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

Role of IGF2BP1 in the chemoresistance induced by hypoxia. HepG2 cells were transfected with RISC free negative control (RF) or siRNA IGF2BP1 (50 nM) during 48 h and then cells were incubated under normoxia (N) or hypoxia (H) with or without etoposide (E) (50 μM) during 16 h. (A) IGF2BP1, cleaved caspase 3 and cleaved PARP were detected in total cell extracts by western blotting analysis, using specific antibodies. Immunodetection of α-tubulin was used to assess the total amount of proteins loaded on the gel. Numbers correspond to the quantification of the abundance of protein of interest normalized to the abundance of α-tubulin (B) 48 h after transfection, the caspase 3/7 activity was assayed by measuring free AFC released from the cleavage of the caspase 3/7 substrate Ac-DEVD-AFC. Results are expressed in fluorescence intensity, as means ± 1 SD (n = 3). ***: (p < 0.001), °, °°°: significantly different from untransfected cells (Cells) (p < 0.05, p < 0.001), $$$: significantly different from pre-miR negative control transfected cells (p < 0.001) for each group N, H, NE, HE respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig8: Role of IGF2BP1 in the chemoresistance induced by hypoxia. HepG2 cells were transfected with RISC free negative control (RF) or siRNA IGF2BP1 (50 nM) during 48 h and then cells were incubated under normoxia (N) or hypoxia (H) with or without etoposide (E) (50 μM) during 16 h. (A) IGF2BP1, cleaved caspase 3 and cleaved PARP were detected in total cell extracts by western blotting analysis, using specific antibodies. Immunodetection of α-tubulin was used to assess the total amount of proteins loaded on the gel. Numbers correspond to the quantification of the abundance of protein of interest normalized to the abundance of α-tubulin (B) 48 h after transfection, the caspase 3/7 activity was assayed by measuring free AFC released from the cleavage of the caspase 3/7 substrate Ac-DEVD-AFC. Results are expressed in fluorescence intensity, as means ± 1 SD (n = 3). ***: (p < 0.001), °, °°°: significantly different from untransfected cells (Cells) (p < 0.05, p < 0.001), $$$: significantly different from pre-miR negative control transfected cells (p < 0.001) for each group N, H, NE, HE respectively.

Mentions: Since we demonstrated that IGF2BP1 is a target of miR-196b and because miR-196b was shown to modulate hypoxia-induced chemoresistance, we sought whether the chemoresistance induced by hypoxia is due to IGF2BP1 expression inhibition. For that, HepG2 cells were transfected with IGF2BP1 siRNA for 48 h and then incubated under normoxia or hypoxia with or without etoposide, and apoptotic markers were studied by western blot (Figure 8A). IGF2BP1 silencing decreased IGF2BP1 protein abundance in cells incubated under normoxia or hypoxia with or without etoposide 48 h post-transfection. However, IGF2BP1 silencing did not modify the chemoresistance induced by hypoxia. Indeed, no increase in PARP and caspase 3 cleavage was observed. These data were confirmed using a caspase 3/7 activity assay that showed no modification of caspase 3/7 activity when cells were transfected with IGF2BP1 siRNA (Figure 8B).Figure 8


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)

Role of IGF2BP1 in the chemoresistance induced by hypoxia. HepG2 cells were transfected with RISC free negative control (RF) or siRNA IGF2BP1 (50 nM) during 48 h and then cells were incubated under normoxia (N) or hypoxia (H) with or without etoposide (E) (50 μM) during 16 h. (A) IGF2BP1, cleaved caspase 3 and cleaved PARP were detected in total cell extracts by western blotting analysis, using specific antibodies. Immunodetection of α-tubulin was used to assess the total amount of proteins loaded on the gel. Numbers correspond to the quantification of the abundance of protein of interest normalized to the abundance of α-tubulin (B) 48 h after transfection, the caspase 3/7 activity was assayed by measuring free AFC released from the cleavage of the caspase 3/7 substrate Ac-DEVD-AFC. Results are expressed in fluorescence intensity, as means ± 1 SD (n = 3). ***: (p < 0.001), °, °°°: significantly different from untransfected cells (Cells) (p < 0.05, p < 0.001), $$$: significantly different from pre-miR negative control transfected cells (p < 0.001) for each group N, H, NE, HE respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig8: Role of IGF2BP1 in the chemoresistance induced by hypoxia. HepG2 cells were transfected with RISC free negative control (RF) or siRNA IGF2BP1 (50 nM) during 48 h and then cells were incubated under normoxia (N) or hypoxia (H) with or without etoposide (E) (50 μM) during 16 h. (A) IGF2BP1, cleaved caspase 3 and cleaved PARP were detected in total cell extracts by western blotting analysis, using specific antibodies. Immunodetection of α-tubulin was used to assess the total amount of proteins loaded on the gel. Numbers correspond to the quantification of the abundance of protein of interest normalized to the abundance of α-tubulin (B) 48 h after transfection, the caspase 3/7 activity was assayed by measuring free AFC released from the cleavage of the caspase 3/7 substrate Ac-DEVD-AFC. Results are expressed in fluorescence intensity, as means ± 1 SD (n = 3). ***: (p < 0.001), °, °°°: significantly different from untransfected cells (Cells) (p < 0.05, p < 0.001), $$$: significantly different from pre-miR negative control transfected cells (p < 0.001) for each group N, H, NE, HE respectively.
Mentions: Since we demonstrated that IGF2BP1 is a target of miR-196b and because miR-196b was shown to modulate hypoxia-induced chemoresistance, we sought whether the chemoresistance induced by hypoxia is due to IGF2BP1 expression inhibition. For that, HepG2 cells were transfected with IGF2BP1 siRNA for 48 h and then incubated under normoxia or hypoxia with or without etoposide, and apoptotic markers were studied by western blot (Figure 8A). IGF2BP1 silencing decreased IGF2BP1 protein abundance in cells incubated under normoxia or hypoxia with or without etoposide 48 h post-transfection. However, IGF2BP1 silencing did not modify the chemoresistance induced by hypoxia. Indeed, no increase in PARP and caspase 3 cleavage was observed. These data were confirmed using a caspase 3/7 activity assay that showed no modification of caspase 3/7 activity when cells were transfected with IGF2BP1 siRNA (Figure 8B).Figure 8

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