ABSTRACT
Objective To investigate the effect of MicroRNA-9 (miR-9) on cell proliferation and collagen synthesis in hepatic stellate cells (HSCs),and to explore the potential mechanism.Methods HSC-T6 cells were cultured and transfected with miR-9 mimics with lipofectamine 2000.After incubation 48 h,the cells were collected and total proteins and RNAs were extracted.The expression of miR-9 was detected by quantitative real time polymerase chain reaction (qRT-PCR).The protein expression of type Ⅰ collagen and type Ⅲ collagen were measured by Western blot.The methyl thiazolyl tetrazolium (MTT) method was used to asses the proliferation of HSC-T6 cells.The expression of transforming growth factor-β1 receptor 2 (TGFBR2) was detected by qRT-PCR and Western blot.Results Compared to the control group,miR-9 expression in HSCs was increased in the miR-9 mimics group (P ＜ 0.05),type Ⅰ and type Ⅲ collagen protein expression was reduced by (44 ± 2) ％ and (50 ± 3) ％ (P ＜ 0.01),respectively.The proliferation activity of HSCs was decreased by (48 ± 4)％ (P ＜ 0.05).The expression of TGFBR2 was inhibited in the miR-9 mimics group.Conclusions Upregulation of miR-9 plays a role on suppressing cell proliferation and collagen synthesis in HSCs.This process might be mediated by downregulation of TGFBR2.
ABSTRACT
Objective To investigate the effect of miRNA-200b-specific inhibitor on hepatic stellate cells(HSCs) activation,proliferation,and extracellular matrix production.Methods The miRNA-200b-specific inhibitors were designed,synthesized,and transfected into HSCs with lipofectamine 2000.The supernatant and HSCs were collected after incubation for 48 h.The expression of miR-200b was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR).The expression ofα-smooth muscle actin (oα-SMA) protein in HSCs was detected by Western blotting.The cell proliferation was assayed by methyl thiazolyl tetrazolium (MTT) method.Contents of type Ⅲ procollagen and hyaluronic acid in supernatant were determined by radioimmunoassay.Results Compared to the control group,miRNA-200b expression was decreased in the miRNA-200b inhibitor group by 82％ (P ＜ 0.01),α-SMA protein expression was reduced in the miRNA-200b inhibitor group by (19 ± 3) ％ (P ＜ 0.05),and the activity of HSCs proliferation was reduced by(33 ± 5)％ (P ＜0.01),and the contents of type Ⅲ procollagen and hyaluronic acid in supernatant were reduced in miRNA-200b inhibitor group by (35 ± 4)％ and (31 ± 2)％,respectively(P ＜0.01).Conclusions The miRNA-200b-specific inhibitor could significantly reduce the expression of miRNA-200b,and inhibit HSC proliferation,activation,and extracellular matrix production.
ABSTRACT
ObjectiveTo observe the influence of lipopolysaccharide (LPS) on collagen metabolism of normal human skin fibroblasts and its biological role in the formation of hypertrophic scar.Methods Fibroblasts were isolated and cultured in vitro,and then exposed to different doses of LPS (0.005,0.01,0.05,0.1,0.5,1.0 μg/ml) from E.coli.055:B5 respectively.The expression of proccllagen type Ⅰ,Ⅲand collagenase mRNAs was tested by RT -PCR.Fibroblasts from hypertrophic scar tissue obtained from the same patients in the same culture passage were used as control.ResultsCompared with control group,the expression of procollagen typeⅠ,Ⅲ mRNAs in normal skin fibroblasts increased (0.323 ± 0.041,0.303 ± 0.063,0.391 ± 0.071,0.344 ± 0.086,0.488 ± 0.059,0.401 ± 0.087,0.616 ± 0.107,0.434 ±0.084,0.823 ±0.092,0.542 ± 0.082),while the expression of collagenase mRNAs of normal skin fibroblasts depressed(0.598 ± 0.068,0.556 ± 0.049,0.441 ± 0.043,0.372 ± 0.083,0.260 ± 0.027 ).When LPS was set to the concentration of 0.005 μg/ml,it showed a concentration dependent manner.However,when the concentration of LPS was set to 0.5 μg/ml,the expression of procollagen type Ⅰ,Ⅲ and collagenase mRNAs of normal skin fibroblasts began to decrease (0.451 ± 0.063,0.374 ± 0.072,0.360 ± 0.062).When the concentration of LPS was set to 1.0 μg/ml,the expression of procollagen type Ⅰ,Ⅲ mRNAs (0.162 ± 0.025,0.171 ± 0.061 )were inhibited and the expression of collagenase mRNAs began to increase (0.444 ±0.114).When the concentration of LPS was set to 0.1 μg/ml,the expression of procollagen type Ⅰ,Ⅲ and collagenase mRNAs of normal skin fibroblasts(0.823 ±0.092,0.542 ±0.082,0.260 ±0.027)was similar to that of hypertrophic scar tissue fibroblasts(0.829 ±0.049,0.569 ±0.038,0.277 ±0.059).ConclusionsThis result supported that LPS may be an important factor in collagen metabolism of normal skin fibroblasts and it plays an important role in hypertrophic scar formation.