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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

H19 overexpression. a Left panel: Real-time PCR performed on HUVECs 18 h post-transfection. Data were normalized for β-actin and ΔΔct expressed as fold of induction pH19 vs. pEmpty **p < 0.01; *p < 0.05. Right panel: ELISA assay for VEGF level in supernatant from HUVECs 18 h after transfection. ***p < 0.001. b Left Panel: Phase contrast (20×) of tubulogenesis assay performed 18 h after transfection. Right panel: quantification of matrigel assay expressed as length of cable as arbitrary unit*p<0.05. c FACS analysis for ICAM expression in HUVEC transfected cells. d Left Panel: Phase contrast micrographs (20×) showing the adhesion of CD90 + cells on HUVEC monolayer transfected with pEmpty or pH19. Right Panel Quantification was calculated by counting the number of adherent CD90+ cells (violet) per field. **p < 0.01
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Fig4: H19 overexpression. a Left panel: Real-time PCR performed on HUVECs 18 h post-transfection. Data were normalized for β-actin and ΔΔct expressed as fold of induction pH19 vs. pEmpty **p < 0.01; *p < 0.05. Right panel: ELISA assay for VEGF level in supernatant from HUVECs 18 h after transfection. ***p < 0.001. b Left Panel: Phase contrast (20×) of tubulogenesis assay performed 18 h after transfection. Right panel: quantification of matrigel assay expressed as length of cable as arbitrary unit*p<0.05. c FACS analysis for ICAM expression in HUVEC transfected cells. d Left Panel: Phase contrast micrographs (20×) showing the adhesion of CD90 + cells on HUVEC monolayer transfected with pEmpty or pH19. Right Panel Quantification was calculated by counting the number of adherent CD90+ cells (violet) per field. **p < 0.01

Mentions: To investigate a possible role of H19 as mediator of pro-angiogenic and adhesive stimuli in HUVECs, we transfected endothelial cells with the entire sequence of the lncRNA H19 (pH19). As shown in Fig. 4a, H19 overexpression in HUVECs induced a transcriptional modulation similar to that obtained after CD90 + exo treatment. Real-time PCR indicated that the over expression of H19 induced a significant increase in the VEGF and ICAM1 transcripts, while, no modulation compared to controls was observed for the transcription of VEGF-R1, VCAM and VE-cadherin (Fig. 4a right panel). The ELISA assay (Fig. 4a left panel) found, for the first time to our knowledge, a substantial increase in VEGF release induced by lncH19. Moreover, a rise in the number and length of tubes was found in HUVECs transfected with pH19 (Fig. 4b), while FACS analysis (Fig. 4c) indicated an increase in the number of ICAM-1-expressing cells induced by H19 overexpression, thus explaining the more adhesive phenotype of HUVECs. The adhesion assay, in fact, revealed a two-fold increase in adhering CD90+ cells when HUVECs were transfected with pH19 (Fig. 4d).Fig. 4


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)

H19 overexpression. a Left panel: Real-time PCR performed on HUVECs 18 h post-transfection. Data were normalized for β-actin and ΔΔct expressed as fold of induction pH19 vs. pEmpty **p < 0.01; *p < 0.05. Right panel: ELISA assay for VEGF level in supernatant from HUVECs 18 h after transfection. ***p < 0.001. b Left Panel: Phase contrast (20×) of tubulogenesis assay performed 18 h after transfection. Right panel: quantification of matrigel assay expressed as length of cable as arbitrary unit*p<0.05. c FACS analysis for ICAM expression in HUVEC transfected cells. d Left Panel: Phase contrast micrographs (20×) showing the adhesion of CD90 + cells on HUVEC monolayer transfected with pEmpty or pH19. Right Panel Quantification was calculated by counting the number of adherent CD90+ cells (violet) per field. **p < 0.01
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4536801&req=5

Fig4: H19 overexpression. a Left panel: Real-time PCR performed on HUVECs 18 h post-transfection. Data were normalized for β-actin and ΔΔct expressed as fold of induction pH19 vs. pEmpty **p < 0.01; *p < 0.05. Right panel: ELISA assay for VEGF level in supernatant from HUVECs 18 h after transfection. ***p < 0.001. b Left Panel: Phase contrast (20×) of tubulogenesis assay performed 18 h after transfection. Right panel: quantification of matrigel assay expressed as length of cable as arbitrary unit*p<0.05. c FACS analysis for ICAM expression in HUVEC transfected cells. d Left Panel: Phase contrast micrographs (20×) showing the adhesion of CD90 + cells on HUVEC monolayer transfected with pEmpty or pH19. Right Panel Quantification was calculated by counting the number of adherent CD90+ cells (violet) per field. **p < 0.01
Mentions: To investigate a possible role of H19 as mediator of pro-angiogenic and adhesive stimuli in HUVECs, we transfected endothelial cells with the entire sequence of the lncRNA H19 (pH19). As shown in Fig. 4a, H19 overexpression in HUVECs induced a transcriptional modulation similar to that obtained after CD90 + exo treatment. Real-time PCR indicated that the over expression of H19 induced a significant increase in the VEGF and ICAM1 transcripts, while, no modulation compared to controls was observed for the transcription of VEGF-R1, VCAM and VE-cadherin (Fig. 4a right panel). The ELISA assay (Fig. 4a left panel) found, for the first time to our knowledge, a substantial increase in VEGF release induced by lncH19. Moreover, a rise in the number and length of tubes was found in HUVECs transfected with pH19 (Fig. 4b), while FACS analysis (Fig. 4c) indicated an increase in the number of ICAM-1-expressing cells induced by H19 overexpression, thus explaining the more adhesive phenotype of HUVECs. The adhesion assay, in fact, revealed a two-fold increase in adhering CD90+ cells when HUVECs were transfected with pH19 (Fig. 4d).Fig. 4

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