目的

KRT5基因是Dowling-Degos病（DDD）的致病基因，但仅在角质形成细胞表达的KRT5导致色素异常的机制未知。本课题拟以人永生化角质形成细胞（HaCaT）、MNT-1黑素瘤细胞、人原代黑素细胞、DDD患者皮损为研究对象，应用慢病毒shRNA及CRISPR/Cas9技术构建KRT5基因沉默及定点突变的HaCaT细胞模型，通过与MNT-1黑素瘤细胞及原代黑素细胞共培养，高通量测序构建RNA表达谱，筛查差异蛋白，探究Notch信号通路及黑素代谢相关分子表达的改变，同时应用Notch抑制剂及激动剂证实黑素代谢相关重要分子的改变，并在具有KRT5基因突变的DDD患者皮损中进一步验证Notch信号通路及黑素代谢重要分子的表达改变，明确KRT5基因突变导致DDD色素异常的机制。

方法

1. 应用慢病毒shRNA和CRISPR/Cas9技术分别获得KRT5基因表达改变的HaCaT细胞，分别应用qPCR和WB方法检测细胞中Notch配体及Notch下游因子表达水平的改变。

2. 将KRT5基因表达改变的HaCaT细胞分别与原代黑素细胞或MNT-1黑素瘤细胞共培养，通过高通量测序构建RNA表达谱，并qPCR和WB方法检测黑素细胞Notch信号通路及黑素代谢相关分子表达的改变，通过NaOH裂解法、透射电镜、免疫荧光等方法检测黑素细胞黑素生成的情况。

3. 原代黑素细胞或MNT-1黑素瘤细胞单独培养，或与KRT5基因改变的HaCaT细胞共培养，应用DAPT或sJAG1多肽抑制Notch信号通路，丙戊酸（VPA）激活Notch信号通路，慢病毒过表达Notch信号通路下游N1ICD分子等方法，干预Notch信号通路，qPCR、WB、免疫荧光等方法验证Notch信号通路对黑素细胞黑素代谢的影响。

4. 在具有KRT5基因突变DDD患者皮损中，通过免疫组化的方法验证Notch信号通路及黑素生成相关分子表达水平的变化。

结果

1. 在慢病毒shRNA稳定敲降KRT5基因的HaCaT细胞模型中，各干扰组KRT5 mRNA相对表达量均显著降低（均P＜0.05）；各干扰组KRT5 蛋白相对表达量降低，其中sh-KRT5-3组蛋白表达量降低最明显；Sh-KRT5-3组Jag1表达量降低，N1ICD表达量下降。在CRISPR/Cas9技术构建KRT5基因c.1delA突变的HaCaT细胞模型中，Sanger测序结果示实验组HaCaT细胞KRT5基因第一外显子起始密码子发生杂合突变（c.1delA），实验组KRT5蛋白表达量降低，Jag1表达量降低，N1ICD、Hes1表达量降低。

2. 慢病毒沉默KRT5基因的HaCaT细胞与MNT-1黑素瘤细胞共培养72h后，流式分选获得MNT-1细胞进行转录组测序，KEGG分析显示Notch信号转导与黑色素代谢相关因子存在差异趋势。实验组MNT-1细胞Notch信号通路下游因子Hes1 mRNA水平较对照组下降，差异具有统计学意义（P＜0.05）。实验组N1ICD蛋白表达量降低，Hes1蛋白表达量降低。实验组黑素转运相关因子Fascin1 mRNA表达水平较对照组明显下降，差异具有统计学意义（P＜0.01）。实验组黑素合成相关因子MITF、TYR、TYRP1、TYRP2蛋白表达量升高，黑素转运相关因子MYO5A、Rab27A蛋白表达量升高，黑素转运相关因子Fascin1蛋白表达量下降。透射电镜显示与sh-KRT5共培养的MNT-1黑素瘤细胞中3期和4期黑素小体增多。CRISPR/Cas9编辑KRT5基因c.1delA的HaCaT细胞与HEMn共培养72h后，免疫荧光分析显示，与实验组c.1delA HaCaT细胞共培养的HEMn细胞的N1ICD的表达量下降，代表黑素含量的Pmel17表达量增加。

3. WB结果显示，在20μM sJAG1条件下刺激HEMn细胞，细胞中N1ICD和Hes1蛋白的水平降低，TYR蛋白表达升高，Fascin1蛋白表达降低。在10μM DAPT处理的HEMn细胞中，N1ICD和Hes1蛋白表达量降低，TYR蛋白表达量升高，Fascin1蛋白表达量降低。在sh-KRT5组HaCaT细胞与MNT-1细胞共培养体系的培养基中加入0.1mM VPA，分选获得的MNT-1黑素瘤细胞中MITF、TYR、Rab27A表达下降，Fascin1的表达逆转至与sh-NC组相近的水平。与c.1delA HaCaT细胞共培养的HEMn细胞，0.1 mM VPA刺激组的Pmel17荧光表达强度接近与对照组水平。

4. 两例具有KRT5基因突变的DDD患者皮损中，免疫组化结果显示表皮中Notch配体Jag1表达量降低，Notch信号通路活化的胞内段N1ICD表达量降低，Notch信号通路下游Hes1表达量降低，且与黑素转运相关分子Fascin1表达量降低，黑素生成相关分子Pmel17表达量升高。

结论

1. 慢病毒沉默KRT5基因的HaCaT细胞模型，以及KRT5基因第一外显子起始密码子杂合突变（c.1delA）导致KRT5蛋白单倍体功能不足的HaCaT细胞模型均构建成功。角质形成细胞KRT5通过干扰Notch配体Jag1蛋白的表达，影响角质形成细胞Notch信号下游N1ICD和转录因子Hes1的表达。

2. 角质形成细胞KRT5基因的改变通过抑制Notch信号通路进而活化色素生成相关蛋白TYR、TYRP1、TYRP2和MITF，导致黑素细胞黑素合成增加，并通过影响Fascin1蛋白的表达进而影响黑素小体的转运。

3. sJAG1多肽与DAPT均可通过抑制黑素细胞Notch信号通路，促进黑素产生、抑制黑素转运。使用VPA激活Notch信号通路可以逆转角质形成细胞KRT5基因的改变对黑素细胞黑素代谢的影响。过表达N1ICD可激活黑素合成并影响黑素转运。

4. 角质形成细胞KRT5通过影响Notch信号通路而引起黑素细胞黑素代谢的异常。

Objective

KRT5 gene is a pathogenic gene for Dowling-Degos disease (DDD), but the mechanism of KRT5 expressed only in keratinocytes leads to pigmentation abnormalities is unknown. In this study, HaCaT cells, MNT-1 melanoma cells, human primary melanocytes, and lesions of DDD patients were selected as research objects, and lentivirus shRNA and CRISPR/Cas9 technology were used to construct HaCaT cell models of KRT5 gene silencing and site-directed mutation. By co-culture with MNT-1 melanoma cells and primary melanocytes, RNA-seq were constructed by high-throughput sequencing, and differential proteins related to Notch signaling pathway and melanin metabolism were screened to explore the changes. Notch inhibitors and agonists were used to confirm the changes of important molecules related to melanin metabolism. The Notch signaling pathway and molecules of melanin metabolism were verified in the skin lesions of DDD patients with KRT5 gene mutation, to clarify mechanism of DDD pigment abnormality caused by KRT5 gene mutation.

Methods

1. HaCaT cells with altered expression of KRT5 were obtained by lentivirus shRNA and CRISPR/Cas9, and the expression levels of Notch ligand and Notch downstream were detected by qPCR and WB.

2. HaCaT cells with altered expression of KRT5 were co-cultured with primary melanocytes or MNT-1 melanoma cells, respectively. RNA-seq were constructed by high-throughput sequencing, and changes of Notch signaling pathway and melanin-metabolisation-related molecules were detected by qPCR and WB. The melanin production was detected by NaOH lysis, transmission electron microscopy and immunofluorescence.

3. Notch signaling pathway inhibitor DAPT or sJAG1 peptide and activator valproic acid (VPA), and lentivirus overexpression of N1ICD molecules were used to intervene Notch signaling pathway when primary melanocytes or MNT-1 melanoma cells were cultured or co-cultured with HaCaT cells with KRT5 gene modification. qPCR, WB and immunofluorescence were used to verify the impact on melanin metabolism of melanocytes.

4. In lesions of DDD patients with KRT5 gene mutation, immunohistochemical methods were used to verify the changes of Notch signaling pathway and melanin production-related molecules.

Results

1. In HaCaT cell model of KRT5 knockdown by lentivirus shRNA, KRT5 mRNA in all interference groups was significantly decreased (P＜0.05), the relative expression of KRT5 protein decreased in all interference groups, and Sh-KRT5-3 decreased most obviously. Jag1 and N1ICD expression were decreased in sh-KRT5-3 group. In HaCaT cell model with c.1delA mutation of KRT5 gene constructed by CRISPR/Cas9, Sanger sequencing showed that heterozygous mutation (c.1delA) occurred in initiation codon of first exon of KRT5 gene in experimental group, the expression of KRT5 protein in experimental group was decreased, and the expression of Jag1, N1ICD and Hes1 decreased.

2. HaCaT cell model of KRT5 knockdown by lentivirus shRNA were co-cultured with MNT-1 melanoma cells for 72h, and MNT-1 cells were obtained by flow cytometry for transcriptomic sequencing. KEGG analysis showed a trend of difference between Notch signal and melanin metabolism. Hes1 mRNA in MNT-1 cells in experimental group was lower than that in control group, and the difference was statistically significant (P＜0.05).  The expression of N1ICD protein and Hes1 protein decreased in experimental group.  Expression of Fascin1 mRNA in experimental group was significantly lower than that in control group, with statistical significance (P＜0.01). The expression of MITF, TYR, TYRP1, TYRP2, MYO5A and Rab27A protein increased, and Fascin1 protein decreased in experimental group. Transmission electron microscopy showed stage 3 and 4 melanosomes were increased in MNT-1 cells co-cultured with sh-KRT5. Expression of N1ICD decreased and Pmel17 increased in HEMn cells co-cultured with KRT5 c.1delA HaCaT cells.

3. In HEMn cells stimulated with 20μM sJAG1, the expression levels of N1ICD and Hes1 decreased, TYR increased, and Fascin1 decreased. In HEMn cells treated with 10μM DAPT, the expression levels of N1ICD and Hes1 decreased, TYR increased, and Fascin1 decreased. When 0.1mm VPA was added in co-culture system of HaCaT cells and MNT-1 cells in sh-KRT5 group, the expressions of MITF, TYR and Rab27A in the selected MNT-1 melanoma cells decreased, while the expression of Fascin1 reversed to a similar level to that of sh-NC group. In HEMn cells co-cultured with KRT5 c.1delA HaCaT cells, fluorescence expression intensity of Pmel17 in 0.1mM VPA group was close to control group.

4. In lesions of the two DDD patients with KRT5 gene mutation, immunohistochemical showed the expression of Notch ligand Jag1 decreased, N1ICD and Hes1 decreased, Fascin1decreased, while Pmel17 increased.

Conclusion

1. The HaCaT cell models of KRT5 silenced by lentivirus and KRT5 haploid deficiency with c.1delA were successfully constructed. Keratinocyte KRT5 affected the expression of N1ICD and transcription factor Hes1 by interfering the expression of Notch ligand Jag1.

2. The alteration of KRT5 in keratinocytes led to the increase of melanin synthesis by inhibiting Notch signaling pathway and activating pigment production-related proteins TYR, TYRP1, TYRP2 and MITF, and affects melanin transport by affecting Fascin1.

3. Both sJAG1 and DAPT promoted melanin production and inhibit melanin transport by inhibiting the Notch signaling pathway of melanocyte. Activation of Notch signaling by VPA reverses the effects of keratinocyte KRT5 alteration on melanin metabolism in melanocytes. Overexpression of N1ICD activated melanin synthesis and affect melanin transport.

4. Keratinocyte KRT5 led to abnormal melanin metabolism by influencing Notch signaling pathway.