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. 2019 May;19(5):3537-3547.
doi: 10.3892/mmr.2019.10025. Epub 2019 Mar 14.

Astaxanthin Inhibits Proliferation and Induces Apoptosis of LX‑2 Cells by Regulating the miR‑29b/Bcl‑2 Pathway

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Free PMC article

Astaxanthin Inhibits Proliferation and Induces Apoptosis of LX‑2 Cells by Regulating the miR‑29b/Bcl‑2 Pathway

Shanshan Zhu et al. Mol Med Rep. .
Free PMC article

Abstract

The aim of the present study was to investigate the role of microRNAs (miRNAs/miRs) in the anti‑fibrotic effect of astaxanthin (AST), using the human hepatic stellate cell (HSC) line LX‑2 as the research model. LX‑2 cells were treated with various concentrations of AST (10, 20 and 40 µM) for 24 or 48 h. miR‑29b was selected based on existing literature, and its targeting gene B cell lymphoma (Bcl)‑2 was predicted by TargetScan and miRanda databases for further analysis. Interactions between miR‑29b and Bcl‑2 in the AST treated LX‑2 cells were evaluated using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot analysis. MTT analysis was used to analyze cell viability. Overexpression of miR‑29b decreased the expression of Bcl‑2 in AST‑treated LX‑2 cells, and silencing of it had the opposite effect. Additionally, Annexin V‑fluorescein isothiocyanate/propidium iodide double staining and flow cytometry were used to evaluate the cell apoptosis, and overexpression of miR‑29b increased cell apoptosis rates in AST‑treated LX‑2 cells; however, silencing of it had the opposite effect. RT‑qPCR and western blotting demonstrated that AST induced LX‑2 cells apoptosis which may be by regulating miR‑29b, as indicated by inhibited Bcl‑2 expression levels and elevated Bax and Caspase‑3 expression levels. These results highlight an important role of miR‑29b in the AST modulating LX‑2 cells proliferation and apoptosis and implicate a potential mechanism of miR‑29b and AST preventing liver fibrosis.

Figures

Figure 1.
Figure 1.
AST elevates the expression of miR-29b. LX-2 cells were treated with various concentration of AST (10, 20, 40 µM) or the vehicle (DMSO) for (A) 24 or (B) 48 h. *P<0.05, **P<0.01, ***P<0.001 vs. DMSO. AST, astaxanthin; DMSO, dimethyl sulfoxide; miR, microRNA.
Figure 2.
Figure 2.
The expression efficiency of miR-29b. LX-2 cells were transfected with (A) miR-29b mimics or (B) miR-29b inhibitors for 48 h. Results are expressed as the mean ± standard deviation of fold. **P<0.01, ****P<0.0001 vs. Con miR or Con Inh group. AST, astaxanthin; Con, control; Inh, inhibitor; miR, microRNA.
Figure 3.
Figure 3.
AST inhibits the LX-2 cells proliferation by regulating miR-29b. (A) LX-2 cells were treated with various concentration of AST (5, 10, 20, 40 and 80 µM) or the vehicle (DMSO) for 12, 24, 48 and 72 h. **P<0.01, ***P<0.001, ****P<0.0001 vs. DMSO. (B) LX-2 cells were transfected with either miR-29b mimics or miR-29b mimic negative control or miR-29b inhibitors or miR-29b inhibitor negative control for 48 h, and then treated with AST (40 µM) or the vehicle for 48 h. Data are expressed as the mean ± standard deviation. **P<0.01, ****P<0.0001 vs. Con miR or Con Inh group. AST, astaxanthin; Con, control; DMSO, dimethyl sulfoxide; Inh, inhibitor; miR, microRNA.
Figure 4.
Figure 4.
AST inhibits the expression of α-SMA and Col1a1 in LX-2 cells. After treating cells with AST or the vehicle for 48 h, mRNA and protein expression levels of (A) α-SMA and (B) Col1a1 were measured by RT-qPCR and western blotting. Band densities of α-SMA (C) and Col1a1 (D) were quantified using Quantity One software. Data are expressed as the mean ± standard deviation. *P<0.05, ***P<0.001 vs. DMSO. α-SMA, α-smooth muscle actin; AST, astaxanthin; Col1a1, collagen α-1(I) chain; DMSO, dimethyl sulfoxide; miR, microRNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.
Figure 5.
Figure 5.
AST inhibits the expression of α-SMA and Col1a1 in LX-2 cells. LX-2 cells were transfected with either miR-29b mimics or miR-29b mimic negative control or miR-29b inhibitors or miR-29b inhibitor negative control for 48 h, and treated with AST (40 µM) or the vehicle for 48 h. (A and B) Protein and mRNA expression levels of α-SMA and (C and D) protein and mRNA expression levels of Col1a1 as measured by RT-qPCR and western blotting. Data are presented as the mean ± standard deviation. *P<0.05, ***P<0.001, ****P<0.0001 vs. Con miR or Con Inh group; #P<0.05 vs. miR-29b or anti-miR-29b group. α-SMA, α-smooth muscle actin; AST, astaxanthin; Col1a1, collagen α-1(I) chain; Con, control; DMSO, dimethyl sulfoxide; Inh, inhibitor; miR, microRNA.
Figure 6.
Figure 6.
AST inhibits the expression levels of apoptosis-associated proteins in LX-2 cells. (A) TargetScan and miRanda databases predicted that the 3′-UTR of Bcl-2 contain putative binding sites for miR-29b. After treated with AST or the vehicle for 24 and 48 h, mRNA and protein expression levels of (B) Bcl-2, (C) Bax and (D) Caspase-3 were investigated by RT-qPCR and western blotting. Data are expressed as the mean ± standard deviation. *P<0.05, ***P<0.001 vs. DMSO. Bax, Bcl-2-associated X protein; Bcl, B cell lymphoma; DMSO, dimethyl sulfoxide; miR, microRNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; UTR, untranslated region.
Figure 7.
Figure 7.
AST regulates the expression levels of apoptosis-associated proteins by regulating miR-29b in LX-2 cells. LX-2 cells were transfected with either miR-29b mimics or miR-29b mimic negative control or miR-29b inhibitors or miR-29b inhibitor negative control for 48 h, and then treated with AST (40 µM) or the vehicle for 48 h. mRNA and protein expression levels of (A and B) Bcl-2, (C and D) Bax and (E and F) Caspase-3 were investigated by RT-qPCR and western blotting. Data are expressed as the mean ± standard deviation. *P<0.05, ***P<0.001, ****P<0.0001 vs. Con miR or Con Inh group. #P<0.05; ##P<0.01 vs. miR-29b or anti-miR-29b group. AST, astaxanthin; Bax, Bcl-2-associated X protein; Bcl, B cell lymphoma; Inh, inhibitor; miR, microRNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.
Figure 8.
Figure 8.
AST promotes LX-2 cells apoptosis by regulating miR-29b. LX-2 cells were transfected with either miR-29b mimics or miR-29b mimic negative control or miR-29b inhibitors or miR-29b inhibitor negative control for 48 h, and treated with AST (40 µM) or the vehicle for 48 h. (A and B) Apoptosis of LX-2 cells and (C and D) its quantification, as measured by flow-cytometry. Data are expressed as the mean ± standard deviation. *P<0.05, **P<0.01, ****P<0.0001 vs. Con miR or Con Inh group; ##P<0.01, ###P<0.001 vs. miR-29b or anti-miR-29b group. AST, astaxanthin; Inh, inhibitor; miR, microRNA.
Figure 9.
Figure 9.
Schematic illustration showing the regulation of AST on liver fibrosis. AST reduces PI3K/Akt activity and Bcl-2 by elevating expression levels of miR-29b, which in turn increases Bax and Caspase-3 activity promoting HSCs apoptosis. AST, astaxanthin; Bax, Bcl-2-associated X protein; Bcl, B cell lymphoma; miR, microRNA; PI3K, phosphoinositide 3-kinase.

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