Effect Of Double-Stranded RNA On Cell Apoptosis And HBV Replication As Well As Effects Of Cell Adhesion On Drugs Induced Cell Apoptosis

Studies have shown that long double-stranded RNA could trigger the activation of interferon (interferon, IFN) defensive pathway to activate the PKR that can form a dimer with dsRNA. On one hand, phosphorylation of eIF-2αcan inhibit the protein synthesis and lead to cell apoptosis; On the other hand, the inhibitor of phosphorylation of transcription factor NF-KappaB can activate NF-KappaB ,bring the activation of IFN-related promote factor and result in the increase of IFN transcription. In order to investigate the mechanism of long double-stranded RNA induced-cell apoptosis, long double-stranded RNA and short double-stranded RNA was transfected into HepG2.215 cells. Quantitative RT-PCR was detected the IFN expression and DNA Fragmentation ELISA was detected the apoptosis. The results showed that the long double-stranded RNA increased levels of IFN expression and apoptosis in the cells, while the short double-stranded RNA did not cause the increased levels of IFN expression and apoptosis. RNA interference is endogenous or exogenous double-stranded RNA (dsRNA) that degradates of homologous mRNA specificlly to silencing the expression of target genes in the cellular .dsRNA is cut into 21-23nt of the short-chain dsRNA by Dicer, which is called small interfering RNA (siRNA), then form the RISC with Ribozyme to identify and degradate homologous mRNA in the cells. We selected the dsRNA and siRNA to transfect the HepG2.215 cells. HBsAg and HBeAg in the supernatant were measured by ELISA. HBsAg and HBeAg were decreased compared with the control group, suggesting that RNAi could inhibit HBV replication.In the transfected process as described previously, we found the situation of tightly adherent cells had an impact on the apoptosis in the following transfected process. Therefore we hypothesized that strength of cell adhesion could affect the cell apoptosis. To confirm that hypothesis, IFN and 5 - fluorouracil were chosen to stimulate HepG2 and HEK293 cells treated by different adhesion respectively. It was found that culture plate would enhance the growth capacity of adherent cells after precoated by polylysine and the cells respond to drug sensitivity, proliferation inhibition rate and apoptosis rate were decreased. When fibronectin (Fn), an important component of the Fn antibody blocking matrix, was added to the unadherent cells, the cells respond to drug sensitivity, proliferation inhibition rate and apoptosis rate were increased. However, PKR levels were increased by Western in the groups by IFN treatment, but there were no significant difference among the groups.We make the conclusion by previous studies and our experiment:1.long double-stranded RNA can activate PKR system that results in apoptosis and inhibits HBV replication by RNAi. 2. short double-stranded RNA can not activate PKR system that results in no apoptosis but inhibits HBV replication by RNAi.3. interference with cell - matrix adhesion can enhance interferon, 5 - fluorouracil sensitivity in order to inhibit cell proliferation and contribute to apoptosis.4.IFN can enhance the phosphorylation of PKR, but not affected by adhesion.