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lncRNA NBR2 engages a metabolic checkpoint by regulating AMPK under energy stress

View Article: PubMed Central - PubMed

ABSTRACT

Long noncoding RNAs (lncRNAs) have emerged as critical regulators in various cellular processes. However, the potential involvement of lncRNAs in kinase signaling remains largely unknown. AMP-activated protein kinase (AMPK) acts as a critical sensor of cellular energy status. Here we show that lncRNA NBR2 (neighbor of BRCA1 gene 2) is induced by the LKB1-AMPK pathway under energy stress. Upon energy stress, NBR2 in turn interacts with AMPK and promotes AMPK kinase activity, thus forming a feed-forward loop to potentiate AMPK activation during energy stress. Depletion of NBR2 attenuates energy stress-induced AMPK activation, resulting in unchecked cell cycling, altered apoptosis/autophagy response, and increased tumor development in vivo. NBR2 is down-regulated and its low expression correlates with poor clinical outcomes in some human cancers. Together, our study uncovers a mechanism coupling lncRNAs with metabolic stress response, and provides a broad framework to further understand the regulation of kinase signaling by lncRNAs.

No MeSH data available.


Energy stress induces NBR2 expression through the LKB1-AMPK pathway(a, b) Various cell lines were cultured in 0 or 25 mM glucose-containing medium (a), or 0 or 5 mM 2DG-containing medium (b) for 12–24 hours, and then subjected to real-time PCR analysis to measure NBR2 expression (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (c, d) Hela or A549 cells stably expressing EV (empty vector) or Lkb1 expression vectors were cultured in 25 or 0 mM glucose-containing medium, and then subjected to real-time PCR (c) (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test) and Western blotting analyses (d). (e) MDA-MB-231 cells treated with 100 µM A769662 were subjected to real-time PCR analysis to measure NBR2 (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (f) MDA-MB-231 cells were treated with 20 µM Compound C in 25 or 0 mM glucose-containing medium for 24 hours, and then subjected to real-time PCR analysis to measure NBR2 expression (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (g) MDA-MB-231 cells transfected with AMPKα or control (Ctrl) siRNA were cultured in 25 or 0 mM glucose-containing medium for 24 hours, and then subjected to real-time PCR analysis to measure NBR2 (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). Source data for a, b, c, e, f, g can be found in Supplementary Table 1. Unprocessed original scans of blots are shown in Supplemental Fig. 8.
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Figure 1: Energy stress induces NBR2 expression through the LKB1-AMPK pathway(a, b) Various cell lines were cultured in 0 or 25 mM glucose-containing medium (a), or 0 or 5 mM 2DG-containing medium (b) for 12–24 hours, and then subjected to real-time PCR analysis to measure NBR2 expression (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (c, d) Hela or A549 cells stably expressing EV (empty vector) or Lkb1 expression vectors were cultured in 25 or 0 mM glucose-containing medium, and then subjected to real-time PCR (c) (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test) and Western blotting analyses (d). (e) MDA-MB-231 cells treated with 100 µM A769662 were subjected to real-time PCR analysis to measure NBR2 (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (f) MDA-MB-231 cells were treated with 20 µM Compound C in 25 or 0 mM glucose-containing medium for 24 hours, and then subjected to real-time PCR analysis to measure NBR2 expression (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (g) MDA-MB-231 cells transfected with AMPKα or control (Ctrl) siRNA were cultured in 25 or 0 mM glucose-containing medium for 24 hours, and then subjected to real-time PCR analysis to measure NBR2 (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). Source data for a, b, c, e, f, g can be found in Supplementary Table 1. Unprocessed original scans of blots are shown in Supplemental Fig. 8.

Mentions: Real-time PCR revealed that glucose starvation induced NBR2 expression in different cancer cell lines, except Hela and A549 cells, which are Lkb1 deficient (Fig. 1a). Treatment with the glucose analog 2-deoxy-glucose (2DG), another energy stress inducer that inhibits hexokinase and blocks glycolysis, yielded similar results (Fig. 1b). Importantly, re-expression of Lkb1 in these Lkb1-deficient cells restored energy stress-induced NBR2 expression (Fig. 1c, d). In addition, treatment of A769662 (an AMPK activator) induced NBR2 expression (Fig. 1e), while AMPK inactivation by compound C (an AMPK inhibitor) treatment or siRNA-mediated AMPKα knockdown significantly attenuated glucose starvation-induced NBR2 expression (Fig. 1f, g, and Supplemental Fig. 2). Together, our results revealed that energy stress induces NBR2 expression at least partly through the LKB1-AMPK pathway.


lncRNA NBR2 engages a metabolic checkpoint by regulating AMPK under energy stress
Energy stress induces NBR2 expression through the LKB1-AMPK pathway(a, b) Various cell lines were cultured in 0 or 25 mM glucose-containing medium (a), or 0 or 5 mM 2DG-containing medium (b) for 12–24 hours, and then subjected to real-time PCR analysis to measure NBR2 expression (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (c, d) Hela or A549 cells stably expressing EV (empty vector) or Lkb1 expression vectors were cultured in 25 or 0 mM glucose-containing medium, and then subjected to real-time PCR (c) (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test) and Western blotting analyses (d). (e) MDA-MB-231 cells treated with 100 µM A769662 were subjected to real-time PCR analysis to measure NBR2 (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (f) MDA-MB-231 cells were treated with 20 µM Compound C in 25 or 0 mM glucose-containing medium for 24 hours, and then subjected to real-time PCR analysis to measure NBR2 expression (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (g) MDA-MB-231 cells transfected with AMPKα or control (Ctrl) siRNA were cultured in 25 or 0 mM glucose-containing medium for 24 hours, and then subjected to real-time PCR analysis to measure NBR2 (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). Source data for a, b, c, e, f, g can be found in Supplementary Table 1. Unprocessed original scans of blots are shown in Supplemental Fig. 8.
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Figure 1: Energy stress induces NBR2 expression through the LKB1-AMPK pathway(a, b) Various cell lines were cultured in 0 or 25 mM glucose-containing medium (a), or 0 or 5 mM 2DG-containing medium (b) for 12–24 hours, and then subjected to real-time PCR analysis to measure NBR2 expression (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (c, d) Hela or A549 cells stably expressing EV (empty vector) or Lkb1 expression vectors were cultured in 25 or 0 mM glucose-containing medium, and then subjected to real-time PCR (c) (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test) and Western blotting analyses (d). (e) MDA-MB-231 cells treated with 100 µM A769662 were subjected to real-time PCR analysis to measure NBR2 (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (f) MDA-MB-231 cells were treated with 20 µM Compound C in 25 or 0 mM glucose-containing medium for 24 hours, and then subjected to real-time PCR analysis to measure NBR2 expression (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (g) MDA-MB-231 cells transfected with AMPKα or control (Ctrl) siRNA were cultured in 25 or 0 mM glucose-containing medium for 24 hours, and then subjected to real-time PCR analysis to measure NBR2 (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). Source data for a, b, c, e, f, g can be found in Supplementary Table 1. Unprocessed original scans of blots are shown in Supplemental Fig. 8.
Mentions: Real-time PCR revealed that glucose starvation induced NBR2 expression in different cancer cell lines, except Hela and A549 cells, which are Lkb1 deficient (Fig. 1a). Treatment with the glucose analog 2-deoxy-glucose (2DG), another energy stress inducer that inhibits hexokinase and blocks glycolysis, yielded similar results (Fig. 1b). Importantly, re-expression of Lkb1 in these Lkb1-deficient cells restored energy stress-induced NBR2 expression (Fig. 1c, d). In addition, treatment of A769662 (an AMPK activator) induced NBR2 expression (Fig. 1e), while AMPK inactivation by compound C (an AMPK inhibitor) treatment or siRNA-mediated AMPKα knockdown significantly attenuated glucose starvation-induced NBR2 expression (Fig. 1f, g, and Supplemental Fig. 2). Together, our results revealed that energy stress induces NBR2 expression at least partly through the LKB1-AMPK pathway.

View Article: PubMed Central - PubMed

ABSTRACT

Long noncoding RNAs (lncRNAs) have emerged as critical regulators in various cellular processes. However, the potential involvement of lncRNAs in kinase signaling remains largely unknown. AMP-activated protein kinase (AMPK) acts as a critical sensor of cellular energy status. Here we show that lncRNA NBR2 (neighbor of BRCA1 gene 2) is induced by the LKB1-AMPK pathway under energy stress. Upon energy stress, NBR2 in turn interacts with AMPK and promotes AMPK kinase activity, thus forming a feed-forward loop to potentiate AMPK activation during energy stress. Depletion of NBR2 attenuates energy stress-induced AMPK activation, resulting in unchecked cell cycling, altered apoptosis/autophagy response, and increased tumor development in vivo. NBR2 is down-regulated and its low expression correlates with poor clinical outcomes in some human cancers. Together, our study uncovers a mechanism coupling lncRNAs with metabolic stress response, and provides a broad framework to further understand the regulation of kinase signaling by lncRNAs.

No MeSH data available.