[Apoptosis of human keloid fibroblast induced by small interfering RNA-mediated CyclinD1 gene silencing].

OBJECTIVE

To study the inhibition effect of CyclinD1 specific small interfering RNA(siRNA) on CyclinD1 gene expression in human keloid fibroblast, investigating the effect of CyclinD1 specific siRNA (siRNA-CyclinD1) on the cell cycle, multiplication and apoptosis.

METHODS

According to the principle of siRNA design, siRNA-CyclinD1 was designed and the keloid fibroblast were transfected. RT-PCR was used to examine CyclinD1 mRNA expression. Flow cytometry was used to examine cell cycle. The apoptotic rate was analyzed by using Annexin V-FITC/PI kit. The DNA gragmentation were measured by DNA ladder analysis.

RESULTS

After transfection, the expression of CyclinD1 mRNA decreased remarkably. Twenty-four, forty-eight and seventy-two hours after transfection, the radio of G1 stage cell was (59.80 +/- 3.06)%, (66.01 +/- 4.03)% and (67.43 +/- 5.35)%, all significantly higher than in the control group (54.50 +/- 5.35)%; the radio of S stage cell was (18.40 +/- 1.42)%, (17.21 +/- 1.76)% and (11.07 +/- 1.00)%, significantly lower than in the control group (22.33 +/- 1.49)%; the proportion of the cells in G1 stage increased and those in the S stage decreased in the keloid fibroblast transfected with siRNA-CyclinD1. The apoptotic rate of the siRNA-CyclinD1 group was (7.82 +/- 0.45)%, (15.71 +/- 1.06)%, (18.32 +/- 1.08)%, all significantly higher than in the control group (0.68 +/- 0.12)%, and the DNA gragmentation can be seen remarkably.

CONCLUSIONS

Chemically synthesized siRNA- CyclinD1 effectively inhibits. The expression of CyclinD1 in keloid fibroblast thus arresting the cell cycle at G1 stage and enhancing cell apoptosis. Our study provided a preliminary results in seaching of a RNAi therapy of keloid.