ABSTRACT
Objective To determine the expression of caspase-14 in skin lesions of patients with chronic actinic dermatitis (CAD),and to explore the effect of ultraviolet B (UVB) radiation on its mRNA and protein expression in HaCaT cells.Methods In 2016,skin samples were collected from lesions of 10 patients with CAD (test group),10 patients with eczema (positive control group) and from normal skin of 10 healthy controls after cosmetic surgery (negative control group) in the Department of Dermatology,First Affiliated Hospital of Kunming Medical University.Immunohistochemical staining was performed to determine the expression of caspase-14 in the normal skin,CAD and eczema lesions.Cultured HaCaT cells were divided into several groups:UVB groups irradiated with 0,30,60,90 mJ/cm2 UVB separately,and 5-AzaC groups irradiated with 0,30,60,90 mJ/cm2 UVB separately followed by the treatment with the methylase inhibitor 5-AzaC for 24 hours.Then,the cells were collected,and real-time fluorescence-based quantitative PCR (RT-PCR) and Western blot analysis were conducted to determine the mRNA and protein expression of caspase-14 respectively in HaCaT cells in the UVB groups and 5-AzaC groups.Statistical analysis was carried out with SPSS22.0 software by using chi-square test for the comparison of rates,and t test and two-factor analysis of variance for the comparison of means.Results In the CAD and eczema lesions,caspase-14 was mainly expressed in the spinous and granular layers,but not in the stratum comeum.However,caspase-14 was markedly expressed in the stratum corneum of the normal skin tissues.Of the 10 CAD samples,5 were positive for caspase-14,and 9 of 10 normal skin samples were positive for caspase-14.The positive rate of caspase-14 significantly differed between the two above groups (x2 =7.30,P ＜ 0.05).RT-PCR and Western blot analysis showed significant changes in the mRNA and protein expression of caspase-14 in HaCaT cells after irradiation with different doses of UVB (F =87.54,23.46,both P ＜ 0.05),which showed a decreasing trend along with the increase in the dose of UVB.After exposure to 0,30,60 and 90 mJ/cm2 UVB,the mRNA and protein expression of caspase-14 was significantly higher in the 5-AzaC groups than in the UVB groups (all P ＜ 0.05).Conclusions In CAD lesions,the expression of caspase-14 markedly decreased,and was absent in the stratum corneum.UVB radiation can downregulate the mRNA and protein expression of caspase-14 in HaCaT cells.
ABSTRACT
Objective To study the effects of semaphorin 5A (SEMA5A) gene silencing by lentivirus?mediated short hairpin RNA(shRNA)on biological activity of malignant melanoma cell line A375. Methods Two pairs of interference sequences for SEMA5A gene(shRNA1 and A375?shRNA2)and a pair of control interference sequences were designed to build lentiviral vectors, which were then transfected into HEK293T cells to gain lentivirus. A375 cells were divided into three groups:experimental group(A375?shRNA1 and A375?shRNA2 cells)transfected with the lentivirus containing shRNA1 or shRNA2, negative control group (A375?con cells) transfected with that containing the control shRNA, and blank control group(A375 cells)receiving no transfection. The A375 cells with stable knockdown of SEMA5A gene expression were screened by puromycin. Subsequently, reverse transcription?PCR and Western?blot analysis were performed to detect mRNA and protein expressions of Semaphorin 5A in these cells, and methyl thiazolyl tetrazolium(MTT)assay was applied to evaluate the growth of cells. The scratch assay and invasion assay were conducted to estimate migration and invasion ability of cells. Results The lentivirus containing the SEMA5A?targeting shRNAs or control shRNA was successfully transfected into A375 cells, and stably transfected cells were gained after puromycin selection. The expressions of semaphorin 5A mRNA and protein in the A375?shRNA2 cells were significantly reduced compared with those in the A375?con and A375 cells(all P 0.05). The scratch assay showed that there was no obvious cell migration into the scratch in the experiment group, whereas the scratch was almost covered by cells in the negative control group and blank control group. The invasion assay showed that the number of A375?shRNA2 cells passing through the Transwell chamber was significantly smaller than that of A375 and A375?con cells(both P 0.05). Conclusion The silencing of SEMA5A gene by lentivirus?mediated shRNA could effectively down?regulate the expression of semaphorin 5A, and inhibit the growth, invasion and migration of A375 cells.