Fig 1: NRN1 silencing suppresses in vivo growth of RCC-PDC-derived xenograft tumors. (A) Representative images of xenograft tumor-bearing nude mice at time of sacrifice. (B) Volume of xenograft tumors derived from RCC-PDC1 cells treated with siControl (n = 5, red) or siNRN1 #1 (n = 5, blue). (C) Body weights of mice at time of sacrifice. (D) Representative images of hematoxylin and eosin (HE) staining and NRN1 and CXCR4 IHC staining in dissected xenograft tumors treated with siControl or siNRN1 #1. Scale bars, 50 µm. (E, F) NRN1 (E) and CXCR4 (F) levels in xenograft tumors analyzed by qRT-PCR. Data are shown as mean ± SD, n = 5; *P < 0.05, **P < 0.01 by two-sided Student’s t-test. (G) Schematic representation of oncogenic function of NRN1 and CXCR4 in RCC tumors.
Fig 2: NRN1 and CXCR4 promote RCC-PDC viability. (A–H) NRN1 silencing decreases whereas overexpression increases PDC viability. RCC-PDC1/2 spheroid cultures were transfected with NRN1 (siNRN1 #1 and #2) or control (siControl) siRNAs (A–D), and NRN1 or control expression vector (E–H). NRN1 mRNA levels were analyzed by qRT-PCR (A, C, E, G) (n = 3) and spheroid growth was estimated by cell viability assay based on ATP quantification in cell lysates (B, D, F, H) (n = 4). Data are shown as means ± SD. *P < 0.05 by two-sided Student’s t-test. (I–L) NRN1 silencing decreases whereas overexpression increases CXCR4 expression. RCC-PDC1/2 were transfected with siRNAs: siNRN1 #1, #2, or siControl (I, J), or expression vectors (NRN1 or control vector) (K, L). (M–P) CXCR4 silencing represses PDC viability. RCC-PDC1 and 2 were transfected with siRNAs targeting CXCR4 (siCXCR4 #1 and #2) or siControl. CXCR4 mRNA levels were analyzed by qRT-PCR (M, O) and spheroid growth was estimated by cell viability assay (N, P). Data are shown as means ± SD, n = 3. *P < 0.05 by two-sided Student’s t-test.
Fig 3: Histological analysis of RCC primary tumors, and their patient-derived cancer models. Hematoxylin and eosin (HE) staining, NRN1 and CXCR4 immunohistochemistry of primary tumor specimens, corresponding patient-derived cancer cell (PDC) spheroid cultures, and their xenograft tumors. Star represents ccRCC tumor portion with eosinophilic cytoplasm. Scale bars, 50 µm.
Fig 4: NRN1 is a poor prognostic factor for patients with RCC. (A) Representative immunohistochemistry (IHC) staining of low (left panel) and high (right panel) expression of NRN1 in RCC tissue sections. Scale bars, 100 µm. (B) Overall survival of 100 patients with clear cell RCC (ccRCC) of low or high NRN1 immunoreactivity was analyzed by Kaplan-Meier method. Statistical significance was evaluated by log-rank test. (C) Overall survival of RCC patients with low or high NRN1 mRNA levels were analyzed by Kaplan-Meier method. NRN1 expression data in TCGA RCC dataset were retrieved from The Human Protein Atlas (n = 845). High and low NRN1 expressions were determined at an expression cut off level as 6.6 fragments per kilobase of exon per million mapped fragments (FPKM) (expression level range: 0.1-204.2, expression median level: 2.9 FPKM, P = 3.4e-4). Statistical significance was evaluated by log-rank test.
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