RESUMO
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 G(0)/G(1) 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.
RESUMO
This study investigated the influence of silencing TRAF6 with shRNA on lipopolysaccharide (LPS)/toll-like receptor (TLR)-4 signaling pathway in vitro. Four plasmids (pGCsi-TRAF6-shRNA1, 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-alpha, IL-1beta and TGF-beta1 in the supernatants, mRNA expressions of TRAF6, IL-6 and COX-2, protein expression of TRAF6 and translocation of NF-kappaB 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-shRNA1, 2 could significantly reduce the production of pro-inflammatory cytokines and mediators including TNF-alpha, IL-1beta, IL-6 and COX-2, and inhibit NF-kappaB nuclear translocation. Moreover, pGCsi-TRAF6-shRNA1, 2 could suppress the release of TGF-beta1 at the protein level. It was concluded that the recombinant plasmid pTRAF6-shRNA can, to some extent, inhibit inflammatory response stimulated by LPS at the initial phase. TRAF6 may become the potential therapeutic target of many inflammation-related diseases.
RESUMO
Quercetin and praziquantel were used to treat mice with hepatic fibrosis due to Schistosoma japonicum infection.Quercetin treatment obviously relieved the degree of hepatic fibrosis,significantly reduced the expression of immediate early gene,tissue inhibitor of metalloproteinase 1(TIMP 1),typesⅠ and Ⅲ collagen compared to the control.The expression of c-jun mRNA,typeⅠ and type Ⅲ collagen were reduced significantly compared to the group treated with praziquantel,whereas no difference in the expression of c-fos mRNA and TIMP1 between the two groups,indicating that quercetin may have better effect on schistosomal liver fibrosis than praziquantel in the long term.