关键词: 化脓性汗腺炎, 基因沉默, 细胞增殖, PSENEN基因, γ分泌酶, HaCaT细胞

Abstract: 【Abstract】 Objective To establish a presenilin enhancer-2 （PSENEN） gene-silenced human immortalized keratinocyte （HaCaT） cell model, and to evaluate the effect of PSENEN gene silencing on the proliferation of and γ-secretase expression in HaCaT cells. Methods Three shRNAs targeting the PSENEN gene were constructed, and inserted into the linearized LV3-pGLV-h1-GFP-puro vector to establish a recombinant lentiviral expression plasmid. After restriction enzyme digestion and sequencing, lentiviral packaging and purification were performed, and lentiviral titer was determined. Cultured HaCaT cells were divided into 5 groups: shRNA1, shRNA2 and shRNA3 groups treated with the lentivirus solutions containing PSENEN gene-targeted shRNA1, shRNA2 and shRNA3 respectively, NC group treated with the lentivirus solution containing a negative control shRNA （shNC）, and blank group treated without lentivirus solution. After transfection, inverted fluorescence microscopy was performed, and transfection efficiency was determined by flow cytometry. Cell counting kit-8 （CCK8） assay was performed to evaluate the effect of PSENEN gene silencing on the proliferation of HaCaT cells, and real-time fluorescence-based quantitative PCR （qPCR） and Western blot analysis were conducted to determine the mRNA and protein expression of PSENEN, nicastrin （NCT）, presenilin-1 （PS1） and anterior pharynx defective 1a （APH1a） genes respectively. Statistical analysis was carried out by using repeated measures analysis of variance, one-way analysis of variance, and least significant difference t test for multiple comparisons. Results Inverted fluorescence microscopy showed that fluorescence was observed in the shRNA1 group, shRNA2 group, shRNA3 group and NC group, and flow cytometry showed that the transfection efficiency was over 98% in the above 4 groups. qPCR and Western blot analysis revealed that the mRNA and protein expression of PSENEN gene significantly decreased in the shRNA1 （0.187 ± 0.010, 0.219 ± 0.097, respectively）, shRNA2 （0.163 ± 0.022, 0.208 ± 0.014, respectively） and shRNA3 （0.174 ± 0.009, 0.185 ± 0.062, respectively） groups compared with the NC group （1.054 ± 0.272, 1.076 ± 0.075, respectively, all P < 0.001）. CCK8 assay showed that the cellular proliferative activity significantly increased in the shRNA1 group compared with the NC group at 0, 12, 36 and 48 hours （all P < 0.05）, and there was no significant difference between the 2 groups at 24 or 60 hours （both P > 0.05）; the cellular proliferative activity was significantly higher in the shRNA2 and shRNA3 groups than in the NC group at 0, 12, 24, 36, 48 and 60 hours （all P < 0.05）. There was no significant difference in the mRNA expression of NCT, PS1 and APH1a genes among the shRNA1 group, shRNA2 group, shRNA3 group, NC group, and blank group （F = 8.168, 4.644, 1.981, respectively, all P > 0.05）, while the relative protein expression level of mature NCT （mNCT）, immature NCT （imNCT）, carboxyl-terminal fragment of PS1 （PS1-CTF） and APH1a significantly differed among the above 5 groups （F = 39.268, 5.929, 27.842, 20.663, respectively, all P ≤ 0.01）. Compared with the NC group, the shRNA1, shRNA2 and shRNA3 groups all showed significantly decreased protein expression of mNCT, PS1-CTF and APH1a （all P < 0.01）, but insignificant changes in imNCT protein expression （all P > 0.05）. Conclusion The PSENEN gene-silenced HaCaT cell model was successfully constructed, and the PSENEN gene silencing could lead to an increase in the cellular proliferative activity of HaCaT cells and a decrease in the protein expression of γ-secretase subunits mNCT, PS1-CTF and APH1a.

Key words: Hidradenitis suppurativa, Gene silencing, Cell proliferation, PSENEN gene, γ-Secretase, HaCaT cells