[Effects of silencing Smad ubiquitination regulatory factor 2 on the function of human hypertrophic scar-derived fibroblasts].
Objective:To explore the effects of silencing Smad ubiquitination regulatory factor 2 (Smurf2) on the secretion of transforming growth factor beta 1 (TGF-β(1)), alpha-smooth muscle actin (α-SMA), and collagen type Ⅰ by human hypertrophic scar-derived fibroblasts.Methods:The human normal skin-derived fibroblasts and hypertrophic scar-derived fibroblasts were cultured with explant culture technique from the normal skin and hypertrophic scar tissue, which was obtained from 9 patients with hypertrophic scars after burn. Two kinds of fibroblasts of the third passage were both divided into 6 groups according to the random number table, with 9 wells in each group. Fibroblasts in blank control group were cultured for 72 h without transfection of any small interfering RNA (siRNA), fibroblasts in negative control group were for cultured for 72 h after transfected with non-target siRNA, fibroblasts in Smurf2 siRNA group were cultured for 72 h after transfected with 100 nmol/L Smurf2 siRNA, fibroblasts in blank control+ TGF-β(1) group were cultured for 72 h without transfection of any siRNA and then treated with 10 ng/mL TGF-β(1) for 6 h, fibroblasts in negative control+ TGF-β(1) group were cultured for 72 h after transfected with non-target siRNA and then treated with 10 ng/mL TGF-β(1) for 6 h, fibroblasts in Smurf2 siRNA+ TGF-β(1) group were cultured for 72 h after transfected with Smurf2 siRNA and then treated with 10 ng/mL TGF-β(1) for 6 h. (1) The protein and mRNA expression levels of Smurf2 of the two kinds of cells in blank control group, negative control group, and Smurf2 siRNA group were assessed by Western blotting and reverse transcription-polymerase chain reaction (RT-PCR), respectively. (2) The content of TGF-β(1) in the cell culture supernatant of the two kinds of cells in blank control group and Smurf2 siRNA group was determined by enzyme-linked immunosorbent assay (ELISA). (3) The protein expression levels of α-SMA of the two kinds of cells in the 6 groups were assessed by Western blotting. The content of collagen type Ⅰ in the cell culture supernatant of the two kinds of cells in the 6 groups was determined by ELISA. (4) The mRNA expression levels of α-SMA and collagen type Ⅰ of the two kinds of cells in the 6 groups were assessed by RT-PCR. The sample numbers of each group in the above experiments were all 9. Data were processed with analysis of variance of factorial design and Bonferroni test.Results:(1) The protein and mRNA expression levels of Smurf2 of the two kinds of cells in Smurf2 siRNA group were significantly lower than those in blank control group and negative control group (withPvalues below 0.05). The protein and mRNA expression levels of Smurf2 of the two kinds of cells in blank control group and negative control group were close (withPvalues above 0.05). (2) The content of TGF-β(1) in the cell culture supernatant of hypertrophic scar-derived fibroblasts in blank control group and Smurf2 siRNA group was respectively (4.34±0.56) and (2.14±0.28) pg/mL, which was significantly higher than (1.52±0.20) and (1.41±0.18) pg/mL of normal skin-derived fibroblasts respectively (withPvalues below 0.05). In hypertrophic scar-derived fibroblasts, the content of TGF-β(1) in the cell culture supernatant in Smurf2 siRNA group was significantly lower than that in blank control group (P<0.05). In normal skin-derived fibroblasts, the content of TGF-β(1) in the cell culture supernatant in Smurf2 siRNA group was close to that in blank control group (P>0.05). (3) The protein expression levels of α-SMA and content of collagen type Ⅰ in the cell culture supernatant of the two kinds of cells in blank control+ TGF-β(1) group were significantly higher than those in blank control group (withPvalues below 0.05). The protein expression levels of α-SMA and content of collagen type Ⅰ in the cell culture supernatant of the two kinds of cells in negative control+ TGF-β(1) group were significantly higher than those in negative control group (withPvalues below 0.05). The protein expression levels of α-SMA and content of collagen type Ⅰ in the cell culture supernatant of the two kinds of cells in Smurf2 siRNA group were close to those in blank control group and negative control group (withPvalues above 0.05). The protein expression levels of α-SMA and content of collagen type Ⅰ in the cell culture supernatant of the two kinds of cells in Smurf2 siRNA+ TGF-β(1) group were significantly lower than those in blank control+ TGF-β(1) group and negative control+ TGF-β(1) group (withPvalues below 0.05). (4) The mRNA expression levels of α-SMA and collagen type Ⅰ of the two kinds of cells in blank control+ TGF-β(1) group were significantly higher than those in blank control group (withPvalues below 0.05). The mRNA expression levels of α-SMA and collagen type Ⅰ of the two kinds of cells in negative control+ TGF-β(1) group were significantly higher than those in negative control group (withPvalues below 0.05). The mRNA expression levels of α-SMA and collagen type Ⅰ of the two kinds of cells in Smurf2 siRNA group were close to those in blank control group and negative control group (withPvalues above 0.05). The mRNA expression levels of α-SMA and collagen type Ⅰ of the two kinds of cells in Smurf2 siRNA+ TGF-β(1) group were significantly lower than those in blank control+ TGF-β(1) group and negative control+ TGF-β(1) group (withPvalues below 0.05).Conclusions:Silencing Smurf2 in human hypertrophic scar-derived fibroblasts can reduce the autocrine of TGF-β(1) and inhibit the TGF-β(1)-induced α-SMA expression and collagen type Ⅰ synthesis.
Department of Burns and Plastic Surgery, Guangzhou Red Cross Hospital, Medical College, Ji'nan University, Guangzhou 510220, China.
Z
Z
Zhang
F
F
Kuang
C L
CL
Liu
B
B
Chen
W B
WB
Tang
X J
XJ
Li
D000199:Actins; D015153:Blotting, Western; D002056:Burns / Q000378:metabolism; D002478:Cells, Cultured; D017439:Cicatrix, Hypertrophic / Q000378:metabolism*; D024042:Collagen Type I; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D012333:RNA, Messenger / Q000378:metabolism; D011897:Random Allocation; D014162:Transfection; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1 / Q000378:metabolism / Q000494:pharmacology*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination
C541116:ACTA2 protein, human; D000199:Actins; D024042:Collagen Type I; D012333:RNA, Messenger; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C471641:SMURF2 protein, human; D044767:Ubiquitin-Protein Ligases
Cicatrix; Fibroblasts; Smad ubiquitination regulatory factor 2; Transforming growth factor beta