Investigation Of The Roles Play By SiRNA Targeting K17 In Keratinocytes

Psoriasis is a familiar chronic inflammatory dermatosis, for which three key factors are certain genetic background,infection and cellular immunity disorder with T-cell playing the key role. Keratin 17 (K17) is the type-1 keratin of Mr46000,whose abnormal expression in Psoriasis vulgaris and interaction with T-cell are attracting more and more attention in recent years. Studies show that, K17 possesses several T-activating sites, through which, the T-cell can be activated, caused into hyperplasia to release cellular factor of Thl family, especially inflammation factor 7-IFN, the expression of K17 can also be increased so as to build up a circuit between Kl 7 and psoriasis. As a result, T-cells are activated in succession, causing keratinocyte's abnormal hyperplasia and K17 expression, and consequently cause cellular immunity reaction toward K17. Therefore, abnormal expression of K17 might be a key factor in the occurrence and development of Psoriasis. Taking K17 gene as a new target site for psoriasis treatment, and blocking out or inhibiting the expression of K17 so to cut off the "K17—T-cell circuit'by means of molecular technologies might be a newdirection for future psoriasis treatment.This paper aims to make use of RNAi to block out the expression of K17 in keratinocytes so to lay foundation for this new target for psoriasis treatment. For RNAi, an oligo RNA targeting K17 gene is designed and synthesized to construct stable vector expressing siRNA, which is put into cell by transfection.Main experiments and results include:I. Constructing human K17 RNAi vector and transfected keratinocyte cell line1. Constructing human K17 RNAi vectorAccording to the requirements of psilencer3.1-Hl neo vector, choose two 19nt target sequences, and synthesize four 65-base hairpin oligo RNAs. The oligo RNAs are annealed respectively with linear vector to form double strand oligo RNA which is sequenced subsequently. The combined plasmid are named psilencer3.1/K17—1 and psilencer3.1/K17—2 respectively.2. Cell transfection and building up keratinocyte cell line with stable expression of siRNAK17 siRNA vectors and negative control are transfected into keratinocyte cell line HaCaT by liposome. Filter cell by G418 48h after transfection. 2 weeks later, single clone is picked out and cultured for 2 months to build up keratinocyte cell line with stable expression of siRNA. The cell lines are named HaCaT-K17~1,HaCaT—K17—2 and HaCaT-neg (negative control)respectively.3. Identifying the transfected cellsResults of RT-ECR and western blotting show, with no remarkable difference at the P-actin expression, high level expression of K17 can be seen in the blank and control groups, while the K17 expression is remarkably reduced in the siRNA transfected groups.II. Effect of siRNA targeting K17 gene on keratinocyte's proliferation and apoptosis1. Effect of siRNA targeting K17 gene on keratinocyte's proliferationFor transfected groups, the cell proliferation is observed to be remarkably reduced by means of blood cell counting and MTT, compared with the normal keratinocyte, while the control group is not affected. The results of cell cycle experiment show that, the percent of G1 phase cells are much higher and the percent of S phase cells are much lower for transfected cells, which is a consequence of Gl blocking.2. Effect of siRNA targeting K17 gene on keratinocyte's apoptosisDouble staining of PI and Annexin V in Flowcytometry experiment shows that, percents of cell death and apoptosis for transfected group both are higher than the control group. Observation through transmission electron microscopy illustrates there are more apoptosis cells and apoptosis bodies. In addition, in cell cycle experiment, sub-diploid peak(apoptosis peak) can be observed in the transfected group.3. Effect of siRNA targeting K17 gene on keratinocyte's morphographyObservation through inverted microscopy shows that, for the transfected group, from the 1st day after transfection, the cells begin to show changes in morphography. Some dark particles occur in cytoplast, and many cells begin to crimple to die. The transfected cells'metabolism is abnormal with less K17 expression.In this study, RNAi is used to contruct successfully siRNA-expressing plasmid and it is proved that in transfected cells, the mRNA and protein expression both are reduced. Based on this, it is proved through cell proliferation and cell cycle assays, keratinocyte's proliferation is remarkably inhibited with apoptosis after K17 gene silencing.It can be seen from the above results, the expression of K17 is closely related to keratinocyte's proliferation, and K17 might have important value...