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CD90+ liver cancer cells modulate endothelial cell phenotype through the release of exosomes containing H19 lncRNA.

Conigliaro A, Costa V, Lo Dico A, Saieva L, Buccheri S, Dieli F, Manno M, Raccosta S, Mancone C, Tripodi M, De Leo G, Alessandro R - Mol. Cancer (2015)

Bottom Line: Experiments of gain and loss of function of H19 showed that this LncRNA plays an important role in the exosome-mediated phenotype of endothelial cells.Our data indicate a new exosome-mediated mechanism by which CSC-like CD90+ cells could influence their tumor microenvironment by promoting angiogenesis.Moreover, we suggest the lncRNA H19 as a putative therapeutic target in hepatocellular carcinoma.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biotecnologie Cellulari ed Ematologia, Sapienza University of Rome, c/o Policlinico Umberto I, V Clinica Medica Viale Regina Elena, Rome, 324-00161, Italy. alice.conigliaro@uniroma1.it.

ABSTRACT

Background: CD90+ liver cancer cells have been described as cancer stem-cell-like (CSC), displaying aggressive and metastatic phenotype. Using two different in vitro models, already described as CD90+ liver cancer stem cells, our aim was to study their interaction with endothelial cells mediated by the release of exosomes.

Methods: Exosomes were isolated and characterized from both liver CD90+ cells and hepatoma cell lines. Endothelial cells were treated with exosomes, as well as transfected with a plasmid containing the full length sequence of the long non-coding RNA (lncRNA) H19. Molecular and functional analyses were done to characterize the endothelial phenotype after treatments.

Results: Exosomes released by CD90+ cancer cells, but not by parental hepatoma cells, modulated endothelial cells, promoting angiogenic phenotype and cell-to-cell adhesion. LncRNA profiling revealed that CD90+ cells were enriched in lncRNA H19, and released this through exosomes. Experiments of gain and loss of function of H19 showed that this LncRNA plays an important role in the exosome-mediated phenotype of endothelial cells.

Conclusions: Our data indicate a new exosome-mediated mechanism by which CSC-like CD90+ cells could influence their tumor microenvironment by promoting angiogenesis. Moreover, we suggest the lncRNA H19 as a putative therapeutic target in hepatocellular carcinoma.

No MeSH data available.


Related in: MedlinePlus

a. LncRNAs expressed in CD90+ cells and their exosomes (left and middle panel). Data are expressed as fold induction compared with Huh7 mix population. Of the 90 lncRNAs analyzed, only those over-expressed more than ten-fold in CD90+ cells were considered. Listed on the right the lncRNA up-regulated in HCC. Right panel: LncRNA Profile in exosomes released by CD90 + Huh7. Data are expressed as fold of induction compared with exosomes from Huh7 parental cells. b H19 analysis. Real-time PCR analysis for H19 expression in exosomes derived from Huh7 or CD90+ cells. Exosomes were treated with RNase and subsequently processed for RNA extraction and retrotrascription. Data were normalized for β-actin and ΔΔct indicated as fold of induction compared with Huh7-derived exosomes. ***p < 0.001. c Real-time PCR for H19 on HUVEC 18 h after treatment with CD90 + exo or Huh7exo. Data were normalized for β-actin and ΔΔct indicated as fold of induction compared with control (untreated cells). ***p < 0.001
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Fig3: a. LncRNAs expressed in CD90+ cells and their exosomes (left and middle panel). Data are expressed as fold induction compared with Huh7 mix population. Of the 90 lncRNAs analyzed, only those over-expressed more than ten-fold in CD90+ cells were considered. Listed on the right the lncRNA up-regulated in HCC. Right panel: LncRNA Profile in exosomes released by CD90 + Huh7. Data are expressed as fold of induction compared with exosomes from Huh7 parental cells. b H19 analysis. Real-time PCR analysis for H19 expression in exosomes derived from Huh7 or CD90+ cells. Exosomes were treated with RNase and subsequently processed for RNA extraction and retrotrascription. Data were normalized for β-actin and ΔΔct indicated as fold of induction compared with Huh7-derived exosomes. ***p < 0.001. c Real-time PCR for H19 on HUVEC 18 h after treatment with CD90 + exo or Huh7exo. Data were normalized for β-actin and ΔΔct indicated as fold of induction compared with control (untreated cells). ***p < 0.001

Mentions: It has been confirmed that dysregulation of lncRNAs is associated with several human tumors and, recently, a contribution of lncRNAs to hepatocarcinogenesis was found [30–32]. In order to clarify the molecular mechanism driving the modifications induced in HUVECs by CD90 + -derived exosomes we did an lncRNA profile study in CD90+ cells and parental Huh7 by analyzing the expression of 90 different lncRNAs. In Fig. 3a (left and middle panel), the RNAs over-expressed in CD90+Huh7 cells compared with Huh7 parental cells with at least a ten-fold increase are listed. Among these, Air, Hotair, LincRNA-ROR, Hulc, and H19 have already been identified as positively correlated with hepatocellular carcinoma [31, 33, 34]. We focused our interest on H19, expression of which has been previously associated with metastasis [35, 36]. In line with recent articles, which have demonstrated that hepatocellular carcinoma cells release exosomes containing lncRNA [25, 37], we investigated the expression in exosomes, of those LncRNAs that we found overexpressed in cells. As shown in Fig. 3a right panel the LncProfiler performed on CD90+ Huh7 and Huh7-derived exosomes evidences that H19 was 10-fold up-regulated in exosomes derived from CD90+ Huh7, compared to parental cell line. The Real-time PCR confirmed that vesicles released by CD90 + cells (both sorted or SkHep cells) are highly enriched in H19 transcript compared with vesicles from Huh7 parental cells (Fig. 3b, S1e). Moreover, treatment with CD90 + -derived exosomes induced in HUVECs an increase in H19 transcript (Fig. 3c, S1f). These data suggest a transport of H19 lncRNA from CD90+ cells to HUVECs, even if we cannot exclude a stimulation of endogenous lncRNA.Fig. 3


CD90+ liver cancer cells modulate endothelial cell phenotype through the release of exosomes containing H19 lncRNA.

Conigliaro A, Costa V, Lo Dico A, Saieva L, Buccheri S, Dieli F, Manno M, Raccosta S, Mancone C, Tripodi M, De Leo G, Alessandro R - Mol. Cancer (2015)

a. LncRNAs expressed in CD90+ cells and their exosomes (left and middle panel). Data are expressed as fold induction compared with Huh7 mix population. Of the 90 lncRNAs analyzed, only those over-expressed more than ten-fold in CD90+ cells were considered. Listed on the right the lncRNA up-regulated in HCC. Right panel: LncRNA Profile in exosomes released by CD90 + Huh7. Data are expressed as fold of induction compared with exosomes from Huh7 parental cells. b H19 analysis. Real-time PCR analysis for H19 expression in exosomes derived from Huh7 or CD90+ cells. Exosomes were treated with RNase and subsequently processed for RNA extraction and retrotrascription. Data were normalized for β-actin and ΔΔct indicated as fold of induction compared with Huh7-derived exosomes. ***p < 0.001. c Real-time PCR for H19 on HUVEC 18 h after treatment with CD90 + exo or Huh7exo. Data were normalized for β-actin and ΔΔct indicated as fold of induction compared with control (untreated cells). ***p < 0.001
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4536801&req=5

Fig3: a. LncRNAs expressed in CD90+ cells and their exosomes (left and middle panel). Data are expressed as fold induction compared with Huh7 mix population. Of the 90 lncRNAs analyzed, only those over-expressed more than ten-fold in CD90+ cells were considered. Listed on the right the lncRNA up-regulated in HCC. Right panel: LncRNA Profile in exosomes released by CD90 + Huh7. Data are expressed as fold of induction compared with exosomes from Huh7 parental cells. b H19 analysis. Real-time PCR analysis for H19 expression in exosomes derived from Huh7 or CD90+ cells. Exosomes were treated with RNase and subsequently processed for RNA extraction and retrotrascription. Data were normalized for β-actin and ΔΔct indicated as fold of induction compared with Huh7-derived exosomes. ***p < 0.001. c Real-time PCR for H19 on HUVEC 18 h after treatment with CD90 + exo or Huh7exo. Data were normalized for β-actin and ΔΔct indicated as fold of induction compared with control (untreated cells). ***p < 0.001
Mentions: It has been confirmed that dysregulation of lncRNAs is associated with several human tumors and, recently, a contribution of lncRNAs to hepatocarcinogenesis was found [30–32]. In order to clarify the molecular mechanism driving the modifications induced in HUVECs by CD90 + -derived exosomes we did an lncRNA profile study in CD90+ cells and parental Huh7 by analyzing the expression of 90 different lncRNAs. In Fig. 3a (left and middle panel), the RNAs over-expressed in CD90+Huh7 cells compared with Huh7 parental cells with at least a ten-fold increase are listed. Among these, Air, Hotair, LincRNA-ROR, Hulc, and H19 have already been identified as positively correlated with hepatocellular carcinoma [31, 33, 34]. We focused our interest on H19, expression of which has been previously associated with metastasis [35, 36]. In line with recent articles, which have demonstrated that hepatocellular carcinoma cells release exosomes containing lncRNA [25, 37], we investigated the expression in exosomes, of those LncRNAs that we found overexpressed in cells. As shown in Fig. 3a right panel the LncProfiler performed on CD90+ Huh7 and Huh7-derived exosomes evidences that H19 was 10-fold up-regulated in exosomes derived from CD90+ Huh7, compared to parental cell line. The Real-time PCR confirmed that vesicles released by CD90 + cells (both sorted or SkHep cells) are highly enriched in H19 transcript compared with vesicles from Huh7 parental cells (Fig. 3b, S1e). Moreover, treatment with CD90 + -derived exosomes induced in HUVECs an increase in H19 transcript (Fig. 3c, S1f). These data suggest a transport of H19 lncRNA from CD90+ cells to HUVECs, even if we cannot exclude a stimulation of endogenous lncRNA.Fig. 3

Bottom Line: Experiments of gain and loss of function of H19 showed that this LncRNA plays an important role in the exosome-mediated phenotype of endothelial cells.Our data indicate a new exosome-mediated mechanism by which CSC-like CD90+ cells could influence their tumor microenvironment by promoting angiogenesis.Moreover, we suggest the lncRNA H19 as a putative therapeutic target in hepatocellular carcinoma.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biotecnologie Cellulari ed Ematologia, Sapienza University of Rome, c/o Policlinico Umberto I, V Clinica Medica Viale Regina Elena, Rome, 324-00161, Italy. alice.conigliaro@uniroma1.it.

ABSTRACT

Background: CD90+ liver cancer cells have been described as cancer stem-cell-like (CSC), displaying aggressive and metastatic phenotype. Using two different in vitro models, already described as CD90+ liver cancer stem cells, our aim was to study their interaction with endothelial cells mediated by the release of exosomes.

Methods: Exosomes were isolated and characterized from both liver CD90+ cells and hepatoma cell lines. Endothelial cells were treated with exosomes, as well as transfected with a plasmid containing the full length sequence of the long non-coding RNA (lncRNA) H19. Molecular and functional analyses were done to characterize the endothelial phenotype after treatments.

Results: Exosomes released by CD90+ cancer cells, but not by parental hepatoma cells, modulated endothelial cells, promoting angiogenic phenotype and cell-to-cell adhesion. LncRNA profiling revealed that CD90+ cells were enriched in lncRNA H19, and released this through exosomes. Experiments of gain and loss of function of H19 showed that this LncRNA plays an important role in the exosome-mediated phenotype of endothelial cells.

Conclusions: Our data indicate a new exosome-mediated mechanism by which CSC-like CD90+ cells could influence their tumor microenvironment by promoting angiogenesis. Moreover, we suggest the lncRNA H19 as a putative therapeutic target in hepatocellular carcinoma.

No MeSH data available.


Related in: MedlinePlus