miR-149-3p suppresses malignant biological behaviors of cervical cancer HeLa cells via targeting FOXP3 / 中国肿瘤生物治疗杂志

@#Objective: To explore the effects of miR-149-3p on the proliferation, apoptosis, invasion and migration of cervical cancer HeLa cells and the possible mechanisms. Methods: HeLa cells were randomly divided into five groups, including untransfected (HeLa) group, mimic-scramble group (the negative control of miR-149 mimic), miR-149 mimic group, FOXP3 over-expression (pc-FOXP3) group, and co-transfection (mimic+pc-FOXP3) group. The targeted relationship of miR-149-3p and FOXP3 was verified by luciferase assay. The expressions of miR-149-3p and FOXP3 mRNA were tested by quantitative real-time reverse transcription PCR (qRT-PCR). The protein levels of FOXP3 were measured by Western blotting. The proliferation was detected by CCK-8; the apoptosis was tested by flow cytometry, the cell invasion was measured by transwell invasion assay and cell migration was detected by scratch assay. Results: The luciferase assay showed that miR-149-3p could target combine with FOXP3. Compared with untransfected group, the expression of miR-149-3p was increased while mRNA level of FOXP3 was decreased in miR-149 mimic group (all P<0.01). Moreover, the protein level of FOXP3 in miR-149 mimic group was lower than that in untransfected group (P<0.01), while the protein level of FOXP3 in pcFOXP3 group was higher than that in untransfected group (P<0.01); Compared with pc-FOXP3 group, the protein levels of FOXP3 in mimic+pc-FOXP3 group were reduced (P<0.01). The proliferation in miR-149 mimic group was lower than that in untransfected group (P<0.01), while the proliferation in pc-FOXP3 was higher than that in untransfected group (P<0.01); compared with pc-FOXP3 group, the proliferation in mimic+pc-FOXP3 group was decreased (P<0.01). The apoptosis rate of HeLa cells in miR-149 mimic group was higher than that in untransfected group (P<0.01), while the apoptosis rate in pc-FOXP3 was lower than that in untransfected group (P< 0.01); compared with pc-FOXP3 group, the apoptosis in mimic+pc-FOXP3 group was elevated (P<0.01). The number of invasive cells per field and wound healing rate in miR-149 mimic group was lower than those in untransfeccted group (P<0.01) while the invasive cells and wound healing rate in pc-FOXP3 group was higher than those in untransfeceted group (P<0.01); compared with pc-FOXP3 group, the number of invasive cells per field and wound healing rate in mimic+pc-FOXP3 group was reduced (P<0.01). Conclusion: miR-149-3p inhibits proliferation, invasion and migration and promotes apoptosis of cervical cancer HeLa cells via targeting FOXP3.

Invasion and metastasis inhibition of Foxp3 on ovarian cancer cells Skov3 and its mechanism / 中国免疫学杂志

Objective:To investigate the effect of Foxp3 on the invasion and metastasis of the ovarian cancer cells Skov 3 and the related mechanism.Methods: Plasmid overexpressing Foxp 3 gene was constructed and transfected into Skov 3 cells using liposome.Proliferation,invasion,RT-PCR and Western blot analysis were done to observe the expression of Foxp 3 gene in Skov3 cells and the invasion and metastasis ability.Also,MMP-2 and MMP-9 gene expression was detected.Results:Overexpression of Foxp3 was confirmed in Skov3 cells after Foxp3 plasmid transfection.Compared with the empty vector group , overexpression of Foxp3 gene significantly inhibited the migration and invasion of Skov 3 cells.Meanwhile , the expression level of MMP-2 and MMP-9 genes were decreased.Conclusion:Upregulation of FoxP3 gene expression can inhibit cell proliferation ,invasion and migration in ovarian cancer Skov3 cells through downregulating MMP-2 and MMP-9 gene expression.This study provided experimental basis for further research on Foxp3 gene and gene therapy for ovarian cancer.

An association study of single nucleotide polymorphisms of the FOXP3 intron-1 and the risk of Psoriasis vulgaris.

Psoriasis vulgaris (PV) is a common autoimmune disease that involves the dysfunction of CD4+CD25+ regulatory T cells. FOXP3 is a key transcription factor in the development and function of CD4+CD25+ regulatory T cells. Previous studies have demonstrated a genetic association between the FOXP3 gene and some autoimmune diseases. To elucidate the association between the FOXP3 gene and the risk of PV, 408 patients diagnosed with PV and 363 age and sex-matched healthy controls from a cohort of the Chinese majority Han population were recruited. Four single nucleotide polymorphisms (rs2232365, rs3761547, rs3761548 and rs3761549) of the FOXP3 gene were analyzed using the polymerase chain reaction and ligase detection reaction. The major allele of three single nucleotide polymorphisms (SNPs — rs2232365 A, rs3761547 A and rs3761549 C) were associated with an increased risk of PV in a clinical subgroup of female patients, who were less than 40 yrs of age, had a family history of the disease and did not have disease complications (p < 0.05 for all parameters). The haplotype was structured between rs3761547 and rs3761549. An increased risk of PV was observed in haplotype A/A-T/T (p = 0.0055; adjusted OR = 3.188; 95% CI = 0.4354-23.34) and A/G-C/C (p = 0.0082; adjusted OR = 1.288; 95% CI = 0.1529-10.85) between rs3761547 and rs3761549. A synergistic effect was found among the three SNPs. Subjects with the rs2232365AA- rs3761547 AG + GG genotype were more susceptible to PV (p = 0.0393; OR = 2.90; 95% CI = 1.05-7.97). No correlation was found between rs3761548 and the onset of PV. Therefore, the FOXP3 polymorphisms appear to contribute to the risk of psoriasis among the Chinese majority Han population. These findings may aid in our understanding of the pathogenesis of psoriasis

FOXP3 gene polymorphisms and hepatocellular carcinoma / 中华普通外科杂志

Objective To investigate the correlation between single nucleotide polymorphisms (SNPs) of FOXP3 gene and the susceptibility of hepatocellular carcinoma (HCC). Methods Two SNPs rs2280883 and rs3761549 of FOXP3 gene in 392 HCC patients and 372 healthy controls were analyzed by Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI-TOF).Results At rs3761549,C allele frequency was significantly higher ( OR =1.32,95％ CI 1.03 -1.70,P =0.027) in HCC patients than healthy controls.Compared with healthy controls,HCC patients had higher frequencies of TT genotype (79.6％ ) at rs2280883 or CC genotype (77.6％) at rs3761549 of FOXP3 gene.Patients carrying rs2280883 TT genotype ( OR =1.53,95％ CI 1.10 - 2.14,P ＜ 0.00001 ) or rs3761549 CC genotype ( OR =1.92,95％ CI 1.39 - 2.64,P ＜ 0.00001 ) were more susceptible to HCC.Stratified analysis showed that rs3761549 CC genotype was significantly associated with higher incidence of portal vein tumor thrombus ( x2 =5.578,P =0.018 ),and rs3761549 TT/CT genotype was significantly associated with higher rate of tumor recurrence in HCC patients (x2 =6.561,P =0.010).Conclusions FOXP3 gene polymorphisms at rs2280883 and rs3761549 might be associated with increased susceptibility to HCC. rs3761549,CC genotype and TT/CT genotype were respectively associated with higher incidence of portal vein tumor thrombus and tumor recurrence in HCC patients.

T Regulatory Cell Responses to Immunization with a Soluble Egg Antigen in Schistosoma mansoni-Infected Mice

The aim of the study is to characterize the phenotypes of CD4+ CD25+ T regulatory cells within the liver granulomas and association with both Foxp-3 gene expression and splenic cytokines. Naive C57BL/6 mice were intravenously injected with multiple doses of the soluble egg antigen (SEA) 7 days before cercarial infection. The immunized and infected control groups were sacrificed 8 and 16 weeks post-infection (PI). Histopathology, parasitological parameters, splenic phenotypes for T regulatory cells, the FOXP-3 expression in hepatic granuloma using real-time PCR, and the associated splenic cytokines were studied. Histopathological examination of the liver revealed remarkable increase in degenerated ova within hepatic granuloma which decreased in diameter at weeks 8 and 16 PI (P<0.01). The percentage of T regulatory cells (CD4+ CD25+) increased significantly (P<0.01) in the immunized group compared to the infected control at weeks 8 and 16 PI. The FOXP-3 expression in hepatic granulomas increased from 10 at week 8 to 30 fold at week 16 PI in the infected control group. However, its expression in the immunized group showed an increase from 30 at week 8 to 70 fold at week 16 PI. The splenic cytokine levels of pro-inflammatory cytokines, IFN-gamma, IL-4, and TNF-alpha, showed significant decreases (P<0.05) compared to the infected control group. In conclusion, the magnitude and phenotype of the egg-induced effects on T helper responses were found to be controlled by a parallel response within the T regulatory population which provides protection in worm parasite-induced immunopathology.

Expressions of RORγt and Foxp3 in peripheral blood of children with atopic dermatitis / 中华皮肤科杂志

ObjectiveTo detect the mRNA expression levels of RORγt and Foxp3 genes in peripheral blood mononuclear cells(PBMCs) of children with atopic dermatitis(AD),and to explore their possible roles in the development of AD.MethodsSixty-three children with AD and 54 age-matched normal children were eligible for this study.Real-time fluorescence-based quantitative PCR was carried out to measure the mRNA expressions of RORγt and Foxp3 genes in PBMCs of these subjects.ResultsThe relative expression levels of Foxp3 and RORγt mRNA were significantly higher in children with AD compared with the normal controls (2.4060 ± 0.3355 vs.1.1852 ± 0.4189,t =17.50,P＜ 0.01; 6.9130 ± 0.2046 vs.2.7501 ± 0.2518,t =98.63,P ＜ 0.01 ).A statistical increase was observed in the level of RORγt mRNA in children with severe AD compared with children with mild and moderate AD(7.1203 ± 0.1056 vs.6.8046 ± 0.1731 and 6.8655 ± 0.3671,t =6.61,5.23,both P ＜ 0.01).No significant difference was noted in the expression of RORγt mRNA between children with mild and moderate AD,or in the Foxp3 mRNA expression among children with mild,moderate and severe AD.The severity of AD was positively correlated with the level of RORγt mRNA (r =0.62,P ＜ 0.01 ),but uncorrelated with the level of Foxp3 mRNA(r =0.04,P ＞ 0.05).ConclusionsThe high expressions of RORγt and Foxp3 genes may be involved in the pathogenesis of AD,and the severity of AD is positively correlated with the expression intensity of RORγt gene.

Detection of peripheral CD4+CD25+ regulatory T (Treg) cells in patients with atopic dermatitis / 中华皮肤科杂志

Objective To explore the action mechanism and clinical significance of CD4+CD25+ regulatory T (Treg) cells in the development of atopic dermatitis (AD). Methods Peripheral blood mononuclear cells (PBMC) were obtained from 46 patients with AD and 20 normal human controls. Flow cytometry was performed to detect the number of CD4+CD25+ Treg cells, real-time fluorescence PCR assay to measure the Foxp3 mRNA level in PBMC, ELISA to determine the serum levels of IL-2, IL-4, IL-10 and IFN-γ. Results A statistical decrease was observed in the percentages of peripheral CD4+CD25+ Treg cells among CD3+ T cells and CD4+ T cells in AD patients compared with normal controls (t' = 3.775, 4.533, both P＜ 0.01 ), and in the percentage of peripheral CD4+CD25+ Treg cells among CD3+ T cells in patients with acute AD compared with those with chronic AD (t = 2.217, P ＜ 0.05), but no significant difference was noted between patients with acute AD and those with subacute AD or between those with subacute AD and those with chronic AD in the percentage of peripheral CD4+CD25+ Treg cells among CD3+ T cells (t = 1.558, 0.49, both P ＞ 0.05). The mRNA level of Foxp3 in PBMC from AD patients was statistically decreased compred with that from normal controls (z =-2.368, P ＜ 0.05 ). The count of CD4+CD25+ Treg cells was positively correlated with serum levels of IL-2 and IL-10 (r = 0.512, 0.494, both P ＜ 0.05), but had no significant correlation with serum levels of IL-4 and IFN-γ (r = -0.110, -0.237, both P ＞ 0.05). Conclusions In AD patients, there is a decrease in the count of CD4+CD25+ Treg cells and in the level of Foxp3 mRNA, which may suppress the proliferation of and secretion of Foxp3 mRNA by Th2 cells, lead to Th2 predominance, participate in the development of AD.