ABSTRACT
This study examined the effect of subcellular localization of P21 on the proliferation and apoptosis of HepG2 cells.The coding genes of the wild and the mutant P21 were amplified by mega primer PCR from the plasmid pCEP-WAF1 which contains human P21 cDNA in the nuclear localizational signal (NLS) sequence,and then inserted into the eukaryotic expression vector pDsRed1-C1.The recombinants were transfected into HepG2 cells.The transcription and expression of P21 were determined by RT-PCR and fluorescence microscopy.The cell proliferation was measured by MTT,and the cell cycle and apoptosis of HepG2 cells by flow cytometry.The results of restriction analysis,DNA sequencing and fluorescence microscopy confirmed the construction of the wild and the mutant P21 in the eukaryotic expression plasmid.The plasmid containing the mutant P21 was found to accelerate cell proliferation and the wild P21 plasmid to inhibit cell proliferation.Cell cycle analysis showed that the cell ratio of G0/G1 in the wild type group was significantly increased as compared with that in the mutant type group,and cell apoptosis analysis revealed that the apoptosis rate in the wild type group was much higher than that in the mutant type group.It was concluded that the subcellular localization of P21 may contribute to the development of hepatic cancer.
ABSTRACT
This study investigated the influence of silencing TRAF6 with shRNA on lipopolysac-charide (LPS)/toll-like receptor (TLR)-4 signaling pathway in vitro. Four plasmids (pGCsi-TRAF6-shRNA 1, 2, 3, 4) containing different shRNA sequences were designed and synthesized. The proliferation of RAW264.7 cells after transfected with these plasmids was measured by MTT assay. Inflammatory cellular models were established by LPS stimulation. Levels of TNF-α, IL-1β and TGF-β1 in the supernatants, mRNA expressions of TRAF6, IL-6 and COX-2, protein expression of TRAF6 and translocation of NF-κB were assayed by ELISA, real-time quantitative PCR and Western blotting, respectively. The results showed that the TRAF6 gene knockdown by RNAi hardly inhibited the proliferation of RAW264.7 cells within 72 h. The mRNA and protein expression of TRAF6 was lower in the TRAF6-shRNA1, 2 groups than in the TRAF6-shRNA3, 4 groups. Therefore, pGCsi-TRAF6-shRNA1, 2 were selected for the subsequent experiments. Our results still showed that pGCsi-TRAF6-shRNA 1, 2 could significantly reduce the production of pro-inflammatory cyto-kines and mediators including TNF-α, IL-1β, IL-6 and COX-2, and inhibit NF-κB nuclear transloca-tion. Moreover, pGCsi-TRAF6-shRNA1, 2 could suppress the release of TGF-β1 at the protein level. It was concluded that the recombinant plasmid pTRAF6-shRNA can, to some extent, inhibit inflam-matory response stimulated by LPS at the initial phase. TRAF6 may become the potential therapeutic target of many inflammation-related diseases.