Effect of microRNA-181b-5p on the proliferation and invasion of cutaneous melanoma cells and its mechanisms / 中华皮肤科杂志

Objective:To explore whether microRNA (miRNA) -181b-5p inhibits the proliferation and invasion of cutaneous melanoma cells by targeting pleckstrin (PLEK) .Methods:Bioinformatics methods were used to analyze cutaneous melanoma-associated core genes; dual-luciferase reporter assay was performed to verify the targeted interaction between miRNA-181b-5p and PLEK. Oligo RNA and small interfering RNA (siRNA) were used to regulate the expression of miRNA-181b-5p and PLEK in A375 cells respectively in this experiment, and A375 cells were divided into the following groups in detail: mimic negative control group, miRNA-181b-5p mimic group, inhibitor negative control group, miRNA-181b-5p inhibitor group, PLEK siRNA group, siRNA negative control group, miRNA-181b-5p inhibitor + control siRNA co-transfection group and miRNA-181b-5p inhibitor + PLEK siRNA3 co-transfection group. After 48-hour treatment, qPCR was performed to determine the mRNA expression of miRNA-181b-5p and PLEK in A375 cells, Western blot analysis to determine the PLEK protein expression, and Transwell assay to assess the invasive ability of A375 cells; after additional 24-96 hours of culture, cell counting kit-8 (CCK8) assay was conducted to assess the proliferative ability of A375 cells.Results:PLEK was the core gene for cutaneous melanoma. PLEK expression in the cutaneous melanoma in situ tissues was significantly higher than that in the paracancerous tissues ( P = 0.031) , but lower than that in the metastatic tissues ( P = 0.001) . Compared with human epidermal melanocytes HEMa-LP, the mRNA and protein expression of PLEK significantly increased in A375 cells (mRNA: 3.884 ± 0.156 vs. 0.997 ± 0.010, t = 18.48, P < 0.001; protein: 2.840 ± 0.301 vs. 1.029 ± 0.094, t = 5.47, P = 0.005) , but the miRNA-181b-5p expression significantly decreased in A375 cells (0.333 ± 0.042 vs. 0.967 ± 0.069, t = 7.83, P = 0.001) . Dual-luciferase reporter assay showed targeted binding of miRNA-181b-5p to PLEK. Compared with the mimic negative control group, the miRNA-181b-5p mimic group showed significantly decreased survival rate of A375 cells (48 hours: t = 7.96, P = 0.015; 72 hours: t = 7.50, P = 0.002; 96 hours: t = 7.96, P = 0.001) , and significantly decreased invasive ability of A375 cells ( t = 5.07, P = 0.007) ; on the contrary, the survival rate and invasive ability of A375 cells were significantly higher in the miRNA-181b-5p inhibitor group than in the inhibitor negative control group (survival rate: 24 hours, t =5.38, P = 0.013; 48 hours, t = 5.36, P = 0.013; 72 hours, t =7.63, P = 0.005; 96 hours, t = 5.99, P = 0.004; invasive ability: t = 7.24, P = 0.002) ; compared with the siRNA negative control group, the proliferative and invasive ability of A375 cells significantly decreased in the PLEK siRNA group (proliferative ability: 48, 72, 96 hours, P = 0.015, 0.011, 0.001, respectively; invasive ability: t = 4.93, P = 0.008) ; compared with the miRNA-181b-5p inhibitor + control siRNA co-transfection group, the miRNA-181b-5p inhibitor + PLEK siRNA co-transfection group showed significantly decreased proliferation rate and invasive ability of A375 cells (proliferation rate: 24, 48, 72, 96 hours, P = 0.042, 0.042, 0.037, 0.017, respectively; invasive ability: t = 8.52, P = 0.001) . Conclusion:miRNA-181b-5p can inhibit the proliferation and invasion of cutaneous melanoma A375 cells, likely by down-regulating the PLEK expression.

Expression and the role of HIF-1alpha and VEGF in human nasal epithelial cells / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To observe the expression and the role of hypoxic inducible factor-1alpha and VEGF induced by infection factor, inflammatory factor and hypoxia in primary human nasal epithelial cell (HNEC).@*METHOD@#Epithelial cells of nasal polyps (NP) and inferior turbinate (IT) were cultured without serum under stimulus of LPS10, 100, 1000 microg/L, IL-1beta 20 microg/L and hypoxia for 3 h, 6 h, 9 h, respectively. The expression of HIF-1alpha and VEGF protein and mRNA derived from epithelial cells was detected by immunocytochemistry and in situ hybridization.@*RESULT@#1) Under hypoxia, EPO mRNA was expressed intensely in epithelial cells from NP and IT, and there was no significant difference between both of them. This result suggested that EPO might be regarded as a hypoxic mark; 2) The expression of HIF-1alpha and VEGF increased under the stimulus of infection factor, inflammatory factor and hypoxia with the increasing of time and concentration, especially in hypoxia (P<0.05); 3) The expression of HIF-1alpha and VEGF expressed more intensively in epithelial cells from NP than IT under all conditions (P<0. 05); 4) There was positive correlation between the expression of HIF-1alpha and VEGF (r=0.870, P<0.05).@*CONCLUSION@#Epithelial cells actively produce vast HIF-1alpha and VEGF under infection factor, inflammatory factor and hypoxia. HIF-1alpha and VEGF are involved in the formation of nasal polyps.

Expression and correlation of MMP-1 ,TNF-α in cholesteatoma / 中国基层医药

Objective To observe the expression of MMP-1, TNF-a in cholesteatoma and to determine their roles in the destruction of bone and their correlation. Methods Immunohistochemical method and the computer image quantitative analysis were used to examine the expression of TNF-α and MMP-1 in 22 cases of chotesteatomamiddle ear and 20 cases of normal external acoustic meatus skin. Results Positive stainings of MMP-1 and TNF-α were both localized in cytoplasm. The MMP-1 positive cells were found in all strata of cholesteatoma epithelium and active multiplication stromal cell. TNF-α was expressed in both epithlium and stromal cells. The results of the computer image quantitative analysis showed that the mean optical density of MMP-1 (0. 2013±0. 0106) and TNF-α (0.3852±0.0318) in cholesteatoma were higher than that in normal skin epithelial tissue( P＜0.05 ). Conclusion (1)MMP-1 and TNF-α are overexpressed in cholesteatoma. (2)MMP-1 and TNF-α have a correlation in their expression. (3)MMP-1 and TNF-α are both observed in stromal cells which indicates that stromal cells play an irnportant role in bone destruction.

Inhibiting the expression of proinflammatory factors in human nasal epithelial cells by dexamethasone / 中国耳鼻咽喉头颈外科

OBJECTIVE To observe the inhibition of the expression of HIF-1? and VEGF induced by LPS in primary human nasal epithelial cell (HNEC) by dexamethasone. METHODS Epithelial cells of nasal polyps (NP) and inferior turbinate (IT) were cultured without serum under stimulus of LPS 100 ng/ml, IL-1? 20 ng/ml, LPS 100 ng/ml +dexamethasone 13 ng/ml and IL-1? 20 ng/ml+ dexamethasone 13 ng/ml for 3h, 6h and 9h respectively. The expression of HIF-1?, VEGF protein and mRNA derived from epithelial cells was detected by immunocytochemistry and situ hybridization. RESULTS ①The expression of HIF-1? and VEGF increased under the stimulus of LPS and IL-1? with the increasing of time and concentration, especially under LPS 100 ng/ml for 6h (P