in tissues of patients with/without lymph node metastasis was detected by qRT-PCR and western blot. *p < 0.05, compared with para-cancer, or LN(-) group. BC, bladder cancer; DEGs, differentially expressed genes; LN, lymph node metastasis.

Figure 2. Low expression of NR2F1 suppressed inflammation and vascular mimicry in BC cells.

(A) the expression of NR2F1 in BC cells (HT-1376, 5637, J82, and T24) and human normal bladder epithelium (SV-HUC-1) were assessed by qRT-PCR and western blot. (B) the transfection efficiency was measured by qRT-PCR and western blot. (C-D) CCK-8 and colony-forming assays were used to test cell proliferation. (E) the ability of vascular mimicry was evaluated through tube formation experiment. (F) cell invasion ability was tested by transwell assay. (G) the levels of TNF-α, IL-6, and IL-1β were measured by ELISA. (H) the expression of Snail, E-cadherin, and VEGFA was examined by western blot. Data were expressed as mean ± standard deviation, and each experiment was repeated three times. *p < 0.05, compared with SV-HUC-1 or sh-NC group. BC, bladder cancer.

Figure 3. LINC00663 positively modulated NR2F1.

(A-B) qRT-PCR was used to measure the expression of LINC00663 in BC tissues and cells. (C) FISH assay was used to detect the localization of LINC00663 in BC cells. (D) the binding of LINC00663 and NR2F1 was verified by dual-luciferase reporter gene assay. (E) the correlation between LINC00663 and NR2F1 was analysed by Pearson. (F) after 5673 cells were transfected with sh-LINC00663, the expression of NR2F1 was evaluated by qRT-PCR and western blot. Data were expressed as mean ± standard deviation, and each cellular experiment was repeated three times. *p < 0.05, compared with para-cancer, SV-HUC-1 or sh-NC group. BC, bladder cancer.

Figure 4. Knockdown of LINC00663 inhibited inflammation and vascular mimicry in BC cells by decreasing NR2F1 expression.

After 5637 cells were transfected with sh-LINC00663 and oe-NR2F1, (A) the transfection efficiency was tested with qRT-PCR and western blot. (B-C) cell proliferation was evaluated by CCK-8 and colony-forming assays. (D) the ability of vascular mimicry was assessed by tube formation experiments. (E) cell invasion was detected by transwell assay. (F) the levels of TNF-α, IL-6, and IL-1β were measured by ELISA. (G) the expression of Snail, E-cadherin, and VEGFA was examined by western blot. Data were shown as mean ± standard deviation, and each assay was repeated three times. *p < 0.05, compared with the sh-LINC00663 + oe-NC group; ^#p < 0.05, compared with the sh-LINC00663 + oe-NC group. BC, bladder cancer.

Figure 5. LINC00663 modulated NR2F1 expression through EBF1 and affected inflammation and vascular mimicry in BC cells.

Figure 5. (Continued). (A) RIP assay was used to verify the binding between LINC00663 and EBF1. (B) the binding sites between EBF1 and NR2F1 were predicted by the JASPAR website. (C-E) the binding relationship between EBF1 and NR2F1 was verified by dual-luciferase reporter gene assay and ChIP assay (groups of site 1/2/3 referred to qPCR primers designed according to sequences of site 1/2/3 of NR2F1). (F) the binding between LINC00663 and NR2F1 was verified by ChIP assay. After BC cells were transfected with sh-LINC00663 and oe-EBF1, (G) the expression of NR2F1 was measured by qRT-PCR and western blot. (HI) cell proliferation was tested by CCK-8 and colony-forming assays. (J) the ability of vascular mimicry was examined by tube formation experiments. (K) cell invasion was measured by transwell assay. (L) the levels of TNF-α, IL-6, and IL-1β were measured by ELISA. (M) the expression of Snail, E-cadherin, and VEGFA was examined by western blot. Data were exhibited as mean ± standard deviation, and each experiment was run in triplicate. *p < 0.05, compared with IgG, oe-NC, or Distal group; ^#p < 0.05, compared with sh-NC or sh-NC + oe-NC group; ^&p < 0.05, compared with the sh-LINC00663 + oe-NC group. BC, bladder cancer.

Figure 6. Silencing LINC00663 repressed tumour growth by reducing NR2F1 expression.

(A) xenograft tumour extracted from nude mice. (B) the weight of tumour was analysed.(C) the growth curve was used to evaluate the growth rate. (D) the expression of LINC00663, NR2F1, Snail, VEGFA, and E-cadherin was measured by qRT-PCR or western blot. (E) the levels of TNF-α, IL-6, and IL-1β were tested by ELISA. (F) IHC assay was used to measure the expression of Ki67. (G) IHC assay was used to assess the change of MVD. Data were expressed as mean ± standard deviation, n = 6. *p < 0.05, compared with sh-NC+ oe-NC group; ^#p < 0.05, compared with the sh-LINC00663 + oe-NC group. MVD, microvessel density; IHC, Immunohistochemistry.

Data availability statement

The datasets used or analysed during the current study are available from the corresponding author on reasonable request.