Effect of decorin gene overexpression on cell cycle and apoptosis of QBC939 cells

Abstract

Objective To investigate the effects of decorin(DCN)gene overexpression on cell cycle and apoptosis of human cholangiocarcinoma cells. Methods The pEGFP-DCN plasmids and pEGFP-N1 control plasmids were transfected into the QBC939 cells using Lipofectamine 2000. The mRNA and protein expression of decorin in QBC939 cells transfected with plasmid overexpressing decorin was detected by real-time reverse transcriptase-polymerase chain reaction(RT-qPCR) and Western blotting respectively. The growth of QBC939 cells was detected by using colony-forming growth assays. Cell proliferation activity was detected by methyl thiazol tetrazolium(MTT). Cell cycle and apoptosis of cells were analyzed by flow cytometry(FCM). The expression of apoptosis-related protein, cysteinyl aspartate-specific protease-3(Caspase-3)and B cell lymphoma/leukemia-2(bcl-2) was detected by Western blotting. Results Compared to the control plasmids, RT-qPCR showed the mRNA expression of decorin in the pEGFP-DCN plasmids was increased by 32.34 fold.Western blotting showed the protein expression of decorin was increased by 2 fold. The growth of QBC939 cells was inhibited in cells transfected with pEGFP-DCN as compared to the empty vector. MTT assay showed a decrease of QBC939 cells proliferation in cells transfected with pEGFP- DCN as compared to the empty vector. Cell cycle analysis presented an obvious cell- cycle arrest at the G1-G0 phase and a decreased G2- S phase in cells transfected with pEGFP- DCN as compared to the negative control group. Flow cytometry detected an increase of the apoptotic cells in cells treated with pEGFP-DCN as compared to the empty vector.Western blotting identified that cleaved Caspase- 3 was significantly increased,and bcl- 2 was decreased in cells transfected with pEGFP- DCN(0.787±0.068 vs.1.276±0.157). Conclusion The overexpression of DCN gene could significantly promote the proliferation of cholangiocarinoma cells, which may be associated with the overexpression of Caspase-3 and downexpression of bcl-2. Key words: Cholangiocarcinoma; Decorin; Cell cycle; Apoptosis