FOXP3 gene is associated with susceptibility to ischemic stroke in the Chinese population.

AIM: Immunoinflammatory response plays an important role in the pathophysiological process of ischemic stroke (IS). Forkhead box P3 (FOXP3) is a master regulator for immune cells. Polymorphisms of FOXP3 gene might contribute to the susceptibility of IS. This study aimed to explore the association between FOXP3 gene polymorphisms (rs3761548 and rs2232365) and IS susceptibility in the Chinese Han population. METHODS: Polymerase chain reaction and Sanger sequencing were used to detect the genotype of FOXP3 gene rs3761548 and rs2232365 polymorphisms. RESULTS: Smoking, diabetes mellitus (DM), and HBP histories, higher TG and HDL-C levels were more frequently observed in IS patients than in controls. In comparison with rs3761548 GG genotype, GT genotype (OR = 1.573, 95 %CI = 1.030-2.402; adjusted: OR = 1.736, 95 %CI = 1.070-2.817) and GT + TT vs. GG model (OR = 1.581, 95 %CI = 1.0449-2.382; adjusted: OR = 1.720, 95 %CI = 1.074-2.755) of rs3761548 polymorphism was significantly correlated with elevated ischemic stroke susceptibility both at prior and after adjusted by smoking, HBP, DM, TG and HDL-C. Recessive model of rs2232365 polymorphism could elevate the susceptibility of ischemic stroke (OR = 11.962, 95 %CI = 1.144-3.3363; adjusted: OR = 1.876, 95 %CI = 1.016-3.463). Besides, rs3761548 dominant model (OR = 2.757, 95 %CI = 1.379-5.552; adjusted: OR = 2.601, 95 %CI = 1.268-5.336) and rs2232365 recessive model (OR = 3.103, 95 %CI = 1.463-6.583; adjusted: OR = 3.545, 95 %CI = 1.600-7.855) were related to the severity of ischemic stroke. CONCLUSION: FOXP3 gene rs3761548 and rs2232365 polymorphisms were risk factors for susceptibility and severity of IS.

Reversal of multidrug resistance by magnetic Fe3O4 nanoparticle copolymerizating daunorubicin and MDR1 shRNA expression vector in leukemia cells.

In many instances, multidrug resistance (MDR) is mediated by increasing the expression at the cell surface of the MDR1 gene product, P-glycoprotein (P-gp), a 170-kD energy-dependent efflux pump. The aim of this study was to investigate the potential benefit of combination therapy with magnetic Fe(3)O(4) nanoparticle [MNP (Fe(3)O(4))] and MDR1 shRNA expression vector in K562/A02 cells. For stable reversal of "classical" MDR by short hairpin RNA (shRNA) aiming directly at the target sequence (3491-3509, 1539-1557, and 3103-3121 nucleotide) of MDR1 mRNA. PGC silencer-U6-neo-GFP-shRNA/MDR1 called PGY1-1, PGY1-2, and PGY1-3 were constructed and transfected into K562/A02 cells by lipofectamine 2000. After transfected and incubated with or without MNP (Fe(3)O(4)) for 48 hours, the transcription of MDR1 mRNA and the expression of P-gp were detected by quantitative real-time PCR and Western-blot assay respectively. Meanwhile intracellular concentration of DNR in K562/A02 cells was detected by flow cytometry (FCM). PGC silencer-U6-neo-GFP-shRNA/MDR1 was successfully constructed, which was confirmed by sequencing and PGY1-2 had the greatest MDR1 gene inhibitory ratio. Analysis of the reversal ratio of MDR, the concentration of daunorubicin (DNR) and the transcription of MDR1 gene and expression of P-gp in K562/A02 showed that combination of DNR with either MNP (Fe(3)O(4)) or PGY1-2 exerted a potent cytotoxic effect on K562/A02 cells, while combination of MNP (Fe(3)O(4)) and PGY1-2 could synergistically reverse multidrug resistance. Thus our in vitro data strongly suggested that a combination of MNP (Fe(3)O(4)) and shRNA expression vector might be a more sufficient and less toxic anti-MDR method on leukemia.

[Construction and identification of Mdr-1-shRNA eukaryotic expression vector].

This study was purposed to construct and identify the short hairpin RNA (shRNA) eukaryotic expression vector for targeting gene mdr-1 which may play an important role in K562/A02. Short hairpin RNA (shRNA) aiming at the target sequence was to synthesized, the 3491-3509, 1539-1557and 3103-3121 nucleotide of mdr-1 mRNA were selected as targets. The selected nucleotides were cloned in the plasmid pGCSilencer-U6-neo-GFP respectively, and the resultant recombinant plasmids were named as pGY1-1, pGY1-2 and pGY1-3. The sequences of the recombinant plasmids were identified by DNA sequencing and PCR electrophoresis. The recombinant plasmids were transfected into the cell line K562/A02 by lipofection. After being transfected for 48 hours, the inhibition of mdr-1 mRNA was detected by real time-PCR, and P-gp expression was detected by Western blot. The results showed that the specific oligonucleotide was cloned into the vector successfully, and the expression of mdr-1 mRNA and P-gp in K562/A02 cells was reduced after transfecting the recombinant plasmid, as compared to the control group. It is concluded that the shRNA eukaryotic expression vector has been successfully established which can inhibit the expression of mdr-1 mRNA, setting up the basis to futher explore the effects of mdr-1 on cell line of K562/A02.