LINC00173 facilitates tumor progression by stimulating RAB1B-mediated PA2G4 and SDF4 secretion in nasopharyngeal carcinoma

Abstract

An increasing number of studies have found that long non-coding RNA (lncRNA) play important roles in driving the progression of nasopharyngeal carcinoma (NPC). Our microarray screening revealed that expression of the lncRNA long intergenic non-protein coding RNA 173 (LINC00173) was upregulated in NPC. However, its role and mechanism in NPC have not yet been elucidated. In this study, we demonstrate that high LINC00173 expression indicated a poor prognosis in NPC patients. Knockdown of LINC00173 significantly inhibited NPC cell proliferation, migration and invasion in vitro. Mechanistically, LINC00173 interacted and colocalized with Ras-related protein Rab-1B (RAB1B) in the cytoplasm, but the modulation of LINC00173 expression did not affect the expression of RAB1B at either the mRNA or protein levels. Instead, relying on the stimulation of RAB1B, LINC00173 could facilitate the extracellular secretion of proliferation-associated 2G4 (PA2G4) and stromal cell-derived factor 4 (SDF4; also known as 45-kDa calcium-binding protein) proteins, and knockdown of these proteins could reverse the NPC aggressive phenotype induced by LINC00173 overexpression. Moreover, in vivo LINC00173-knockdown models exhibited a marked slowdown in tumor growth and a significant reduction in lymph node and lung metastases. In summary, LINC00173 serves as a crucial driver for NPC progression, and the LINC00173-RAB1B-PA2G4/SDF4 axis might provide a potential therapeutic target for NPC patients.

Keywords: LINC00173; PA2G4; RAB1B; SDF4; nasopharyngeal carcinoma.

Fig. 1

LINC00173 is upregulated and related to poor prognosis in nasopharyngeal carcinoma (NPC). (A) LINC00173 expression in NPC from GEO Dataset (GSE126683, normal = 3 cases, NPC = 3 cases). (B) LINC00173 expression in freshly frozen NPC tissues (n = 20) and normal nasopharynx tissues (n = 16). (C) LINC00173 expression in NPC cell lines and normal nasopharyngeal epithelial cell line NP69 (n = 3). (D–F) Kaplan–Meier analysis of overall survival (D), disease‐free survival (E) and distant metastasis‐free survival (F) in NPC patients (high LINC00173 expression, n = 107; low LINC00173 expression, n = 107). Relative LINC00173 expression in NPC tissues of 214 patients were measured by RT‐qPCR assay. High and low LINC00173 expression groups were divided using the median expression value. (G–I) Kaplan–Meier analysis of overall survival (G), disease‐free survival (H) and distant metastasis‐free survival (I) according to the prognostic prediction model, low risk [early tumor‐node‐metastasis (TNM) stage and low LINC00173 expression, n = 68], intermediate risk (advanced TNM stage or high LINC00173 expression, n = 107) and high risk groups (advanced TNM stage and high LINC00173 expression, n = 39). The data of (A–C) were shown as mean ± SD, and the P‐values were determined by Student's t‐test (*P < 0.05; **P < 0.01). The data of (D–I) were measured by the Kaplan–Meier method, and the P‐values were determined by the log‐rank test.

Fig. 2

LINC00173 promotes nasopharyngeal carcinoma (NPC) cells proliferation, migration and invasion in vitro. (A,B) Cell proliferation curves (A) and clonogenic assays (B) of HK1 and SUNE1 cells transfected with shCtrl or shLIN00173 (sh0173) plasmid (n = 3). (C,D) Cell proliferation curves (C) and clonogenic assays (D) of HK1 and SUNE1 cells transfected with vector or LIN00173 overexpression (OE) plasmid (n = 3). (E) Transwell migration and invasion assays of HK1 and SUNE1 cells transfected with shCtrl or sh0173 vector plasmids (n = 3). Scale bar: 100 μm. The migrated or invaded cells were counted. (F) Transwell migration and invasion assays of HK1 and SUNE1 cells transfected with vector or LIN00173 OE plasmids (n = 3). Scale bar: 100 μm. Data are presented as mean ± SD. P‐values were determined by Student's t‐test (*P < 0.05; **P < 0.01).

Fig. 3

LINC00173 directly interacts with RAB1B. (A) Biotin‐labeled LINC00173 sense (SS) and anti‐sense (AS) RNA pulled down samples were subjected to SDS/PAGE electrophoresis and silver stained (n = 3). RAB1B is a potential interactive candidate of LINC00173. (B) RNA pulldown with western blot (WB) analysis of the interactions of LINC00173 and RAB1B in HK1 and SUNE1 cells (n = 3). (C) RNA immunoprecipitation (RIP) with anti‐RAB1B antibody in HK1 and SUNE1 cells revealed RAB1B protein bound to LINC00173 RNA. Relative enrichment of LINC00173 levels interacted with RAB1B proteins was normalized to Input extraction (10% total extraction without immunoprecipitation, n = 3). (D) Agarose gel electrophoresis showed the mount of LINC00173 in the above RIP samples (n = 3). GAPDH was used as a negative control. (E) Nucleocytoplasmic separation and RT‐qPCR assays detected LINC00173 expression in cell nucleus and cytoplasm (n = 3). (F) Confocal imaging of LINC00173 and RAB1B in HK1 and SUNE1 cells was visualized by RNA‐FISH and immunofluorescence assays (n = 3). Scale bar: 50 μm. (G) Relative levels of LINC00173 and RAB1B in HK1 and SUNE1 cells after transfection with shLINC00173 or LINC00173 overexpression plasmids (n = 3). (H) Relative levels of RAB1B protein in HK1 and SUNE1 cells after transfection with shLINC00173 or LINC00173 overexpression plasmids (n = 3). Data of (C and G) are presented as mean ± SD. P‐values were determined by Student's t‐test (*P < 0.05; **P < 0.01).

Fig. 4

LINC00173 facilitates PA2G4 and SDF4 secretion in a RAB1B‐mediated manner. (A) Workflows of mass spectrometry (MS) analysis for LINC00173 regulated specific secreted proteins. (B) Top10 enriched proteins of concentrated supernatants in shCtrl and shLINC00173 (sh0173) group. (C) The peptide peak spectrum of PA2G4 and SDF4 proteins according to the MS analysis. (D) Western blot (WB) assay detected the RAB1B, PA2G4 and SDF4 expression in the lysate and concentrated supernatants of HK1 and SUNE1 cells transfected with sh0173 plasmid (n = 3). (E) WB assay detected the RAB1B, PA2G4 and SDF4 expression in lysate and concentrated supernatants of HK1 and SUNE1 cells transfected with sh0173 plasmid and treated with Exo‐1 (n = 3). (F) WB assay detected the RAB1B, PA2G4 and SDF4 expression in lysate and concentrated supernatants of HK1 and SUNE1 cells cotransfected with sh0173 and RAB1B overexpression (OE) plasmids (n = 3). The gels were stained with Coomassie blue to determine the total protein consistent loading in each lane.

Fig. 5

Knockdown of PA2G4 or SDF4 reverses LINC00173‐mediated nasopharyngeal carcinoma (NPC) progression. (A) Cell proliferation curves of HK1 and SUNE1 cells cotransfected with LINC00173 overexpression (0173‐OE) and shPA2G4 plasmids (n = 3). (B) Cell proliferation curves of HK1 and SUNE1 cells cotransfected with 0173‐OE and shSDF4 plasmids (n = 3). (C) Clonogenic assays of HK1 and SUNE1 cells cotransfected with 0173‐OE and shPA2G4 plasmids (n = 3). (D) Clonogenic assays of HK1 and SUNE1 cells cotransfected with 0173‐OE and shSDF4 plasmids (n = 3). (E) Transwell migration and invasion assays of HK1 and SUNE1 cells cotransfected with 0173‐OE and shPA2G4 plasmids (n = 3). Scale bar: 100 μm. (F) Transwell migration and invasion assays of HK1 and SUNE1 cells cotransfected with 0173‐OE and shSDF4 plasmids (n = 3). Scale bar: 100 μm. Data are presented as mean ± SD. P‐values were determined by Student's t‐test (*P < 0.05; **P < 0.01).

Fig. 6

Knockdown of LINC00173 inhibits nasopharyngeal carcinoma (NPC) tumor growth and metastasis in vivo. (A) Tumor growth curves of nude mice injected with stable LINC00173 knockdown (sh0173) or shCtrl SUNE1 cells (n = 8). (B) Representative xenograft tumor images from shCtrl and sh0173 groups (n = 8). (C) Average weight of tumors for shCtrl and sh0173 group (n = 8). (D) Representative inguinal lymph node and foot pad tumor images of lymphatic metastasis model for shCtrl and sh0173 groups (n = 8). (E) Representative inguinal lymph node images for shCtrl and sh0173 groups (n = 8). (F) Average volume of inguinal lymph nodes for the sh0173 and shCtrl group (n = 8). (G) H&E staining for foot pad primary tumors (n = 8). Scale bar: 50 μm. (H) Pan‐cytokeratin staining for the inguinal lymph nodes from both groups (n = 8). Scale bars: 2 mm and 500 μm. (I) Pearson's χ² test for metastatic ratio of inguinal lymph nodes in the sh0173 and shCtrl group (n = 8). (J) Representative images of metastatic nodules in lung metastasis model (n = 8). Scale bar: 1 cm. (K) H&E staining of lung tissues from shCtrl and sh0173 groups (n = 8). Scale bars: 5 mm, 2 mm and 500 μm. (L) Average number of metastatic nodules for the sh0173 and shCtrl group (n = 8). (M) Graphical abstract of LINC00173 function and mechanism in NPC. P‐values of (A, C, F and L) were determined by Student's t‐test; data are presented as mean ± SD (*P < 0.05; **P < 0.01; ***P < 0.001). The P‐value of (I) was determined by Pearson's χ² test (*P < 0.05).