Exosomal long noncoding RNA LNMAT2 promotes lymphatic metastasis in bladder cancer
Changhao Chen, … , Rufu Chen, Tianxin Lin
Changhao Chen, … , Rufu Chen, Tianxin Lin
Published October 8, 2019
Citation Information: J Clin Invest. 2020;130(1):404-421. https://doi.org/10.1172/JCI130892.
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Exosomal long noncoding RNA LNMAT2 promotes lymphatic metastasis in bladder cancer

Abstract

Patients with bladder cancer (BCa) with clinical lymph node (LN) metastasis have an extremely poor prognosis. VEGF-C has been demonstrated to play vital roles in LN metastasis in BCa. However, approximately 20% of BCa with LN metastasis exhibits low VEGF-C expression, suggesting a VEGF-C–independent mechanism for LN metastasis of BCa. Herein, we demonstrate that BCa cell–secreted exosome-mediated lymphangiogenesis promoted LN metastasis in BCa in a VEGF-C–independent manner. We identified an exosomal long noncoding RNA (lncRNA), termed lymph node metastasis-associated transcript 2 (LNMAT2), that stimulated human lymphatic endothelial cell (HLEC) tube formation and migration in vitro and enhanced tumor lymphangiogenesis and LN metastasis in vivo. Mechanistically, LNMAT2 was loaded to BCa cell–secreted exosomes by directly interacting with heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1). Subsequently, exosomal LNMAT2 was internalized by HLECs and epigenetically upregulated prospero homeobox 1 (PROX1) expression by recruitment of hnRNPA2B1 and increasing the H3K4 trimethylation level in the PROX1 promoter, ultimately resulting in lymphangiogenesis and lymphatic metastasis. Therefore, our findings highlight a VEGF-C–independent mechanism of exosomal lncRNA-mediated LN metastasis and identify LNMAT2 as a therapeutic target for LN metastasis in BCa.

Authors

Changhao Chen, Yuming Luo, Wang He, Yue Zhao, Yao Kong, Hongwei Liu, Guangzheng Zhong, Yuting Li, Jun Li, Jian Huang, Rufu Chen, Tianxin Lin

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Figure 6

Direct interaction of LNMAT2 with hnRNPA2B1.

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Direct interaction of LNMAT2 with hnRNPA2B1. (A) RNA pull-down assay usi...
(A) RNA pull-down assay using LNMAT2 sense and antisense RNAs in 5637 cells, followed by silver staining. Red arrows indicate hnRNPA2B1. (B) MS identification of LNMAT2-binding proteins. RNA pull-down and Western blot with 5637 cell nuclear extract (C) or purified recombinant hnRNPA2B1 (D) confirmed that LNMAT2 was associated with hnRNPA2B1. (E) Fluorescence assessment of LNMAT2 and hnRNPA2B1 colocalization in 5637 cells. Scale bar: 5 μm. (F) RIP analysis using the anti-hnRNPA2B1 antibody revealing that LNMAT2 interacted with hnRNPA2B1 in 5637 cells. Negative control, IgG; nonspecific control, U1. Statistical significance was assessed using 2-tailed Student’s t test. (G and H) Serial deletions of LNMAT2 were used in RNA pull-down assays to identify regions required for LNMAT2 and hnRNPA2B1 interaction. (I) POSTAR2 prediction of sequence motifs of hnRNPA2B1 binding sites. (J) RNAalifold predicted that LNMAT2 would have 4 stable stem-loop structures. The inset (framed in red) indicates the hnRNPA2B1 binding stem-loop structures in LNMAT2. (K) RIP assays performed after site-directed mutagenesis of 1930–1960 nt of LNMAT2 in 5637 cells. Statistical significance was assessed using 2-tailed Student’s t test. Error bars represent the SD of 3 independent experiments. *P < 0.05; **P < 0.01.