LincRNA01703 Facilitates CD81+ Exosome Secretion to Inhibit Lung Adenocarcinoma Metastasis via the Rab27a/SYTL1/CD81 Complex

Abstract

Metastasis, a major cause of cancer-related mortality worldwide, frequently occurs early in the diagnosis of lung adenocarcinoma (LUAD). However, the precise molecular mechanisms governing the aggressive metastatic behavior of LUAD remain incompletely understood. In this study, we present compelling evidence indicating that the long noncoding RNA linc01703 is significantly downregulated in metastatic lung cancer cells. Intriguingly, in vivo experiments revealed that Linc01703 exerted a profound inhibitory effect on lung cancer metastasis without discernible impact on the in vitro proliferation or invasion capacities of LUAD cells. Mechanistically, Linc01703 enhanced the interaction between Rab27a, SYTL1, and CD81, consequently promoting the secretion of CD81⁺ exosomes. These exosomes, in turn, suppressed the infiltration of immune cells within the tumor microenvironment, thereby impeding LUAD metastasis. Importantly, our analysis of lung cancer tissues revealed a correlation between reduced CD81 expression and an unfavorable patient prognosis. Collectively, our findings suggest that Linc01703 functions as a metastasis suppressor by facilitating the secretion of CD81⁺ exosomes through the formation of the Rab27a/SYTL1/CD81 complex.

Keywords: CD81; exosomes; long noncoding RNA; lung cancer; metastasis.

Figure 1

Linc01703 expression is decreased in metastatic lung cancer cells. (A) Indicated A549 cells (1 × 10⁶) were injected via ventricle, and representative bioluminescent images of metastasis are shown. (B) Relative fold change of Linc01703 in A549-PR and A549-HM cells. (C) The relative expression of U6 (nuclear control) and β-Actin (cytoplasmic control) and the expression of Linc01703 were analyzed by using qRT-PCR in the nuclear and cytoplasmic fractions. (D) The images of PCR products from the 5′-RACE and 3′-RACE procedures. (E,F) The sequence of the full length of Linc01703. Blue: C, Black: G, Red: T, Green: A. (G) The full length of Linc01703 was cloned into pLenti with an ATG-start codon and a C-terminal Flag peptide in three expression patterns. An anti-Flag antibody was used to probe transcribed proteins. The uncropped blots are shown in File S1. PTEN with a Flag tag served as a positive control. Results are presented as mean ± SD, ** p < 0.01.

Figure 2

Linc01703 inhibits lung cancer metastases in vivo. (A) Relative fold change of Linc01703 in Vector and Linc01703-overexpressing cells of A549 and H1975. (B) MTT assay of A549-Vector and A549-Linc01703 cells. (C) Representative graphs and quantification of indicated cells analyzed via Transwell assays. Scale bar: 100 μm. (D) Representative graphs and the statistical results of the wound healing assay in A549-Vector and A549-Linc01703 cells. Wound closures were photographed at 0 and 24 h after wounding. Scale bar: 100 μm. The percentage of wound closure was based on statistics in Image J. (E) Indicated A549 cells (1 × 10⁶) were injected via tail vein (E,F) or spleen (G,I), and representative bioluminescent images (BLI) (E,G), tumor picture (F upper panel; H upper panel), H&E staining (F lower panel; H lower panel, Scale bar: 100 μm), and liver weight (I) of metastasis are shown. (n = 5 per group). Results are presented as mean ± SD derived from three independent experiments, * p < 0.05, ** p < 0.01.

Figure 3

Linc01703 promotes the release of CD81⁺ exosomes. (A,B) Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the 199 upregulated genes in A549-Linc01703 compared with A549-Vector genes. (C) Gene set enrichment analysis (GSEA) of the correlation between Linc0170 and extracellular exosome genes using the TCGA lung cancer database. (D) Concentration of exosomes in A549-Vector and A549-Linc01703 cells. (E) The effect of Linc01703 overexpression on CD9, CD63, and CD81 in whole cell lysis (WCL) and exosome (EXO). The uncropped blots are shown in File S1. ** p < 0.01.

Figure 4

Linc01703 inhibits lung cancer metastasis and affects immune cell infiltration through CD81⁺ exosomes. (A) The expression of CD81 in the indicated cells. The uncropped blots are shown in File S1. (B,C) Indicated A549 cells (1 × 10⁶) were injected via tail vein, and representative bioluminescent images of metastasis are shown (n = 5 per group). (D) Percentage of CD11b⁺Gr-1⁺, B220⁺CD86⁺, CD3⁻CD49⁺, and B220⁺CD80⁺ cells in the tumor microenvironment after exosome treatment. Results are presented as mean ± SD derived from three independent experiments, * p < 0.05, ** p < 0.01, ns: no significance.

Figure 5

Linc01703 promotes the formation of Rab27a/SYTL1/CD81 transport complexes. (A) Gene set enrichment analysis (GSEA) of the correlation between Linc01703 and extracellular exosome genes using genes in A549-Linc01703 and A549-Vector cells. (B) Relative fold change of exosome release genes in A549-Linc01703 and A549-Vector cells. (C) RNA pull-down assay and Western blot assay validation of the binding of Rab27a, Rab27b, SYTL1, SYTL3, and CD81 with Linc01703. (D) The effect of Linc01703 on the binding of Rab27a with CD81 and CD81. (E) The effect of silencing Rab27a or SYTL1 on the expression of CD9, CD63, and CD81 in WCL and EXO. (F) Fluo-4 AM staining of indicated cells depicts intracellular calcium. Scare bar: 50 μm. (G) The effect of Nexinhib 20 (the inhibitor of Rab27a-SYTL1 binding), BAPTA-AM (Ca²⁺ chelator), or BAPTA (Ca²⁺ chelator) on the expression of CD9, CD63, and CD81 on the WCL and EXO of the indicated cells. The uncropped blots are shown in File S1. Results are presented as mean ± SD, * p < 0.05, ** p < 0.01.

Figure 6

CD81 is decreased in lung cancer tissues and correlates with a better prognosis for patients. (A) Analyses of the expression levels of CD81 in patients and control using the TCGA LUAD dataset (two-tailed paired Student’s t-test). (B) Analyses of the expression levels of CD81 in paired LUAD and adjacent non-tumor tissues (n = 59) using the TCGA dataset (two-tailed paired Student’s t-test). (C) A Western blot assay shows the expression of CD81 in paired LUAD and adjacent non-tumor tissues (n = 10). The uncropped blots are shown in File S1. (D) Representative images of IHC from LUAD patients show the protein expression level of CD81. Scale bar: 20 μm. (E) Kaplan–Meier analyses of overall survival (OS) based on the KMPLOT LUAD dataset on CD81 expression. Results are presented as mean ± SD, ** p < 0.01.