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Hypoxia-mediated downregulation of miRNA biogenesis promotes tumour progression.

Rupaimoole R, Wu SY, Pradeep S, Ivan C, Pecot CV, Gharpure KM, Nagaraja AS, Armaiz-Pena GN, McGuire M, Zand B, Dalton HJ, Filant J, Miller JB, Lu C, Sadaoui NC, Mangala LS, Taylor M, van den Beucken T, Koch E, Rodriguez-Aguayo C, Huang L, Bar-Eli M, Wouters BG, Radovich M, Ivan M, Calin GA, Zhang W, Lopez-Berestein G, Sood AK - Nat Commun (2014)

Bottom Line: Here we report a previously unrecognized effect of hypoxia in the downregulation of Drosha and Dicer in cancer cells that leads to dysregulation of miRNA biogenesis and increased tumour progression.Rescue of Drosha by siRNAs targeting ETS1/ELK1 in vivo results in significant tumour regression.These findings provide a new link in the mechanistic understanding of global miRNA downregulation in the tumour microenvironment.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Gynecologic Oncology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA [2] Graduate School of Biomedical Sciences, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA.

ABSTRACT
Cancer-related deregulation of miRNA biogenesis has been suggested, but the underlying mechanisms remain elusive. Here we report a previously unrecognized effect of hypoxia in the downregulation of Drosha and Dicer in cancer cells that leads to dysregulation of miRNA biogenesis and increased tumour progression. We show that hypoxia-mediated downregulation of Drosha is dependent on ETS1/ELK1 transcription factors. Moreover, mature miRNA array and deep sequencing studies reveal altered miRNA maturation in cells under hypoxic conditions. At a functional level, this phenomenon results in increased cancer progression in vitro and in vivo, and data from patient samples are suggestive of miRNA biogenesis downregulation in hypoxic tumours. Rescue of Drosha by siRNAs targeting ETS1/ELK1 in vivo results in significant tumour regression. These findings provide a new link in the mechanistic understanding of global miRNA downregulation in the tumour microenvironment.

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Hypoxia mediated downregulation in Drosha and Dicer results in decreased miRNA levels(a), Heat map showing mature microRNA (miRNA) levels under hypoxic conditions, assessed using miRNA array data. (b) Heat map showing precursor miRNA levels under hypoxic conditions, along with the corresponding mature sense (mature) and antisense (mature*) levels. (c–d)Pri-miRNA and mature miRNA expression levels of significantly altered miRNAs under hypoxia exposure in A2780 cells. (e)Pri-miRNA levels in RNA extracted from nuclear fractionated A2780 cells treated with normoxia and hypoxia. (f–g) Precursor and mature miRNA levels in RNA extracted from cytoplasmic fraction of A2780 cells treated with normoxia and hypoxia. Data are presented as mean ± standard error of the mean of n ≥ 3 experimental groups. *p < 0.05, **p < 0.01, ***p < 0.001 (Student t test).
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Figure 2: Hypoxia mediated downregulation in Drosha and Dicer results in decreased miRNA levels(a), Heat map showing mature microRNA (miRNA) levels under hypoxic conditions, assessed using miRNA array data. (b) Heat map showing precursor miRNA levels under hypoxic conditions, along with the corresponding mature sense (mature) and antisense (mature*) levels. (c–d)Pri-miRNA and mature miRNA expression levels of significantly altered miRNAs under hypoxia exposure in A2780 cells. (e)Pri-miRNA levels in RNA extracted from nuclear fractionated A2780 cells treated with normoxia and hypoxia. (f–g) Precursor and mature miRNA levels in RNA extracted from cytoplasmic fraction of A2780 cells treated with normoxia and hypoxia. Data are presented as mean ± standard error of the mean of n ≥ 3 experimental groups. *p < 0.05, **p < 0.01, ***p < 0.001 (Student t test).

Mentions: We next considered whether Drosha or Dicer downregulation leads to impaired miRNA biogenesis by carrying out miRNA microarray analysis of samples exposed to hypoxia or normoxia. Upon analysis of mature miRNA array data, we observed significant global miRNA downregulation following exposure to hypoxia (Fig. 2a; Supplementary data 1). Analysis of precursor and mature miRNA levels from deep sequencing data revealed significant downregulation of mature miRNAs, compared to their precursors(Fig. 2b; Supplementary data 2). Using qRT-PCR, we analyzed expression of seven significantly downregulated miRNAs from Fig. 2a, in cells exposed to normoxia or hypoxia. There was a significant increase in pri-miRNA levels of six of seven miRNAs that were tested (Fig. 2c). There was also significant downregulation of corresponding mature miRNA levels in response to hypoxia (Fig. 2d), suggesting defective processing machinery under hypoxia conditions. These data were further validated by northern blot analysis of miR-16a and 27a in cancer cells exposed to normoxia and hypoxia (Supplementary Fig. 4a).


Hypoxia-mediated downregulation of miRNA biogenesis promotes tumour progression.

Rupaimoole R, Wu SY, Pradeep S, Ivan C, Pecot CV, Gharpure KM, Nagaraja AS, Armaiz-Pena GN, McGuire M, Zand B, Dalton HJ, Filant J, Miller JB, Lu C, Sadaoui NC, Mangala LS, Taylor M, van den Beucken T, Koch E, Rodriguez-Aguayo C, Huang L, Bar-Eli M, Wouters BG, Radovich M, Ivan M, Calin GA, Zhang W, Lopez-Berestein G, Sood AK - Nat Commun (2014)

Hypoxia mediated downregulation in Drosha and Dicer results in decreased miRNA levels(a), Heat map showing mature microRNA (miRNA) levels under hypoxic conditions, assessed using miRNA array data. (b) Heat map showing precursor miRNA levels under hypoxic conditions, along with the corresponding mature sense (mature) and antisense (mature*) levels. (c–d)Pri-miRNA and mature miRNA expression levels of significantly altered miRNAs under hypoxia exposure in A2780 cells. (e)Pri-miRNA levels in RNA extracted from nuclear fractionated A2780 cells treated with normoxia and hypoxia. (f–g) Precursor and mature miRNA levels in RNA extracted from cytoplasmic fraction of A2780 cells treated with normoxia and hypoxia. Data are presented as mean ± standard error of the mean of n ≥ 3 experimental groups. *p < 0.05, **p < 0.01, ***p < 0.001 (Student t test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4215647&req=5

Figure 2: Hypoxia mediated downregulation in Drosha and Dicer results in decreased miRNA levels(a), Heat map showing mature microRNA (miRNA) levels under hypoxic conditions, assessed using miRNA array data. (b) Heat map showing precursor miRNA levels under hypoxic conditions, along with the corresponding mature sense (mature) and antisense (mature*) levels. (c–d)Pri-miRNA and mature miRNA expression levels of significantly altered miRNAs under hypoxia exposure in A2780 cells. (e)Pri-miRNA levels in RNA extracted from nuclear fractionated A2780 cells treated with normoxia and hypoxia. (f–g) Precursor and mature miRNA levels in RNA extracted from cytoplasmic fraction of A2780 cells treated with normoxia and hypoxia. Data are presented as mean ± standard error of the mean of n ≥ 3 experimental groups. *p < 0.05, **p < 0.01, ***p < 0.001 (Student t test).
Mentions: We next considered whether Drosha or Dicer downregulation leads to impaired miRNA biogenesis by carrying out miRNA microarray analysis of samples exposed to hypoxia or normoxia. Upon analysis of mature miRNA array data, we observed significant global miRNA downregulation following exposure to hypoxia (Fig. 2a; Supplementary data 1). Analysis of precursor and mature miRNA levels from deep sequencing data revealed significant downregulation of mature miRNAs, compared to their precursors(Fig. 2b; Supplementary data 2). Using qRT-PCR, we analyzed expression of seven significantly downregulated miRNAs from Fig. 2a, in cells exposed to normoxia or hypoxia. There was a significant increase in pri-miRNA levels of six of seven miRNAs that were tested (Fig. 2c). There was also significant downregulation of corresponding mature miRNA levels in response to hypoxia (Fig. 2d), suggesting defective processing machinery under hypoxia conditions. These data were further validated by northern blot analysis of miR-16a and 27a in cancer cells exposed to normoxia and hypoxia (Supplementary Fig. 4a).

Bottom Line: Here we report a previously unrecognized effect of hypoxia in the downregulation of Drosha and Dicer in cancer cells that leads to dysregulation of miRNA biogenesis and increased tumour progression.Rescue of Drosha by siRNAs targeting ETS1/ELK1 in vivo results in significant tumour regression.These findings provide a new link in the mechanistic understanding of global miRNA downregulation in the tumour microenvironment.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Gynecologic Oncology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA [2] Graduate School of Biomedical Sciences, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA.

ABSTRACT
Cancer-related deregulation of miRNA biogenesis has been suggested, but the underlying mechanisms remain elusive. Here we report a previously unrecognized effect of hypoxia in the downregulation of Drosha and Dicer in cancer cells that leads to dysregulation of miRNA biogenesis and increased tumour progression. We show that hypoxia-mediated downregulation of Drosha is dependent on ETS1/ELK1 transcription factors. Moreover, mature miRNA array and deep sequencing studies reveal altered miRNA maturation in cells under hypoxic conditions. At a functional level, this phenomenon results in increased cancer progression in vitro and in vivo, and data from patient samples are suggestive of miRNA biogenesis downregulation in hypoxic tumours. Rescue of Drosha by siRNAs targeting ETS1/ELK1 in vivo results in significant tumour regression. These findings provide a new link in the mechanistic understanding of global miRNA downregulation in the tumour microenvironment.

Show MeSH
Related in: MedlinePlus