Long noncoding RNA

© Biotarget. All rights reserved. Biotarget 2019;3:5 biotarget.amegroups.com Since Malat1 was identified as a prognostic marker for poor clinical outcomes in non-small cell lung cancer (NSCLC) patients, number of Malat1 studies have revealed its roles in human cancers (1,2). Recently, we demonstrated unexpected Malat1 function in suppressing breast cancer metastasis using genetic mouse and cell line models of breast cancer (3). Here, I review the controversies in Malat1 studies conducted by ourselves and others by comparing research designs and interpreting the results. Previously, Malat1 was shown to co-localize with nuclear speckles enriched in pre-mRNA splicing factors. In vitro siRNA study revealed that Malat1 interacts with splicing factors in the nucleus and regulates pre-mRNA splicing (4). Malat1 genetic knockout mice were generated from independent research groups and exhibited no apparent phenotypic abnormalities (5,6). Surprisingly, unlike in vitro results, there were no significant changes in alternative splicing or global gene expression due to genetic loss of Malat1 (3,6). Arun et al. (7) generated Malat1 knockout mice by deleting 3 kb genomic locus spanning both upstream and downstream of transcription start site and crossed them to Polyomavirus middle T antigen (PyMT)-induced mouse mammary tumor model. Although Malat1 loss did not substantially alter primary tumorigenesis, mammary tumors of Malat1 PyMT mice exhibited cystic phenotype compared to Malat1 PyMT animals. Furthermore, lung metastasis was significantly suppressed by Malat1 loss. In addition to genetic ablation of Malat1, PyMT mice were treated with antisense oligonucleotides (ASOs). Compared to complete genetic Malat1 knockout, ASOs led to ~60% reduction in Malat1 levels but caused stronger suppression in mammary tumor growth and comparable inhibition in lung metastasis. Targeting Malat1 with less-effective ASOs demonstrated more evident tumor-suppressive outcome, which obviously raises a question of off-target effects of ASOs. We utilized Malat1 mouse knockout model generated by targeted insertional inactivation (5) and crossed them to PyMT mice (3). Unexpectedly, however, we observed dramatic induction of lung metastasis in Malat1 PyMT mice while there was no noticeable difference in mammary tumors compared to Malat1 PyMT mice; cystic and highgrade tumor areas were similarly found in both groups. Instead, we could see consistent increase in circulating tumor cells from the peripheral blood of Malat1 PyMT mice, which implies that Malat1 loss affects certain stage of metastatic process. This surprising result against the current dominant paradigm of Malat1’s pro-metastatic function prompted us to restore Malat1 in Malat1 PyMT mice using transgenic animals to determine if metastasispromoting function is attributed to Malat1 loss. When Malat1 was re-expressed, metastatic phenotype was dramatically reversed. Furthermore, considering higher Malat1 levels in Malat1-restored PyMT mice compared to Malat1 PyMT mice, Malat1-re-expression resulted in less metastasis than Malat1, which implies dose-dependent effect of Malat1 in lung metastasis suppression. Using more aggressive PyMT mice on FVB/N background, we overexpressed Malat1 by crossing them to Malat1 transgenic mice on FVB/N and observed that lung metastasis was significantly suppressed by transgenic overexpression of Malat1. In addition, we inactivated MALAT1 in MDA-MB-231 Letter to the Editor