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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.

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miRNA expression profiling in HepG2 cells incubated under normoxia or hypoxia with or without etoposide. Relative expression of 132 miRNAs was quantified by TLDA (TaqMan Low Density Array) in cells incubated under normoxia (N) or hypoxia (H) with or without etoposide (E) (50 μM) during 16 h. (A) Ratio of miRNA expression between cells incubated with etoposide under hypoxia in comparison to normoxia has been calculated. Four miRNAs were detected with an induction fold higher than 1.5 and seven miRNAs with a fold induction lower than - 1.5. Data were normalized to RNU44 and RNU48 endogenous controls using the threshold cycle method. (B) miRNA-565, −196b and −210 expression was monitored by conventional TaqMan RT-qPCR in HepG2 cells exposed to normoxia (N) or hypoxia (H) with or without etoposide (E) (50 μM) during 16 h. The relative expression of miRNA was determined using the threshold cycle method by normalizing to endogenous RNU44 (means ± 1SD, n = 3). ***: significantly different from normoxia (p < 0.001), #, ###: significantly different from hypoxia (p < 0.05, p < 0.001), $$$: significantly different from normoxia with etoposide (p < 0.001).
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Fig1: miRNA expression profiling in HepG2 cells incubated under normoxia or hypoxia with or without etoposide. Relative expression of 132 miRNAs was quantified by TLDA (TaqMan Low Density Array) in cells incubated under normoxia (N) or hypoxia (H) with or without etoposide (E) (50 μM) during 16 h. (A) Ratio of miRNA expression between cells incubated with etoposide under hypoxia in comparison to normoxia has been calculated. Four miRNAs were detected with an induction fold higher than 1.5 and seven miRNAs with a fold induction lower than - 1.5. Data were normalized to RNU44 and RNU48 endogenous controls using the threshold cycle method. (B) miRNA-565, −196b and −210 expression was monitored by conventional TaqMan RT-qPCR in HepG2 cells exposed to normoxia (N) or hypoxia (H) with or without etoposide (E) (50 μM) during 16 h. The relative expression of miRNA was determined using the threshold cycle method by normalizing to endogenous RNU44 (means ± 1SD, n = 3). ***: significantly different from normoxia (p < 0.001), #, ###: significantly different from hypoxia (p < 0.05, p < 0.001), $$$: significantly different from normoxia with etoposide (p < 0.001).

Mentions: In order to explore the role of miRNAs in chemoresistance induced by hypoxia, the expression level of 365 miRNAs was compared in HepG2 cells exposed to normoxia or hypoxia with or without etoposide during 16 h. We identified 11 miRNAs for which expression was dysregulated with a fold change higher than 1.5 in cells incubated under normoxia with etoposide when compared to cells incubated under hypoxia with etoposide. The expression of 4 miRNAs was significantly up-regulated in cells incubated under hypoxia with etoposide compared to cells incubated under normoxia with etoposide (hsa-miR-220, −219, −210, −193a) while the expression of 7 miRNAs was significantly down-regulated in cells incubated under hypoxia compared to cells incubated under normoxia in the presence of etoposide (fold change > −1.5) (Figure 1A). To confirm the results of the TaqMan low-density array (TLDA) analysis, RT-qPCR assays were performed to evaluate miRNAs differentially expressed (Figure 1B). Among the 11 differentially expressed miRNAs identified by TLDA, changes in expression for only 3 miRNAs were validated. Etoposide significantly increased the miR-565 and miR-196b expression and hypoxia significantly decreased the expression of these two miRNAs induced by etoposide. On the contrary, hypoxia exposure with etoposide significantly increased miR-210 expression compared to cells incubated under normoxia with etoposide (Figure 1B). These results were consistent with the results obtained with the TLDA analysis.Figure 1


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)

miRNA expression profiling in HepG2 cells incubated under normoxia or hypoxia with or without etoposide. Relative expression of 132 miRNAs was quantified by TLDA (TaqMan Low Density Array) in cells incubated under normoxia (N) or hypoxia (H) with or without etoposide (E) (50 μM) during 16 h. (A) Ratio of miRNA expression between cells incubated with etoposide under hypoxia in comparison to normoxia has been calculated. Four miRNAs were detected with an induction fold higher than 1.5 and seven miRNAs with a fold induction lower than - 1.5. Data were normalized to RNU44 and RNU48 endogenous controls using the threshold cycle method. (B) miRNA-565, −196b and −210 expression was monitored by conventional TaqMan RT-qPCR in HepG2 cells exposed to normoxia (N) or hypoxia (H) with or without etoposide (E) (50 μM) during 16 h. The relative expression of miRNA was determined using the threshold cycle method by normalizing to endogenous RNU44 (means ± 1SD, n = 3). ***: significantly different from normoxia (p < 0.001), #, ###: significantly different from hypoxia (p < 0.05, p < 0.001), $$$: significantly different from normoxia with etoposide (p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: miRNA expression profiling in HepG2 cells incubated under normoxia or hypoxia with or without etoposide. Relative expression of 132 miRNAs was quantified by TLDA (TaqMan Low Density Array) in cells incubated under normoxia (N) or hypoxia (H) with or without etoposide (E) (50 μM) during 16 h. (A) Ratio of miRNA expression between cells incubated with etoposide under hypoxia in comparison to normoxia has been calculated. Four miRNAs were detected with an induction fold higher than 1.5 and seven miRNAs with a fold induction lower than - 1.5. Data were normalized to RNU44 and RNU48 endogenous controls using the threshold cycle method. (B) miRNA-565, −196b and −210 expression was monitored by conventional TaqMan RT-qPCR in HepG2 cells exposed to normoxia (N) or hypoxia (H) with or without etoposide (E) (50 μM) during 16 h. The relative expression of miRNA was determined using the threshold cycle method by normalizing to endogenous RNU44 (means ± 1SD, n = 3). ***: significantly different from normoxia (p < 0.001), #, ###: significantly different from hypoxia (p < 0.05, p < 0.001), $$$: significantly different from normoxia with etoposide (p < 0.001).
Mentions: In order to explore the role of miRNAs in chemoresistance induced by hypoxia, the expression level of 365 miRNAs was compared in HepG2 cells exposed to normoxia or hypoxia with or without etoposide during 16 h. We identified 11 miRNAs for which expression was dysregulated with a fold change higher than 1.5 in cells incubated under normoxia with etoposide when compared to cells incubated under hypoxia with etoposide. The expression of 4 miRNAs was significantly up-regulated in cells incubated under hypoxia with etoposide compared to cells incubated under normoxia with etoposide (hsa-miR-220, −219, −210, −193a) while the expression of 7 miRNAs was significantly down-regulated in cells incubated under hypoxia compared to cells incubated under normoxia in the presence of etoposide (fold change > −1.5) (Figure 1A). To confirm the results of the TaqMan low-density array (TLDA) analysis, RT-qPCR assays were performed to evaluate miRNAs differentially expressed (Figure 1B). Among the 11 differentially expressed miRNAs identified by TLDA, changes in expression for only 3 miRNAs were validated. Etoposide significantly increased the miR-565 and miR-196b expression and hypoxia significantly decreased the expression of these two miRNAs induced by etoposide. On the contrary, hypoxia exposure with etoposide significantly increased miR-210 expression compared to cells incubated under normoxia with etoposide (Figure 1B). These results were consistent with the results obtained with the TLDA analysis.Figure 1

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