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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Research Article Oncology

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 7

LNMAT2 is packaged into exosomes in an hnRNPA2B1-dependent manner and transported to HLECs.

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LNMAT2 is packaged into exosomes in an hnRNPA2B1-dependent manner and t...
(A) qRT-PCR analysis of LNMAT2 expression in exosomes secreted by hnRNPA2B1 knockdown cells. Statistical significance was assessed using 1-way ANOVA followed by Dunnett’s tests. (B) qRT-PCR analysis of LNMAT2 expression in BCa cell–secreted exosomes. Statistical significance was assessed using 1-way ANOVA followed by Dunnett’s tests. (C) The exosome/cell ratio of RNAs in 5637 cells obtained by qRT-PCR. Statistical significance was assessed using 1-way ANOVA followed by Dunnett’s tests. (D) qRT-PCR analysis of RNA levels in exosomes secreted by hnRNPA2B1 knockdown 5637 cells. Statistical significance was assessed using 1-way ANOVA followed by Dunnett’s tests. (E) Schematic illustration of exosome internalization assays and representative images of HLEC fluorescence after incubation with PKH67-labeled (green) BCa cell exosomes. Scale bar: 5 μm. qRT-PCR analysis of LNMAT2 expression in HLECs treated with PBS, 5637-EXO, UM-UC-3-EXO (F) or 5637-EXOsi-NC, 5637-EXOsi-LNMAT2#1, or 5637-EXOsi-LNMAT2#2 (G). Statistical significance was assessed using 1-way ANOVA followed by Dunnett’s tests for multiple comparisons. (H) qRT-PCR confirming the LNMAT2 knockout. Statistical significance was assessed using 2-tailed Student’s t test. Representative images (I) and quantification of tube formation (J) and Transwell migration (K) by HLECs (LNMAT2-KO or LNMAT2-WT) after treating with 5637-EXOsi-NC or 5637-EXOsi-LNMAT2#1. Scale bars: 100 μm. Statistical significance was assessed using 2-tailed Student’s t test. GAPDH was used as an internal control for qRT-PCR analysis in A–H. Error bars represent the SD of 3 independent experiments. *P < 0.05; **P < 0.01.