Background H9c2 cell line is mononucleated myoblast derived from embryonic rat heart tissue. Activities of TGF-β1, MMP-2 and MMP-9 increase in H9c2 cells after treatment with fibrosis stimuli. MicroRNA （miRNA）, a k...Background H9c2 cell line is mononucleated myoblast derived from embryonic rat heart tissue. Activities of TGF-β1, MMP-2 and MMP-9 increase in H9c2 cells after treatment with fibrosis stimuli. MicroRNA （miRNA）, a kind of endogenous small non-coding RNA, participates in cardiac fibrosis. In the present study, expressions of fibrosis-related genes and microRNAs in TGF-β1treated H9c2 cells were investigated. Methods Expressions of fibrosis-associated genes, including Co13al, α-SMA, FN1, CTGF and TSP-1, were measured in TGF-β1 treated H9c2 cells by quantitative reverse transcription and PCR （qRT- PCR）. Level of α-SMA in H9c2 cells was demonstrated by fluorescence immunohistochemistry （FIHC） assay. Expressions of mature miR-16, -21a, -29b in H9c2 cells were determined by qRT-PCR assay. Activations of Smad3 and NF-kB signaling in TGF-β1-treated H9c2 cells were studied by dual luciferase assay. Expressions of Col3al, α-SMA, FN1, CTGF and TSP-1 were detected in H9c2 cells with adenovirus-mediated overexpression of miR-21a. Results qRT-PCR assay showed that ot-SMA, FN1, CTGF, TSP-1, but not Co13al, were up-regulated in TGF-β1treated H9c2 cells. FIHC result also revealed that α-SMA was increased in TGF-β1-treated H9c2 cells. Consistently, dual luciferase assay showed that Smad3 and NF-kB signaling proteins were activated in TGF-β1-treated H9c2 cells, miR-21a, but not miR- 16 and -29b, was significantly up-regulated. Additionally, over-expression of miR-21a significantly increases mRNA expressions of α-SMA, FN1, CTGF and TSP-1 in H9c2 cells. Conclusions MiR-21a is up-regulated in TGF-β1 treated H9c2 cells, and may contribute to up-regulations of fibrosis-associated genes.展开更多
基金supported by grants from the National Natural Science Foundation of China(No.81270222/81302779/81370295/81470439)Natural Science Foundation of Guangdong Province(No.2014A030313635/S2011020005911/S2012010009453)
文摘Background H9c2 cell line is mononucleated myoblast derived from embryonic rat heart tissue. Activities of TGF-β1, MMP-2 and MMP-9 increase in H9c2 cells after treatment with fibrosis stimuli. MicroRNA （miRNA）, a kind of endogenous small non-coding RNA, participates in cardiac fibrosis. In the present study, expressions of fibrosis-related genes and microRNAs in TGF-β1treated H9c2 cells were investigated. Methods Expressions of fibrosis-associated genes, including Co13al, α-SMA, FN1, CTGF and TSP-1, were measured in TGF-β1 treated H9c2 cells by quantitative reverse transcription and PCR （qRT- PCR）. Level of α-SMA in H9c2 cells was demonstrated by fluorescence immunohistochemistry （FIHC） assay. Expressions of mature miR-16, -21a, -29b in H9c2 cells were determined by qRT-PCR assay. Activations of Smad3 and NF-kB signaling in TGF-β1-treated H9c2 cells were studied by dual luciferase assay. Expressions of Col3al, α-SMA, FN1, CTGF and TSP-1 were detected in H9c2 cells with adenovirus-mediated overexpression of miR-21a. Results qRT-PCR assay showed that ot-SMA, FN1, CTGF, TSP-1, but not Co13al, were up-regulated in TGF-β1treated H9c2 cells. FIHC result also revealed that α-SMA was increased in TGF-β1-treated H9c2 cells. Consistently, dual luciferase assay showed that Smad3 and NF-kB signaling proteins were activated in TGF-β1-treated H9c2 cells, miR-21a, but not miR- 16 and -29b, was significantly up-regulated. Additionally, over-expression of miR-21a significantly increases mRNA expressions of α-SMA, FN1, CTGF and TSP-1 in H9c2 cells. Conclusions MiR-21a is up-regulated in TGF-β1 treated H9c2 cells, and may contribute to up-regulations of fibrosis-associated genes.
