The Research Of DcR3siRNA Induce The Apoptosis Of Human Hepatocellular Carcinoma Cells Via Activating Caspase-dependent Mitochondrial Pathway

ObjectivesTo investigate the mechanism of vitro inhibition and apoptosis induced by DcR3siRNA on human hepatocellular carcinoma HepG2cells.MethodsqRT-PCR was used to detect DcR3expression of hepatocellular carcinoma cells.The proliferation of HepG2cells was detected by MTT assay. Light microscopy wasused to detect the morphological changes by DcR3siRNA on human hepatocellularcarcinoma HepG2cells. Cells were treated with rhodamine123（Rh123） and PIpyridine（PI）double staining, cell mitochondrial membrane potential and apoptosisrate was measured by flow cytometry （FCM）. The activity of Caspase-9was detectedby Caspase-9Colorimetric Assay Kit after the human hepatocellular carcinomaHepG2cells treated with the DcR3siRNA. Western blot method was used to detectthe expression of proteins related to mitochondrial apoptosis signal transductionpathway, such as Cytochrome c, Caspase-9, Caspase-3, Bax and Bcl-2in HepG2cellsafter treated with the DcR3siRNA.ResultsThe expression of DcR3mRNA was3.76times in the HepG2cells than the L-02cells. DcR3siRNA inhibited proliferation of HepG2cells significantly in a time-anddose-dependent manner was detected by MTT. The OD values were （0.26±0.03）,（0.33±0.03） and （0.35±0.02） after transfect to40μmol·L-1DcR3siRNA at24h,48hand72h; compared with the control group, respectively, the difference wasstatistically significant （p<0.05）; and the OD value were （0.21±0.03）,（0.34±0.03） and（0.38±0.04） after transfect to DcR3siRNA（20μmol·L-1、40μmol·L-1and80 μmol·L-1）at24h,48h,72h and control the NC24h; compared with the control group,respectively, the difference was statistically significant （p<0.05）. Morphologicalobservation had found that cells presented characteristic changes of apoptosis,including cell rounding, cytokinesis strong eosinophilic, karyopyknosis and deeplystained, and nuclear chromatin are accumulated in the periphery of nuclear membrane.Rh123/PI double staining FCM detection showed that the HepG2cells of Rh123fluorescence intensity decreased and apoptosis rate increased after treated by40μmol·L^-1DcR3siRNA for12h,24h and48h in a time-dependent manner, comparedwith the control group, the difference was statistically significant （p<0.05）.40μmol·L-1DcR3siRNA combination of Ac-LEHD-CHO group, Vanilla aldehyde, thespecific inhibitor of Caspase-9, are treated with24h, the Rh123fluorescence intensityand apoptosis rate compared with single use of DcR3siRNA treated group, thedifference was statistically significant （p<0.05）. DcR3siRNA fostered the activity ofCaspase-9in a time-dependent manner, tested by Caspase-9Colorimetric Assay Kit,the active value of the caspase-9were （5.4±0.4）,（7.8±0.5） and （8.9±0.7） times thancontrol group after treated by40μmol·L-1DcR3siRNA for12h,24h and48h, andAc-LEHD-CHO could suppress the activity of Caspase-9by DcR3siRNA. Westernblot detect results show that40μmol·L-1DcR3siRNA promoted the releasing of Cyt c,the expression of Bcl-2protein was decreased, while Ac-LEHD-CHO could blockthese function.ConclusiosDcR3siRNA could inhibit the proliferation of human hepatocellular carcinomaHepG2cells and induce it apoptosis via activated Caspase-dependent mitochondrialsignal transduction pathway.