The Long Non-Coding RNA MALAT1 Modulates NR4A1 Expression through a Downstream Regulatory Element in Specific Cancer Cell Types

  1. Sara Wernig-Zorc
  2. Uwe Schwartz
  3. Paulina Martínez-Rodríguez
  4. Josefa Inalef
  5. Francisca Pavicic
  6. Pamela Ehrenfeld
  7. Gernot Längst
  8. Rodrigo Maldonado

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Abstract

Long non-coding RNAs (lncRNAs) have been shown to modulate gene expression and are involved in the initiation and progression of various cancer types. Despite the wealth of studies describing transcriptome changes upon lncRNA knockdown, there is limited information describing lncRNA-mediated effects on regulatory elements (REs) modulating gene expression. In this study, we investigated how the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) lncRNA regulates primary target genes using time-resolved MALAT1 knockdown followed by parallel RNA-seq and ATAC-seq assays. The results revealed that MALAT1 primarily regulates specific protein-coding genes and a substantial decrease in the accessibility downstream of the NR4A1 gene that was associated with a decreased NR4A1 expression. Moreover, the presence of an NR4A1-downstream RE was demonstrated by CRISPR-i assays to define a functional MALAT1/NR4A1 axis. By analyzing TCGA data, we identified a positive correlation between NR4A1 expression and NR4A1-downstream RE accessibility in breast cancer but not in pancreatic cancer. Accordingly, this regulatory mechanism was experimentally validated in breast cancer cells (MCF7) but not in pancreatic duct epithelial carcinoma (PANC1) cells. Therefore, our results demonstrated that MALAT1 is involved in a molecular mechanism that fine-tunes NR4A1 expression by modulating the accessibility of a downstream RE in a cell type-specific manner.

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  1. Version published to 10.3390/ijms25105515
  2. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/8313271.

    The article is based on the interaction amongst MALAT1 (metastasis-associated lung adenocarcinoma tran- script 1), a long non-coding RNA (lncRNA), and NR4A1, a transcription factor associated with T-cells dysfunction and some fibrotic diseases. RNA-seq of HeLa cells with MALAT1 modulated by Locked Nucleic Acid (LNA) Gapmers have been used to identify targets of this lncRNA. Then ATAC-seq data have shown that these targets had some changes in chromatin accessibility of regulatory elements (RE). This study focused on downstream modulation of some cancer modulators, specifically NR4A1, by MALAT1.

    This work has created a story line that explain to the reader why long non-coding RNAs (lncRNAs) such as MALAT1 may have a major role in some cancer type, like breast cancer and pancreatic carcinoma. The experimental approach was suitable, but I would suggest conducting additional experiments that could yield more refined results and address emerging questions. In my view, the reason for comparing breast and pancreatic cancer cells was not entirely evident. Therefore, providing additional clarification on this matter could enhance the understanding of the readers. I would like to suggest a review of the assertion that MALAT1 does not play a role in regulating PDAC. While there may be some conflicting viewpoints in the literature, the majority of research papers indicate that MALAT1 expression is associated with a poorer prognosis in PDAC patients (Liu et al., 2014; Liu et al., 2017).

    In the course of your research, it has primarily used the PANC-1 cell line as a model for studying pan- creatic ductal adenocarcinoma (PDAC). While the PANC-1 cell line has been widely employed in previous lncRNA studies, I would suggest considering the inclusion of other cell lines, such as AsPC-1, BxPC-3, and MIA PaCa-2. These alternative cell lines exhibit unique features that could provide another approach on PDAC.

    Considering your main objective of investigating the early effects of lncRNA MALAT1 silencing, I would recommend extending the ATAC sample collection to at least 72 hours. It is important to note that lncRNAs are generally present in relatively lower quantities within cells, which takes a longer time to observe signifi- cantly changes in protein levels. By doing so, you could identify some other proteins that could be regulated by MALAT1 and grasping the indirect effects of silencing this lncRNA.

    To explore the role of MALAT1, you might consider employing the CRISPR-Cas13d system as an alternative to CRISPR-dCas9. Cas13d is designed to target and cleave mRNAs without altering DNA, effectively reducing RNA levels. Additionally, a lentiviral system could prove beneficial, as it enables the creation of a stable cell line with a inducible knockdown of MALAT1. On the other hand, dCas9 could be of interest if you plan to conduct HiC to confirm DNA interactions between the MALAT1 locus and other DNA loci.

    In my opinion, your article has explored a novel role of MALAT1 on breast and pancreatic cancers. I made some considerations in order to improve this amazing work your group have developed. I would like to congratulate again for this great and novel work!

    Competing interests

    The author declares that they have no competing interests.

  3. Version published to 10.1101/2023.03.09.531856v1 on bioRxiv