Construction Of A Model For Lentivirus-mediated Short Hairpin RNA Silencing NCT Gene In Human Immortalized Keratinocyte Cell Line

Backgrounds:Acne inversa (AI), also known as hidradenitis suppurativa, is a chronic recurrent inflammatory skin disease. In 2010, Wang et al first identified six independent mutations responsible for familial AI in distinct subunits of y-secretase. To date, in the more than 20 reported y-secretase mutations in AI, most mutations are located in NCT and they are all loss-of-function mutations. The role of loss-of-function NCT mutation in the pathogenesis of AI is not clear, which need our further study.Objective:To construct a lentiviral vector for RNA interference (RNAi) of the NCT Gene and identify the silencing efficiency in the human immortalized keratinocyte cell line (HaCaT cells). To provide a model for a stable silenced NCT Gene human immortalized keratinocyte cell line. To lay the fundation for subsequent research on silencing the expression of NCT gene affecting biological behavior of keratinocytes and its role in the pathogenesis of AI.Methods:1. Three specific sequences of shRNA targeting NCT gene were designed and inserted into the pGLV3/Hl/GFP+Puro vector, so as to construct three recombinant plasmids and confirmed by sequencing.2. The recombinant plasmids with the lentivirus packaging plasmids were co-transfected into 293 T cells to obtain lentivirus particles. Then viral titer was determined.3. Then the lentivirus particles infected HaCaT cells, the cells were divided into control group (uninfected), the negative control group (NC group, infected with LV3-shNC) and interference group (infected with NCT-shRNA 1,2,3). The transfection efficiency was detected by flow cytometry and the silencing efficiency of target gene in HaCaT cells was detected by RT-PCR. At last, we can select the most efficient sequence.Results:1. The NCT-shRNA lentiviral recombinant plasmids were constructed successfully that confirmed by DNA sequencing.2. The recombinant plasmids with the lentivirus packaging plasmids were co-transfected into 293T cells. The titer of recombinant lentivirus particles was about 109TU/ml.3. After the lentivirus particles infected HaCaT cells, the transfection efficiency was greater than 95% detected by flow cytometry. Compared with NC group, the expression of NCT in the interference group was significantly down-regulated at mRNA level. Clearly, NCT-shRNA1 was the most efficient sequence with mRNA level decreased by 75%.Conclusion:1. The lentiviral vectors expressing short hairpin RNA which target human NCT gene are successfully constructed.2. The most interference sequence is filtered out. A stable and effectively reduced NCT gene silenced cell model of HaCaT cells was established successfully.