Long noncoding RNA BLACAT2 promotes bladder cancer–associated lymphangiogenesis and lymphatic metastasis
Wang He, Guangzheng Zhong, Ning Jiang, Bo Wang, Xinxiang Fan, Changhao Chen, Xu Chen, Jian Huang, Tianxin Lin
Wang He, Guangzheng Zhong, Ning Jiang, Bo Wang, Xinxiang Fan, Changhao Chen, Xu Chen, Jian Huang, Tianxin Lin
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Research Article Oncology

Long noncoding RNA BLACAT2 promotes bladder cancer–associated lymphangiogenesis and lymphatic metastasis

Abstract

The prognosis for bladder cancer patients with lymph node (LN) metastasis is dismal and only minimally improved by current treatment modalities. Elucidation of the molecular mechanisms that underlie LN metastasis may provide clinical therapeutic strategies for LN-metastatic bladder cancer. Here, we report that a long noncoding RNA LINC00958, which we have termed bladder cancer–associated transcript 2 (BLACAT2), was markedly upregulated in LN-metastatic bladder cancer and correlated with LN metastasis. Overexpression of BLACAT2 promoted bladder cancer–associated lymphangiogenesis and lymphatic metastasis in both cultured bladder cancer cell lines and mouse models. Furthermore, we demonstrate that BLACAT2 epigenetically upregulated VEGF-C expression by directly associating with WDR5, a core subunit of human H3K4 methyltransferase complexes. Importantly, administration of an anti–VEGF-C antibody inhibited LN metastasis in BLACAT2-overexpressing bladder cancer. Taken together, these findings uncover a molecular mechanism in the lymphatic metastasis of bladder cancer and indicate that BLACAT2 may represent a target for clinical intervention in LN-metastatic bladder cancer.

Authors

Wang He, Guangzheng Zhong, Ning Jiang, Bo Wang, Xinxiang Fan, Changhao Chen, Xu Chen, Jian Huang, Tianxin Lin

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Figure 7

BLACAT2 directly binds to WDR5 protein and regulates VEGF-C expression.

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BLACAT2 directly binds to WDR5 protein and regulates VEGF-C expression. ...
(A) Representative image of silver-stained PAGE gels showing separated proteins that were pulled down using biotin-labeled BLACAT2. In vitro–transcribed antisense sequence of BLACAT2 was used as the nonspecific control. (B) Western blot analysis indicating that BLACAT2 associates with WDR5, as indicated by the pull-down assay with nuclear extracts or in vitro–synthesized WDR5. Antisense BLACAT2 was used as the negative control RNA in the pull-down assay. (C) RT-qPCR analysis of RNA enrichment in the RIP assay using the anti-WDR5 antibody in UM-UC-3 and 5637 bladder cancer cells. Normal IgG was used as the nonspecific control antibody. U1 and HOTTIP were used as negative and positive controls, respectively, for WDR5 binding. (D and E) Serial deletions of BLACAT2 were used in RNA pull-down assays to identify core regions of BLACAT2 that were required for physical interaction with WDR5. (F–I) Site-directed mutagenesis of 100–130 nt of BLACAT2 was performed, and the effects of BLACAT2 mutant overexpression on VEGF-C mRNA expression (F), VEGF-C secretion (G), HLEC migration (H), and HLEC tube formation (I) were evaluated. All experiments were performed with at least 3 biological replicates. Statistical significance was assessed using 1-way ANOVA followed by Dunnett’s tests for multiple comparisons (C, F–I). **P < 0.01.