[Effect of adenovirus-mediated shRNA down-regulates SHP2 expression on the apoptosis of human hepatic stellate cells LX-2].
Zhonghua Gan Zang Bing Za Zhi
; 31(12): 1313-1317, 2023 Dec 20.
Article
em Zh
| MEDLINE
| ID: mdl-38253076
ABSTRACT
Objective:
To investigate the effect of adenovirus-mediated short hairpin RNA (shRNA) downregulating SH2 domain-containing protein tyrosine phosphatase 2 (SHP2) on the apoptosis of human hepatic stellate cells LX-2 cultured in vitro.Methods:
The recombinant adenovirus Ad-shRNA/SHP2 carrying shRNA targeted SHP2 and expressing green fluorescent protein (GFP), and the empty control virus Ad-GFP expressing GFP were transfected into LX-2 cells cultured in vitro. Real-time fluorescence quantitative PCR was used to detect SHP2 mRNA expression in LX-2 cells. Western blot was used to detect the protein expressions of SHP2, Bax, and Bcl-2 in LX-2 cells. TUNEL and annexin-V/propidium iodide dual-labeled flow cytometry were used to detect apoptosis in LX-2 cells. Experimental group (1) Control group LX-2 cells were transfected with DMEM instead of adenovirus; (2) Ad-GFP group transfected with empty virus Ad-GFP; (3) Ad-shRNA/SHP2 group transfected with recombinant adenovirus Ad-shRNA/SHP2. The means between multiple groups were compared using a one-way ANOVA and the LSD test was used for inter group comparisons.Results:
shRNA-targeted SHP2 significantly down-regulated the expression of SHP2 protein and mRNA in LX-2 cells (P < 0.05). The TUNEL and annexin-V/propidium iodide dual-labeled flow cytometry results showed that the apoptosis rate of LX-2 cells in the Ad-shRNA/SHP2 group (12.755%±1.606%, 19.340%±2.505%) (P < 0.05) was significantly higher compared to the control group (3.077%±0.731%, 9.438%±0.804%) and the Ad-GFP group (3.250%±0.851%, 8.893%±1.982%), with no statistically significant difference between the control group and the Ad-GFP group (P > 0.05). Western blot analysis of Bax and Bcl-2 protein expression in LX-2 cells of each group revealed that the Bax protein expression was significantly higher in the Ad shRNA/SHP2 group (2.493 ± 0.203) (P < 0.05) compared to the control group and Ad-GFP group (1.989 ± 0.147, 1.999 ± 0.162), with no statistically significant difference between the control group and the Ad-GFP group (P > 0.05), while the Bcl-2 protein was significantly decreased in the Ad-shRNA/SHP2 group (1.042±0.148) compared with the control group and the Ad-GFP group (1.707±0.146, 1.521±0.142), with no statistically significant difference between the control group and the Ad-GFP group (P > 0.05).Conclusions:
SHP2 expression down-regulation induces apoptosis of human hepatic stellate cells LX-2 in vitro by reducing Bcl-2/Bax.Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Adenoviridae
/
Apoptose
/
RNA Interferente Pequeno
/
Células Estreladas do Fígado
Limite:
Humans
Idioma:
Zh
Revista:
Zhonghua Gan Zang Bing Za Zhi
Assunto da revista:
GASTROENTEROLOGIA
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
China