The effect of p21 on transcription of survivin in hepatocellular carcinoma HepG2 cells and its regulation mechanism / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To observe the inhibitory effect of cyclin-dependent kinase inhibitor p21 on regulation of survivin transcription in human liver cancer HepG cells, and explore the related mechanisms.</p><p><b>METHODS</b>Doxorubicin (DOX) was used to treat HepG cells. Eukaryotic vector pEGFP-C2-p21 was transfected into HepG cells by lipofectamine and positive clones were screened out by G418. The mRNA expression of p21, p53 and survivin were detected by real-time fluorescent quantitative polymerase chain reaction (RQ-PCR). Flow cytometry was used to determine the cell cycle phases, and reverse transcription polymerase chain reaction (RT-PCR) was used to measure the levels of E2F-1 or p300.</p><p><b>RESULTS</b>After treatment with DOX, the expression of p53 and p21 was increased, whereas that of survivin was reduced during 24 hours of the treatment. After transfection the p21 level was 2100.1-fold or 980.9-fold enhanced in comparison with that in HepG2 cells or HepG2-pEGFP cells. Survivin level was markedly down-regulated to 0.5% or 0.6% relative to that in the other two groups, nevertheless, significant p53 changes were not observed. Overexpression of p21 resulted in G1/G0 phase arrest (F = 31.59, P < 0.01), meanwhile, E2F-1 mRNA or p300 mRNA were less expressed compared with that in the other controls (F(E2F-1) = 125.28, P < 0.05; Fp300 = 46.01, P < 0.01).</p><p><b>CONCLUSION</b>p21 could be a potential mediator of survivin suppression at transcription level in HepG2 cells, which might be through the block at G1/G0 phase and down-regulation of transcription factors E2F-1 and p300.</p>

Linkage disequilibrium analysis of -1516, -574 and 4259 single nucleotide polymorphisms in TIM-3 gene / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the frequencies of -1516,-574 and 4259 single nucleotide polymorphisms (SNPs) of T cells immunoglobulin mucin -3(TIM-3) gene in Hubei population and address the question whether they are in linkage disequilibrium(LD) .</p><p><b>METHODS</b>Genotypes and allele frequencies of TIM-3 gene were examined by allele-specific polymerase chain reaction (AS-PCR) methods in 147 healthy Hubei Han individuals. Hardy-Weinberg equilibrium and Two-point LD analyses and haplotype frequencies were evaluated with Arlequin v3.1 software.</p><p><b>RESULTS</b>The allele frequencies of the 3 SNPs were in agreement with Hardy-Weinberg equilibrium. Minor allelic frequencies of TIM-3 -1516G/T,-574T/G and 4259G/T were 8.5%,1.0% and 2.0%,respectively. The dominant haplotypes comprising the three loci were G-G-G(2.0%),G-G-T(88.4%), T-G-T(8.5%) and G-T-T(1.0%). LD analyses revealed that all of the coefficient of linkage disequilibrium (D') were 1.</p><p><b>CONCLUSION</b>The -1516,-574 and 4259 loci of TIM-3 gene are in complete linkage disequilibrium. Our study has provided population genetic data on TIM-3 gene in Chinese Hubei Han population and a basis for searching immune-mediated disease-related TIM-3 haplotype.</p>

Construction, identification and expression of recombinant eukaryotic vector pEGFP-BMI-1 / 中国实验血液学杂志

This study was purposed to construct and identify the mammalian expression vector of pEGFP-BMI-1 and to detect whether it could express in human cervix cancer cell line HeLa. The cDNA fragment of BMI-1 obtained by RT-PCR was inserted into pEGFP-N1. The recombinant plasmid was confirmed by restriction enzyme digestion, PCR and DNA sequencing. pEGFP-BMI-1 was transfected into HeLa cells with lipofectamine 2000. The expression of pEGFP-BMI-1 was determined by EGFP fluorescence and Western blot analysis. SYBR Green I real-time RT-PCR was used to quantitate P16INK4a mRNA. The results showed that the correct construction of the recombinant plasmid pEGFP-BMI-1 has been shown by restriction enzyme digestion, PCR and DNA sequencing. pEGFP-BMI-1 could express BMI-1-EGFP fusion protein in HeLa cells. Real-time RT-PCR showed that P16INK4a mRNA expression was reduced to 9.2%. It is concluded that the vector of pEGFP-BMI-1 has been successfully constructed and it can be expressed in HeLa cells. This work has laid foundations for further study on biological functions and potential application of BMI-1.

Establishment and optimization of real-time PCR with SYBR Green Ⅰ for quantification of survivin / 中华检验医学杂志

Objective To establish real-time PCR with SYBR Green Ⅰ for quantification the gene of survivin.Methods The components and conditions of PCR system were optimally determined by fluorescence intensity,cycle threshold(Ct),melting curve,coefficiency and slope of the standard curve.The means to eliminate the contaminating fluorescence of primer-dimers and the mode for Ct value determination were also optimized.Using the developed PCR system,we quantificated the survivin gene in 43 patients with gastric carcinoma.Results The optimized condition for PCR amplification of survivin were 2 mmol/L of MgCl_2,2.5 U/100 ?l of Taq DNA polymerase,0.2 ?mol/L of primers,and the optimized annealing temperatures for PCR were 58℃.The influence of primer dimmer can be eliminated by setting the fluorescence collecting temperature below the Tm of the specific amplicon by 2℃.The second derivative maximum mode,instead of fit point mode,was a feasible method to determine the Ct value for quantification.The sensitivity of this method was 10 copies/?l,and a good linearity was found from 10~1 to 10~4 copies/?l(r = 0.999 7).The inter-experimental coefficient of variation was 1.13%-1.91%,whereas the coefficient of variation between runs was 3.31%-4.50%.Using the optimized PCR system,we quantificated the gene of survivin,the result indicated that survivin gene was amplified in 13.9% of gastric carcinomas.Conclusions The optimal real-time PCR with SYBR Green Ⅰ,as a cost-effective and feasible DNA quantitative method,is fit for quantification of the survivin with satisfactory repeatability and high sensitivity.