Limits...
miRNA-196b inhibits cell proliferation and induces apoptosis in HepG2 cells by targeting IGF2BP1.

Rebucci M, Sermeus A, Leonard E, Delaive E, Dieu M, Fransolet M, Arnould T, Michiels C - Mol. Cancer (2015)

Bottom Line: Indeed, miR-196b overexpression decreased IGF2BP1 RNA expression and protein level.The IGF2BP1 down-regulation by either miR-196b or IGF2BP1 siRNA led to an increase in apoptosis and a decrease in cell viability and proliferation in normal culture conditions.These results emphasize that the chemoresistance induced by hypoxia is a complex mechanism.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry and Cellular Biology (URBC), NARILIS, University of Namur, 61 rue de Bruxelles, 5000, Namur, Belgium. magali.rebucci@unamur.be.

ABSTRACT

Background: Tumor hypoxia is one of the features of tumor microenvironment that contributes to chemoresistance. miRNAs have recently been shown to play important roles in tumorigenesis and drug resistance. Moreover, hypoxia also regulates the expression of a series of miRNAs. However, the interaction between chemoresistance, hypoxia and miRNAs has not been explored yet. The aim of this study is to understand the mechanisms activated/inhibited by miRNAs under hypoxia that induce resistance to chemotherapy-induced apoptosis.

Methods: TaqMan low-density array was used to identify changes in miRNA expression when cells were exposed to etoposide under hypoxia or normoxia. The effects of miR-196b overexpression on apoptosis and cell proliferation were studied in HepG2 cells. miR-196b target mRNAs were identified by proteomic analysis, luciferase activity assay, RT-qPCR and western blot analysis.

Results: Results showed that hypoxia down-regulated miR-196b expression that was induced by etoposide. miR-196b overexpression increased the etoposide-induced apoptosis and reversed the protection of cell death observed under hypoxia. By a proteomic approach combined with bioinformatics analyses, we identified IGF2BP1 as a potential target of miR-196b. Indeed, miR-196b overexpression decreased IGF2BP1 RNA expression and protein level. The IGF2BP1 down-regulation by either miR-196b or IGF2BP1 siRNA led to an increase in apoptosis and a decrease in cell viability and proliferation in normal culture conditions. However, IGF2BP1 silencing did not modify the chemoresistance induced by hypoxia, probably because it is not the only target of miR-196b involved in the regulation of apoptosis.

Conclusions: In conclusion, for the first time, we identified IGF2BP1 as a direct and functional target of miR-196b and showed that miR-196b overexpression reverses the chemoresistance induced by hypoxia. These results emphasize that the chemoresistance induced by hypoxia is a complex mechanism.

Show MeSH

Related in: MedlinePlus

Effects of miR-196b overexpression or IGF2BP1 silencing on cell morphology, viability and proliferation. HepG2 cells were transfected either with pre-miR negative control (pre-miR CTL-) or pre-miR-196b (50 nM) either with RISC Free negative control (RF) or IGF2BP1 siRNA (50 nM). (A) 72 h after transfection, the cell morphology was observed by phase contrast microscopy. (B) Cell viability assay (MTT assay) was performed 24, 48, 72 and 96 h after transfection of HepG2 cells (means ± 1 SD, n = 3). (C) Cell proliferation assay (BrdU ELISA assay) was performed 72 h after transfection (means ± 1 SD, n = 3). °°, °°°: significantly different from untransfected cells (Cells) (p < 0.01, p < 0.001), $$, $$$: significantly different from pre-miR negative control transfected cells or RF negative control transfected cells (p < 0.01, p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4403945&req=5

Fig5: Effects of miR-196b overexpression or IGF2BP1 silencing on cell morphology, viability and proliferation. HepG2 cells were transfected either with pre-miR negative control (pre-miR CTL-) or pre-miR-196b (50 nM) either with RISC Free negative control (RF) or IGF2BP1 siRNA (50 nM). (A) 72 h after transfection, the cell morphology was observed by phase contrast microscopy. (B) Cell viability assay (MTT assay) was performed 24, 48, 72 and 96 h after transfection of HepG2 cells (means ± 1 SD, n = 3). (C) Cell proliferation assay (BrdU ELISA assay) was performed 72 h after transfection (means ± 1 SD, n = 3). °°, °°°: significantly different from untransfected cells (Cells) (p < 0.01, p < 0.001), $$, $$$: significantly different from pre-miR negative control transfected cells or RF negative control transfected cells (p < 0.01, p < 0.001).

Mentions: We assessed the cell morphology by phase contrast microscopy, 72 h after pre-miR-196b or IGF2BP1 siRNA transfection. miR-196b overexpression reduced the cell number and floating cells were observed. On the other hand, IGF2BP1 silencing seems to change the cell morphology since an increase in cell size was observed (Figure 5A).Figure 5


miRNA-196b inhibits cell proliferation and induces apoptosis in HepG2 cells by targeting IGF2BP1.

Rebucci M, Sermeus A, Leonard E, Delaive E, Dieu M, Fransolet M, Arnould T, Michiels C - Mol. Cancer (2015)

Effects of miR-196b overexpression or IGF2BP1 silencing on cell morphology, viability and proliferation. HepG2 cells were transfected either with pre-miR negative control (pre-miR CTL-) or pre-miR-196b (50 nM) either with RISC Free negative control (RF) or IGF2BP1 siRNA (50 nM). (A) 72 h after transfection, the cell morphology was observed by phase contrast microscopy. (B) Cell viability assay (MTT assay) was performed 24, 48, 72 and 96 h after transfection of HepG2 cells (means ± 1 SD, n = 3). (C) Cell proliferation assay (BrdU ELISA assay) was performed 72 h after transfection (means ± 1 SD, n = 3). °°, °°°: significantly different from untransfected cells (Cells) (p < 0.01, p < 0.001), $$, $$$: significantly different from pre-miR negative control transfected cells or RF negative control transfected cells (p < 0.01, p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4403945&req=5

Fig5: Effects of miR-196b overexpression or IGF2BP1 silencing on cell morphology, viability and proliferation. HepG2 cells were transfected either with pre-miR negative control (pre-miR CTL-) or pre-miR-196b (50 nM) either with RISC Free negative control (RF) or IGF2BP1 siRNA (50 nM). (A) 72 h after transfection, the cell morphology was observed by phase contrast microscopy. (B) Cell viability assay (MTT assay) was performed 24, 48, 72 and 96 h after transfection of HepG2 cells (means ± 1 SD, n = 3). (C) Cell proliferation assay (BrdU ELISA assay) was performed 72 h after transfection (means ± 1 SD, n = 3). °°, °°°: significantly different from untransfected cells (Cells) (p < 0.01, p < 0.001), $$, $$$: significantly different from pre-miR negative control transfected cells or RF negative control transfected cells (p < 0.01, p < 0.001).
Mentions: We assessed the cell morphology by phase contrast microscopy, 72 h after pre-miR-196b or IGF2BP1 siRNA transfection. miR-196b overexpression reduced the cell number and floating cells were observed. On the other hand, IGF2BP1 silencing seems to change the cell morphology since an increase in cell size was observed (Figure 5A).Figure 5

Bottom Line: Indeed, miR-196b overexpression decreased IGF2BP1 RNA expression and protein level.The IGF2BP1 down-regulation by either miR-196b or IGF2BP1 siRNA led to an increase in apoptosis and a decrease in cell viability and proliferation in normal culture conditions.These results emphasize that the chemoresistance induced by hypoxia is a complex mechanism.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry and Cellular Biology (URBC), NARILIS, University of Namur, 61 rue de Bruxelles, 5000, Namur, Belgium. magali.rebucci@unamur.be.

ABSTRACT

Background: Tumor hypoxia is one of the features of tumor microenvironment that contributes to chemoresistance. miRNAs have recently been shown to play important roles in tumorigenesis and drug resistance. Moreover, hypoxia also regulates the expression of a series of miRNAs. However, the interaction between chemoresistance, hypoxia and miRNAs has not been explored yet. The aim of this study is to understand the mechanisms activated/inhibited by miRNAs under hypoxia that induce resistance to chemotherapy-induced apoptosis.

Methods: TaqMan low-density array was used to identify changes in miRNA expression when cells were exposed to etoposide under hypoxia or normoxia. The effects of miR-196b overexpression on apoptosis and cell proliferation were studied in HepG2 cells. miR-196b target mRNAs were identified by proteomic analysis, luciferase activity assay, RT-qPCR and western blot analysis.

Results: Results showed that hypoxia down-regulated miR-196b expression that was induced by etoposide. miR-196b overexpression increased the etoposide-induced apoptosis and reversed the protection of cell death observed under hypoxia. By a proteomic approach combined with bioinformatics analyses, we identified IGF2BP1 as a potential target of miR-196b. Indeed, miR-196b overexpression decreased IGF2BP1 RNA expression and protein level. The IGF2BP1 down-regulation by either miR-196b or IGF2BP1 siRNA led to an increase in apoptosis and a decrease in cell viability and proliferation in normal culture conditions. However, IGF2BP1 silencing did not modify the chemoresistance induced by hypoxia, probably because it is not the only target of miR-196b involved in the regulation of apoptosis.

Conclusions: In conclusion, for the first time, we identified IGF2BP1 as a direct and functional target of miR-196b and showed that miR-196b overexpression reverses the chemoresistance induced by hypoxia. These results emphasize that the chemoresistance induced by hypoxia is a complex mechanism.

Show MeSH
Related in: MedlinePlus