OBJECTIVETo investigate the effect of 5-aza-2-deoxycytidine on the TGF-beta/smad signal transduction pathway in human keloid fibroblasts (KFSs).

METHODSFirstly, immunohistochemical method was used to detect the positive expression rate of phospho-smad2 and phospho-smad3 in the specimens of 15 cases of keloid and 15 cases of normal skin. The keloid fibroblasts were cultured in vitro with 5-aza-2-deoxycytidine(experimental group) or with DMEM (control group). The effect of 5-aza-2-deoxycytidine on the cell cycle and apoptosis of fibroblasts was analysed with flow cytometry ( FCM). Transforming growth factor (TGF)-beta1, Smad7, phospho-smad2 and phospho-smad3 were analyzed by Western Blot, and Immunofluorescence.

RESULTSIt was found that the positive expression of phospho-smad2 and phospho-smad3 in keloid were higher than those in normal skin. The FCM showed that the proportion of cells in G0/G1 stage was increased, and so does the proportion of apoptosis cells in keloid fibroblasts intervened by 5-aza-2-deoxycytidine. The expression of TGF-beta1, phospho-smad2 and phospho-smad3 protein were significantly suppressed while the expression of smad7 protein increased in keloid fibroblasts with 5-aza-2-deoxycytidine. In addition, 5-aza-2-deoxycytidine reversed phosphorylation and nuclear translocation of smad2 and smad3.

CONCLUSIONS5-aza-2-deoxycytidine, methylase inhibitors, inhibits cell proliferation and promotes apoptosis of KFSs, which may be associated with the suppression of TGF-beta/smad signal pathway.

Apoptosis ; drug effects ; Azacitidine ; analogs & derivatives ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Enzyme Inhibitors ; pharmacology ; Female ; Fibroblasts ; drug effects ; metabolism ; Humans ; Keloid ; metabolism ; pathology ; Male ; Signal Transduction ; drug effects ; Smad2 Protein ; metabolism ; Smad3 Protein ; metabolism ; Smad7 Protein ; metabolism ; Transforming Growth Factor beta ; metabolism

Objective To investigate the expression and significance of DNA methyltransferase 1 (DNMT1) in different parts and formation time of keloid fibroblasts.Methods Specimen were collected from fifty-three patients with keloid.The keloids were grouped according to formation time.Immunohistochemistry was used to assay the expression of DNMT1 in normal skin,invasive part,proliferative part and aged part.RT-PCR was adopted to assay the expression of DNMT1 mRNA in each group.Results DNMT1 is mainly distributed in the nucleus of fibroblasts.The expression of DNMT1protein and DNMT1mRNA in keloid (72％) is higher than that in normal skin fibroblasts (8％) (P =0.001).The expression rate of DNMT1 protein and DNMT1 mRNA in shorter formation time group (100％,Score:10.47 ± 1.85) is higher than a longer formation time group(45％,Score:7.71 + 1.80) (P=0.007).The expression rate of DNMT1 protein and DNMT1 mRNA in the invasive part of keloid (100％,Score:10.47 ± 1.85) is higher than the proliferative part (78％,Score:6.63 ± 1.75) and aged part(38％,Score:5.53 ±1.55) (P=0.001).Conclusions DNMT1 plays an important role in keloid.The expression of DNMT1 tends to decrease along with the increase of the formation time in keloid.It is also related to the part of keloid.DNMT1 plays an important role in the process of keloid growth and is closely related to infiltrative growth in keloid.

Objective To investigate the expression and significance of DNA methyltransferase 1 (DNMT1) in different parts and formation time of keloid fibroblasts.Methods Specimen were collected from fifty-three patients with keloid.The keloids were grouped according to formation time.Immunohistochemistry was used to assay the expression of DNMT1 in normal skin,invasive part,proliferative part and aged part.RT-PCR was adopted to assay the expression of DNMT1 mRNA in each group.Results DNMT1 is mainly distributed in the nucleus of fibroblasts.The expression of DNMT1protein and DNMT1mRNA in keloid (72％) is higher than that in normal skin fibroblasts (8％) (P =0.001).The expression rate of DNMT1 protein and DNMT1 mRNA in shorter formation time group (100％,Score:10.47 ± 1.85) is higher than a longer formation time group(45％,Score:7.71 + 1.80) (P=0.007).The expression rate of DNMT1 protein and DNMT1 mRNA in the invasive part of keloid (100％,Score:10.47 ± 1.85) is higher than the proliferative part (78％,Score:6.63 ± 1.75) and aged part(38％,Score:5.53 ±1.55) (P=0.001).Conclusions DNMT1 plays an important role in keloid.The expression of DNMT1 tends to decrease along with the increase of the formation time in keloid.It is also related to the part of keloid.DNMT1 plays an important role in the process of keloid growth and is closely related to infiltrative growth in keloid.