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 8

Exosomal LNMAT2 forms a DNA-RNA triplex with the PROX1 promoter.

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Exosomal LNMAT2 forms a DNA-RNA triplex with the PROX1 promoter. qRT-PCR...
qRT-PCR (A) and Western blot (B) of PROX1 expression in HLECs treated with PBS, 5637-EXOVector, or 5637-EXOLNMAT2. GAPDH was used as an internal control in qRT-PCR. Statistical significance was assessed using 1-way ANOVA followed by Dunnett’s tests. (C) Sequential deletions for evaluating the transcriptional activity of the PROX1 promoter in HLECs treated with 5637-EXOVector or 5637-EXOLNMAT2. Statistical significance was assessed using 1-way ANOVA followed by Dunnett’s tests. (D) Schematic presentation of the predicted LNMAT2 binding sites in the PROX1 promoter. (E) ChIRP of LNMAT2-associated chromatin in HLECs treated with 5637-EXO. Statistical significance was assessed using 2-tailed Student’s t test. (F) ChIRP of LNMAT2-associated chromatin in LNMAT2-WT or LNMAT2-KO HLECs treated with 5637-EXO. Statistical significance was assessed using 2-tailed Student’s t test. (G) FRET of a 1:5 mixture (red) of TFO (black) in LNMAT2 with TTS (blue) in the PROX1 promoter. (H) CD spectrum of a 1:1 mixture of TFO in LNMAT2 with TTS in the PROX1 promoter (red). The sum of the TFO and TTS is shown in blue. Evaluation of WT or LNMAT2 binding site mutated PROX1 promoter in LNMAT2-WT (I) or LNMAT2-KO (J) HLECs, respectively, treated with 5637-EXOVector or 5637-EXOLNMAT2. Statistical significance was assessed using 1-way ANOVA followed by Dunnett’s tests. ChIP-qPCR of hnRNPA2B1 occupancy (K and M) and H3K4me3 (L and N) status in the PROX1 promoter after HLEC incubation with indicated exosomes. Statistical significance was assessed using 2-tailed Student’s t test and 1-way ANOVA followed by Dunnett’s tests for multiple comparisons. Error bars represent the SD of 3 independent experiments. *P < 0.05; **P < 0.01.