研究背景和目的

皮肤衰老是一种由内源性调控机制和外源环境因素刺激共同介导的病理生理过程。常见的临床表现包括皱纹增多、异常色素沉积、皮肤屏障功能破坏、伤口愈合缓慢及良恶性皮肤肿瘤等。紫外线辐照是皮肤外源性衰老最重要因素，也被称为皮肤光老化。非编码RNA（non-coding RNAs，ncRNAs）在维持皮肤结构、皮肤屏障功能及皮肤光老化发病进程中发挥着关键调节作用。 转运RNA衍生小RNA（tRNA-derived small RNAs，tsRNAs）是一类新型的ncRNA，目前tsRNAs在皮肤光老化中的研究鲜有报道，未有就特定tsRNAs的功能进行探索的相关研究。本研究旨在寻找参与调控光老化发生发展过程的关键tsRNAs、明确该tsRNAs的功能，以及通过何种机制参与光老化的调控，并且期望基于此研究可助于发现防治光老化的新型干预靶点。

方法

光老化细胞模型构建及其tsRNAs表达谱的测定

通过UVB辐照人真皮成纤维细胞（human dermal fibroblast，HDF）构建光老化细胞模型，通过细胞衰老b-半乳糖苷酶染色 (senescence-associated b-galactosidase staining，SA-b-gal staining)、荧光定量PCR（qRT-PCR）检测衰老相关分泌表型（senescence associated secretory phenotype，SASP）因子（白介素（interleukin，IL）-1b、-6、-8） mRNA转录水平、蛋白免疫印迹法（western blotting，WB）检测衰老相关标志物（p53、p21、I型胶原蛋白）、EdU掺入实验等方法评估该模型。

通过ncRNA高通量测序及qRT-PCR技术，分析光老化细胞模型中差异tsRNAs表达谱，并筛选差异表达最高的tsRNAs分子。

5’-tiRNA-His-GTG在光老化模型中的生物学功能研究

转染5’-tiRNA-His-GTG模拟物（Mimics）或抑制剂（Inhibitor），观察其对HDF细胞或光老化细胞模型的影响。

通过连续UVB辐照（5周）裸鼠构建光老化小鼠模型，通过苏木精-伊红染色法（hematoxylin-eosin staining，HE染色）、Masson三色染色、经表皮水丢失测定等方法评估模型构建。

利用腺相关病毒构建5’-tiRNA-His-GTG抑制剂，转染该病毒观察其对光老化动物模型的影响。

5’-tiRNA-His-GTG靶向结合并下调核孔蛋白98促进人真皮成纤维细胞光老化

利用蛋白质谱分析光老化细胞模型抑制5’-tiRNA-His-GTG后的蛋白表达谱。

利用生物信息学分析、qRT-PCR、WB、双荧光素酶报告实验检测在HDF细胞中5’-tiRNA-His-GTG与下游靶基因核孔蛋白98（nucleoporin 98, NUP98）之间的调控关系。

构建慢病毒过表达NUP98 HDF细胞模型，观察过表达NUP98对5’-tiRNA-His-GTG过表达细胞模型及光老化细胞模型的影响。

5’-tiRNA-His-GTG靶向结合核孔蛋白98调控c-Jun氨基末端激酶信号通路促进人真皮成纤维细胞光老化

对过表达NUP98 HDF细胞模型的细胞衰老表型进行验证。

使用mRNA转录组测序、生物信息学分析、WB探究过表达NUP98 HDF细胞模型的下游细胞衰老相关的信号通路变化。

使用c-Jun氨基末端激酶（c-Jun N-terminal kinase，JNK）信号通路抑制剂，观察其对5’-tiRNA-His-GTG过表达HDF细胞模型的影响。

结果

光老化细胞模型构建及其tsRNAs表达谱的测定

通过UVB（30mJ/cm²）辐照HDF细胞构建光老化细胞模型，光老化细胞模型中SA-b-gal阳性细胞占比升高、SASP因子IL-1b、-6、-8 mRNA转录水平升高、衰老相关标志物p53、p21升高，I型胶原蛋白降低、细胞增殖能力降低。

光老化细胞模型与对照组之间有265个差异tsRNAs，光老化细胞模型组中差异表达上调的tsRNAs有115个，而表达下调的tsRNAs有150个。5’-tiRNA-His-GTG表达显著升高。

5’-tiRNA-His-GTG在光老化模型中的生物学功能研究

转染5’-tiRNA-His-GTG Mimics诱导HDF细胞发生衰老。在光老化细胞模型中抑制5’-tiRNA-His-GTG可回复HDF细胞的光老化表型。

连续UVB辐照（5周）裸鼠构建光老化小鼠模型，小鼠皮肤皱纹增多，经表皮水丢失程度加重，HE染色中皮肤表皮增厚，Masson三色染色发现真皮内胶原成分减少。小鼠皮肤样本中5’-tiRNA-His-GTG水平升高。

在小鼠皮肤内抑制5’-tiRNA-His-GTG可有效改善连续UVB辐照所导致的皮肤光老化。

5’-tiRNA-His-GTG靶向结合并下调核孔蛋白98促进人真皮成纤维细胞光老化

蛋白质谱分析发现光老化细胞模型抑制5’-tiRNA-His-GTG后42个蛋白质表达水平出现显著性变化。

生物信息学分析、qRT-PCR、WB、双荧光素酶报告实验证明，在HDF细胞中5’-tiRNA-His-GTG靶向结合NUP98 mRNA，进而降低NUP98蛋白水平。

慢病毒过表达NUP98 HDF细胞模型可以显著改善UVB及过表达5’-tiRNA-His-GTG所导致的细胞衰老，即过表达NUP98具有光保护效应。

5’-tiRNA-His-GTG靶向结合核孔蛋白98调控c-Jun氨基末端激酶信号通路促进人真皮成纤维细胞光老化

过表达NUP98后可改善HDF细胞的衰老表型。

过表达5’-tiRNA-His-GTG可上调JNK蛋白磷酸化水平，激活JNK信号通路，过表达NUP98回复了过表达5’-tiRNA-His-GTG所导致的JNK信号通路激活。

使用JNK抑制剂可改善过表达5’-tiRNA-His-GTG所诱导的HDF细胞衰老。

结论

本研究使用UVB辐照构建HDF光老化细胞模型，在模型组中5’-tiRNA-His-GTG表达水平升高，过表达5’-tiRNA-His-GTG可诱导HDF发生细胞衰老。5’-tiRNA-His-GTG通过靶向结合NUP98 mRNA，促进NUP98 mRNA降解，抑制其蛋白表达，进而激活JNK信号通路导致HDF细胞衰老。本研究为光老化的发病提供了新的分子机制和治疗靶点。

Background and Objectives

Skin aging is a pathophysiological process mediated by combined endogenous regulatory mechanisms and exogenous environmental stimuli. Common clinical manifestations include the presence of wrinkles, abnormal pigmentation, disruption of the skin barrier, slow wound healing, and the development of both benign and malignant skin tumors. UV radiation exposure has been identified as the primary factor contributing to exogenous skin aging, also referred to as skin photoaging. Non-coding RNAs (ncRNAs) plays a critical regulatory role in the maintenance of skin structure, skin barrier function, and the pathogenesis of skin photoaging. Transfer RNA-derived small RNAs (tsRNAs) have been identified as a novel class of ncRNAs. However, the existing studies on tsRNAs in skin photoaging are limited in scope, and the functions of specific tsRNAs in skin photoaging remain to be elucidated. The objective of this study is to identify the key tsRNAs involved in the regulation of photoaging, to clarify the functions of these tsRNAs and the mechanisms through which they are involved in the regulation of photoaging, and to identify novel targets for the prevention and treatment of photoaging based on this study.

Methods

Construction of a photoaging cell model and Sequencing of its tsRNAs expression profile.

A photoaging cell model was constructed by exposing human dermal fibroblast (HDF) cells to UVB radiation. This cell model was evaluated by Senescence-associated b-galactosidase staining (SA-b-gal staining), fluorescence quantitative PCR (qRT-PCR) for detection of mRNA expression levels of senescence-associated secreted phenotypic factors (interleukin (IL)-1b, -6, -8), estern blotting (WB) for detection of senescence-associated markers (p53, p21, Collagen type I) and EdU Cell Proliferation Assay.

The expression profiles of differential tsRNAs in the photoaging cell model were analyzed by ncRNA high-throughput sequencing and qRT-PCR. Among these, 5’-tiRNA-His-GTG was identified as the most significantly differential tsRNA.

Biological function of 5’-tiRNA-His-GTG in the photoaging model.

Transfection of 5’-tiRNA-His-GTG Mimics/Inhibitor and observed its effect on HDF cells or photoaging cell models.

A photoaging mouse model was constructed by continuous UVB radiation (5 weeks) in nude mice, and the model was evaluated by hematoxylin-eosin staining (HE staining), Masson's trichrome staining, and trans-epidermal water loss measurement.

An adeno-associated virus was used to construct a 5’-tiRNA-His-GTG inhibitor, and the virus was transfected into the skin of mice to observe its effect on the photoaging mouse model.

5’-tiRNA-His-GTG promotes photoaging in HDF cells by targeting and downregulating nucleoporin 98.

Analysis of protein expression profiles after inhibition of 5’-tiRNA-His-GTG in a photoaging cell model.

Demonstration of the regulatory relationship between 5’-tiRNA-His-GTG and the downstream target gene nucleoporin 98 (NUP98) in HDF cells using bioinformatics analysis, qRT-PCR assay, WB assay, and dual luciferase reporter assay.

Construct lentiviral overexpression of NUP98 HDF cell model and observe the effect of overexpression of NUP98 on 5’-tiRNA-His-GTG overexpression cell model and photoaging cell model.

5’-tiRNA-His-GTG targets NUP98 and activates c-Jun N-terminal kinase pathway to promote fibroblast photoaging.

Validation of the cellular senescence phenotype in the overexpression NUP98 HDF cell model.

Using mRNA transcriptome analysis and WB assay, the changes in the downstream signaling pathways in the overexpression NUP98 HDF cell model were investigated.

Using c-Jun N-terminal kinase (JNK) signaling pathway inhibitor and observing its effect on 5’-tiRNA-His-GTG overexpression cell model.

Results

Construction of photoaging cell model and Sequencing of its tsRNAs expression profile

A photoaging cell model was constructed by exposing HDF cells to UVB (30mJ/cm²) radiation. In the photoaging cell model, the percentage of SA-b-gal staining-positive cells was elevated, the mRNA expression levels of the SASP factors IL-1b, -6, and -8 were elevated, and the senescence-associated protein marker p53 and p21 were elevated, collagen type I was decreased, and cell proliferation was reduced.

There were 265 differential tsRNAs between the photoaging cell model and the control group, with 115 upregulated tsRNAs and 150 downregulated tsRNAs. 5’-tiRNA-His-GTG expression was significantly elevated.

Biological function of 5’-tiRNA-His-GTG in the photoaging model

Transfection of 5’-tiRNA-His-GTG Mimics induced senescence in HDF cells. Inhibition of 5’-tiRNA-His-GTG in a photoaging cell model rescued the photoaging phenotype of HDF cells.

Continuous UVB radiation (5 weeks) in nude mice was used to construct a photoaging mouse model characterized by an increase in skin wrinkles, an increase in the degree of transepidermal water loss, a thickening of the skin epidermis as shown by HE staining and a decrease in collagenous components of the dermis as shown by Masson's trichrome staining.

Inhibition of 5’-tiRNA-His-GTG in mouse skin effectively ameliorates skin photoaging caused by continuous UVB radiation.

5’-tiRNA-His-GTG promotes photoaging in HDF cells by targeting and downregulating nucleoporin 98

Protein profiling revealed that 42 proteins showed significant changes in expression levels after inhibition of 5’-tiRNA-His-GTG in the photoaging cell model.

Bioinformatics analysis, qRT-PCR assay, WB assay, and dual luciferase reporter assay demonstrated that 5’-tiRNA-His-GTG targeted NUP98 mRNA in HDF cells and consequently regulated the protein expression level of NUP98.

Overexpression of NUP98 HDF cell model significantly ameliorated cellular senescence caused by UVB and overexpression of 5’-tiRNA-His-GTG, i.e., overexpression of NUP98 had a photoprotective effect.

5’-tiRNA-His-GTG targets NUP98 and activates c-Jun N-terminal kinase pathway to promote fibroblast photoaging.

Overexpression of NUP98 alleviates the senescence phenotype of HDF cells.

Overexpression of NUP98 inhibited JNK signaling pathway activation caused by overexpression of 5’-tiRNA-His-GTG.

Use of JNK inhibitor alleviated HDF cell senescence induced by overexpression of 5’-tiRNA-His-GTG.

Conclusion

In this study, we used UVB radiation to construct the HDF photoaging cell model, and the expression level of 5’-tiRNA-His-GTG was increased in the model group. Overexpression of 5’-tiRNA-His-GTG induced senescence in HDF cells. 5’-tiRNA-His-GTG induced senescence in HDF cells by targeting NUP98 mRNA, promoting NUP98 mRNA degradation and inhibiting its protein expression, which then activated the JNK signaling pathway. Our findings provide new insights into the molecular mechanisms of photoaging and highlight promising therapeutic targets.