Influence of RNA Interference mediated cyclinD1 Gene Silencing on The Proliferation and G1 Phase Regulators of Fibroblasts Derived From Keloid
- VernacularTitle:RNA干扰Cyclin D1基因表达对瘢痕疙瘩成纤维细胞增殖和G1期调控的影响
- Author:
Dongmei YU
;
Lijun HAO
;
Dongyan WANG
;
Huidong WANG
- Publication Type:Journal Article
- Keywords:
keioid;
RNA interference;
cyclin D1;
CDK4;
pRb;
P16;
cell cycle
- From:
Progress in Biochemistry and Biophysics
2008;35(2):159-169
- CountryChina
- Language:Chinese
-
Abstract:
In order to investigate the effect of sequence-specific small interfering RNA on suppressing cyclin D1 expression and proliferation and cell cycle and expression of G1 phase regulators of fibroblasts derived from keloid, the plasmid expression vector of siRNA targeted against cyclin D1 was constructed and transfected into fibroblasts with LipofectamineTM 2000. The changes of cyclin D1 expression were detected by fluorescent quantitative PCR(FQ-PCR), semi-quantitative RT-PCR. The effect of sequence- specific small interfering RNA in suppressing the proliferation of fibroblasts was detected by MTT assay. Flow cytometry were used for evaluation ofceU cycle. The expression of cyclin D1, CDK4, pRb and P16 was detected by immunohistochemical method. The results showed that: (1) The sequence- specific siRNA effectively suppressed cyclin D1 expression at both mRNA levels with inhibition rate of 63.68% and 92.83% (P<0.01). (2) Significantly inhibited the proliferation of fibroblasts, and changed cell cycle in percentage of G0/G1 phase cells was increased compared with the controls groups in fibroblasts(P < 0.05). (3) 72 h after transfection, the expression of cyclin D1, CDK4 and pRb decreased, and the expression of P16 increased. It can be concluded that the plasmid expression vector for the RNAi against cyclin D1 constructed in the study can effectively and specifically suppress cyclin D1 expression, and progression of G1/S is effected by G1 phase related regulatory protein, and suppresses proliferation of fibroblasts derived fiom keloid.
