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The MALAT-1 noncoding RNA Regulates Cell Mobility by Modulating Gene Expressions

ABSTRACT: MALAT-1, originally identified as a metastasis associated long noncoding transcript in lung adenocarcinoma, is localized to nuclear speckles. However, functional role of MALAT-1 in metastatic characters in cancer cells has not been elucidated. Our current research has revealed that suppression of MALAT-1 expression in lung adenocarcinoma cell line by RNA interference significantly inhibited cellular mobility both in wound healing assay and in migration assay using Boyden's chamber. Moreover, we have identified metastasis/mobility-associated genes among down-regulated genes in the MALAT-1 knockdown cells by DNA microarray analysis. Quantitative analysis of pre-mRNAs has shown that the pre-mRNAs of some genes were suppressed in MALAT-1 knockdown cells but the others were not suppressed even though their matured mRNAs were found to be down-regulated. Our results suggest that MALAT-1 facilitates cell mobility by modulating the expression of metastasis/mobility-associated genes at the transcriptional and post-transcriptional stages.

ORGANISM(S): Homo sapiens

PROVIDER: GSE22085 | GEO | 2011/06/02

SECONDARY ACCESSION(S): PRJNA128933

REPOSITORIES: GEO

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Project description:Background Pancreatic ductal adenocarcinoma (PDAC) tends to undergo distant metastasis, especially liver metastasis, leading to a poor prognosis. Metabolic remodelling and epigenetic reprogramming are two important hallmarks of malignant tumours and participate in regulating PDAC tumorigenesis and metastasis. However, the interaction between these two processes during PDAC metastasis has not been fully elucidated. Methods We performed metabolomics analysis to identify the critical metabolites associated with PDAC liver metastasis and focused on guanidinoacetic acid (GAA). Intracellular GAA content was significantly increased in liver metastatic PDAC cells compared to primary cancer cells in mouse xenograft tumour models. The effects of GAA supplementation and glycine amidinotransferase (GATM) knockdown on PDAC metastasis were assessed by analysing cell migration, filopodia formation, epithelial-mesenchymal transition (EMT), and in vivo metastasis in different cell and animal models. Next, ChIP‒qPCR, 3C‒qPCR, and CRISPRi/dCas9-KRAB experiments were used to validate the “epigenome-metabolome" mechanism. Finally, the results of in vitro approaches, including RNA-seq, CUT&RUN, RT‒qPCR, and western blot analyses, as well as luciferase reporter gene assay and transwell assay, revealed the GAA-c-Myc-HMGA axis and transcription-activating histone modifications reprogramming. Results A high level of intracellular GAA was associated with PDAC liver metastasis. GAA could promote the migration, EMT, and liver metastasis of pancreatic cancer cells in vitro and in vivo. Next, we explored the role of GATM-mediated de novo GAA synthesis in pancreatic cancer metastasis. High expression of GATM was positively correlated with advanced N stage in PDAC. Knockdown of GATM significantly reduced the intracellular level of GAA, suppressed EMT, and inhibited PDAC liver metastasis, and these effects were attenuated by GAA supplementation. Mechanistically, we identified the active enhancers looped to the GATM gene locus that promoted GATM expression and PDAC liver metastasis. Furthermore, we found that GAA promoted cell migration and EMT by regulating c-Myc-mediated high mobility group AT-hook protein expression. Moreover, GAA increased the H3K4me3 modification level by upregulating histone methyltransferases, which induced the transcription of metastasis-related genes, including MYC. Conclusions These findings revealed the critical role of the epigenome-metabolome interaction in regulating PDAC liver metastasis and suggested potential therapeutic strategies targeting GAA metabolism and epigenetic regulatory mechanisms.

Project description:Purpose: Due to its high metastatic proclivity, pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly types of cancer. Therefore, it is imperative to better understand how the disease spreads as it progresses. Using a novel genetically engineered mouse model that allows us to isolate a subpopulation of cancer cells with superior metastatic capacity, we show that this aggressive phenotype correlates exclusively with a strong hypoxia signature. We subsequently identified the novel hypoxia-inducible gene Blimp1, which appears to play a critical role in regulating the hypoxic response upon its induction. Furthermore, genetic ablation of Blimp1 greatly reduces the level of metastasis in a PDAC mouse model. The nature of this Blimp1-regulated hypoxia signature is very unstable, since the seeded metastatic lesions mostly re-adopt similar transcriptomic profiles as the primary tumors. In conclusion, our results offer a potential mechanistic insight into how hypoxia drives metastasis in PDAC. Methods: The liver metastasis cell line 688M was subjected to control or knockdown of Blimp1 before assay. Cells ere validated for knockdown efficiency and then cultured under normoxia and hypoxia (0.5% O2) for 24 hours before preparation for RNA-Seq (total of 4 groups with duplicates, overall 8 samples). Results: For the control knockdown group, 0.5% O2 culture(hypoxia) for 24 hours induced dramatic changes in global gene expression, and Blimp1 knockdown potently mitigated changes of significant amount of genes induced under hypoxia. In brief, cell cycle-related genes that are normally suppressed by hypoxia (which contributes to hypoxia-induced cell cycle arrest) are not suppressed under hypoxia in the Blimp1 knockdown cells. In contrast, ~35% of genes that are induced under hypoxia in the control knockdown cells are instead induced less than 50% (under hypoxia) upon Blimp1 knockdown. Conclusions: Blimp1 is a critical regulator of hypoxic response in pancreatic cancer.

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The MALAT-1 noncoding RNA Regulates Cell Mobility by Modulating Gene Expressions

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