Abstract: 【Abstract】 Objective To evaluate the proliferative activity of and changes in the expression of related differentiation proteins in a stably NCSTN gene-silenced human immortalized keratinocyte cell line HaCaT, and to preliminarily explore the possible mechanism underlying the occurrence of acne inversa. Methods By lentivirus-mediated short hairpin RNA （shRNA）, a NCSTN gene-silenced HaCaT cell model was established （shRNA group）, and other HaCaT cells transfected with empty lentivirus served as a negative control group. Real-time quantitative PCR and Western blot analysis were performed to determine the NCSTN gene-silencing efficiency. Cell counting kit-8 （CCK8） assay was conducted to evaluate the proliferative activity of HaCaT cells, and real-time quantitative PCR and Western blot analysis were performed to determine the mRNA and protein expression of cytokeratins （CK1, CK5, CK7, CK10, CK14, CK16, CK17, CK18, CK19 and CK20） and other differentiation molecules （involucrin and loricrin） respectively in HaCaT cells. Two-independent-sample t test was used to compare the measurement data between two groups. Results NCSTN mRNA and protein expression were significantly lower in the shRNA group （0.42 ± 0.19, 0.30 ± 0.07 respectively） than in the negative control group （1.00 ± 0.34, 1.00 ± 0.26; t = 5.196, 2.637, P < 0.001, < 0.05, respectively）, and the gene-silencing efficiency was 70%. Compared with the negative control group, the shRNA group showed higher cellular proliferative activity, but decreased protein expression of CK16, CK19 and terminal differentiation molecule involucrin（t = 3.787, 3.817, 2.904, P < 0.01, < 0.05, < 0.05, respectively）. Conclusion Stable silencing of NCSTN gene can lead to abnormal proliferation and differentiation of HaCaT cells, which provides new ideas for subsequent exploration of acne inversa caused by NCSTN gene mutation.

Key words: Hidradenitis suppurativa, Gene silencing, Keratinocytes, Cell proliferation, Cell differentiation, Keratins, HaCaT cells, NCSTN gene