Effects of lncRNA AFAP1-AS1 on proliferation and invasion of thyroid cancer cells and its mechanisms / 国际肿瘤学杂志

Objective:To explore the effects of long noncoding RNA (lncRNA) actinfilament-associated protein 1-antisense RNA1 (AFAP1-AS1) on proliferation and invasion of thyroid cancer cells and its mechanisms.Methods:Quantitative real time fluorescence PCR (qRT-PCR) was performed to assess the expression of AFAP1-AS1 in normal thyroid cells and thyroid cancer cells. The thyroid cancer cell line WRO was divided into three groups, AFAP1-AS1 silencing group (AFAP1-AS1-siRNA group), negative control group (NT-siRNA group) and blank control group (Blank group). AFAP1-AS1-siRNA group and NT-siRNA group were transfected with AFAP1-AS1 siRNA and NT-siRNA respectively using Lipofectamine? 3000, and Blank group was treated with PBST. The proliferation ability was measured by CCK-8. The invasion ability was measured by Transwell assay. The expression levels of Rho A, Cyclin D1 and MMP-9 protein were measured by Western blotting.Results:The relative expressions of AFAP1-AS1 in normal thyroid cell line FRTL-5, thyroid cancer cell lines SW579, CAL-62, FRO and WRO were 1.03±0.04, 2.95±0.17, 5.31±0.35, 7.26±0.49 and 9.67±0.53 respectively, and the difference among the five groups was statistically significant ( F=16.932, P=0.027). The expression of AFAP1-AS1 was highest in WRO cells, therefore, the WRO cells were selected for subsequent experiments. The relative expressions of AFAP1-AS1 in AFAP1-AS1-siRNA group, NT-siRNA group and Blank group were 0.23±0.02, 1.02±0.04 and 1.03±0.05 respectively, and the difference among the three groups was statistically significant ( F=13.590, P=0.006). Compared with NT-siRNA group, the expression of AFAP1-AS1 in AFAP1-AS1-siRNA group was significantly lower ( P<0.001). The A450 values in the three groups were 0.68±0.06, 1.17±0.09, 1.22±0.09, and 0.96±0.08, 1.69±0.11, 1.72±0.12 respectively 3 and 4 days after transfection, and the differences were statistically significant ( F=7.318, P=0.016; F=10.351, P=0.004). The differences between AFAP1-AS1-siRNA group and NT-siRNA group 3 and 4 days after transfection were statistically significant ( P=0.043; P=0.013). The numbers of invasive cells in the three groups were 72.8±5.7, 145.6±8.9, 148.4±7.3, and the difference was statistically significant ( F=37.273, P=0.034). The number of invasive cells in AFAP1-AS1-siRNA group was significantly less than that in NT-siRNA group ( P=0.021). The expressions of Rho A protein in the three groups were 0.34±0.03, 1.02±0.04 and 1.04±0.03 respectively, and the difference was statistically significant ( F=9.667, P=0.013). The expression of Rho A protein in AFAP1-AS1-siRNA group was significantly lower than that in NT-siRNA group ( P=0.018). The expressions of Cyclin D1 protein in the three groups were 0.52±0.04, 1.03±0.02 and 1.05±0.04 respectively, with a statistically significant difference ( F=15.464, P=0.010). The expression of Cyclin D1 in AFAP1-AS1-siRNA group was significantly lower than that in NT-siRNA group ( P=0.023). The expressions of MMP-9 protein in the three groups were 0.42±0.04, 1.05±0.03 and 1.02±0.04 respectively, and the difference was statistically significant ( F=10.328, P=0.032). The expression of MMP-9 in AFAP1-AS1-siRNA group was significantly lower than that in NT-siRNA group ( P=0.035). Conclusion:The silencing of lncRNA AFAP1-AS1 can inhibit the proliferation and invasion of thyroid cancer cells, and the mechanism may be related to the down-regulation of Rho A, Cyclin D1 and MMP-9 proteins.

Analysis of TCIRG1 gene mutation in a Chinese family affected with infantile malignant osteopetrosis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To detect potential mutation of the TCIRG1 gene in a boy with infantile malignant osteopetrosis.</p><p><b>METHODS</b>Target sequence capture and next-generation sequencing were applied for the proband and his parents to identify the causative mutation, and Sanger sequencing was used to verify the suspected mutation.</p><p><b>RESULTS</b>The proband manifested at 4 months of age with symptoms including anemia, thrombocytopenia, hepatosplenomegaly, and cephalus quadratus. X-ray revealed generalized increased bone density. A novel compound heterozygous mutation, c.796G to T (p.E266X) and c.1372G to A (p.G458S), were identified in the boy. His father and grandmother also carried the c.796G to T (p.E266X) mutation, and his mother carried the c.1372G to A (p.G458S) mutation. Neither mutation was found in the PubMed and ClinVar databases.</p><p><b>CONCLUSION</b>The novel compound heterozygous mutation c.796G to T (p.E266X) and c.1372G to A (p.G458S) probably underlies the disease in the proband. Above results may enrich the mutation spectrum of the TCIRG1 gene and provide new evidence for the molecular basis of infantile malignant osteopetrosis.</p>

The expression features and clinical significance of CD4+ CD25+ regulatory T cells in elderly patients with acute myelocytic leukemia / 中华老年医学杂志

Objective To investigate the expression features and clinical significance of CD4+ CD25+ regulatory T cells in elderly patients with newly diagnosed acute myelocytic leukemia.Methods Totally 65 patients newly diagnosed as acute myeloid leukemia (AML group) and 72 healthy volunteers (control group) were divided into the elderly group (aged over 60 years) and the young group aged (44.6±2.9) years.The expression of CD4+ CD25+ regulatory T cells was detected by CD4,CD25 and CD127 three-color fluorescein-labeled and multiparameter flow cytometry.The expression features and clinical significance of CD4+ CD25+ regulatory T cells in each group were analyzed.Results After being gated on CD4+ lymphocytes,the expression level of CD4+ CD25+regulatory T cells was significantly increased in AML group as compared to control group [(7.06±2.60) ％ vs.(5.61 ± 1.06) ％,t =4.19,P=0.000].The expression level of CD4 + CD25 + regulatory T cells was higher in elderly AML patients than in elderly controls [(7.55 ± 2.78)％ vs.(5.98 ±1.08) ％,t=3.42,P=0.001].The expression of CD4+ CD25+ regulatory T cells was higher in young AML patients than in young controls [(6.09±1.91)％ vs.(5.14±0.82)％,t=2.21,P=0.036].The expression level of CD4+ CD25+ regulatory T cells was higher in elderly AML patients than in young AML patients [(7.55±2.78)％ vs.(6.09±1.91)％,t=2.19,P=0.032].Conclusions The dual roles of immunosenescence and tumor may cause the excessive accumulation of CD4+ CD25+regulatory T cells in elderly patients with newly diagnosed acute myeloid leukemia.