Transcriptome Analysis Of Metformin Against UVA-induced Skin Photoaging

Ultraviolet radiation is one of the main causes of skin aging caused by exogenous factors.About 95%of the ultraviolet rays in sunlight are long wavelength ultraviolet A(UVA)with wavelengths between 315 and 400 nm.UVA has strong penetration ability and can penetrate the epidermal layer of the skin to reach the dermis,seriously damaging the deep layers of the skin and causing skin photoaging.Serious skin photoaging will lead to skin diseases such as actinic keratosis and malignant melanoma.With the deepening understanding of skin photoaging,the prevention and treatment of skin photoaging are becoming increasingly urgent.Therefore,it is particularly important to deeply analyze the transcriptome differences of skin photoaging and find specific therapeutic drugs to inhibit skin photoaging.The research team published earlier shows that the hypoglycemic drug metformin has a significant effect on inhibiting skin photoaging,which has been confirmed in human foreskin fibroblast photoaging model,mouse photoaging model,and guinea pig photoaging model.Therefore,this study will continue to explore the target genes of metformin inhibiting UVA induced skin photoaging through transcriptome analysis,Provide new targets for the treatment of UVA induced skin photoaging.Purpose:1.Establish a UVA photoaging model of C.elegans and explore its feasibility.2.Using the previously established photoaging model of human foreskin fibroblasts,transcriptome analysis was conducted to explore the mechanism of metformin’s resistance to UVA induced skin photoaging from the perspective of big data.Methods:1.Construct a photo aging model of N2 type C.elegans,control the irradiation time,and set 5 different UVA irradiation dose gradients,which are 0,2.5,7.5,15,and 30J/cm~2,respectively;2.Daily observation and recording of the death status of each group of C.elegans,and statistics of the average and longest lifespan;3.DCFH-DA staining was used to evaluate the ROS levels in each group of C.elegans;4.Fluorescence microscope was used to detect the effect of UVA irradiation on lipofuscin accumulation in C.elegans;5.The RNA of human foreskin fibroblasts was extracted by the trizol method,and the cell sample quality of each group was evaluated by the sequence fragment length and GC content ratio of transcriptome sequencing;6.Redundant sequence analysis,gene coverage statistics and transcriptome sequencing of genome structure distribution were used to analyze the transcriptome differences of human foreskin fibroblasts in control group,UVA group,metformin group and UVA+metformin group;7.Using chromosomal sequencing sequence distribution to analyze the effects of UVA group,metformin group,and UVA combined with metformin treatment group on human foreskin fibroblasts;8.The obtained transcriptome data were analyzed by correlation analysis,gene expression difference analysis,differential gene annotation and function enrichment analysis among samples to obtain the key genes and signal pathways of metformin inhibiting UVA photoaging.Results:1.The statistical results of the lifespan of C.elegans show that the death time of the nematode gradually advances with the increase of UVA irradiation dose,indicating that C.elegans is sensitive to UVA irradiation dose.2.The ROS detection results showed that compared with the control group,the ROS levels of C.elegans were significantly increased in the irradiation dose groups of 0,2.5,7.5,15,and 30 J/cm2,and there was a dose-dependent effect.In the group with a UVA irradiation dose of 30J/cm~2,the ROS level in the body of C.elegans was the highest,indicating that UVA irradiation significantly increased the ROS level in the body of C.elegans in a dose-dependent manner.3.The accumulation of lipofuscin was used to evaluate the photoaging of C.elegans.The results showed that compared with the control group,the level of lipofuscin in the7.5 J/cm~2 irradiation group was significantly increased,and with the increase of UVA irradiation dose,the level of lipofuscin in the C.elegans was significantly increased.The results showed that UVA induced the accumulation of lipofuscin in C.elegans and the occurrence of photoaging in a dose-dependent manner.4.The transcriptome sequencing results show that the total base number and fragment length of each group of samples meet the requirements,and the proportion of base quality above 20(Q20)is more than 98%,indicating that the quality of the sequencing sequence obtained meets the quality requirements of transcriptome analysis.5.GC content analysis of the obtained base sequences revealed that the GC content(GC%)in all four groups of samples ranged from 50%to 52%,indicating a normal distribution of base differences among the groups.6.The results of redundant sequence analysis,gene coverage statistics,and genome structure distribution showed that the content and proportion of redundant sequences in each group of samples were normal.The significant difference in the number of genes detected between the 90%-100%metformin group and each group indicates that metformin has a significant impact on the gene transcription level of human foreskin fibroblasts.The results of genomic structure analysis showed that both UVA irradiation and metformin can cause changes in the number of exons in human foreskin fibroblasts.7.The sequencing results obtained from each group were analyzed from a chromosomal perspective to investigate the effects of UVA and metformin on human foreskin fibroblasts.The results showed that both UVA and metformin showed no significant chromosomal changes in human foreskin fibroblasts.8.Analyzing the number of common and unique expression genes in the four groups of samples using co expression Wayne diagrams,it was found that a total of 18268 genes were co expressed in the four groups of samples,with each group having over 1000unique differentially expressed genes.Evaluate the correlation index between the four groups of samples using the Heatmap map.The results showed that the correlation between the UVA+metformin group and the control group was higher than that of the UVA group,indicating that metformin has a certain role in reversing UVA induced photoaging.9.The results of differential gene expression analysis showed that there was a significant difference in gene expression in the UVA+metformin group,with a total of3838 genes upregulated compared to the control group.It suggests that metformin regulates the damage effect of UVA on human foreskin fibroblasts at the gene transcription level.10.The results of protein interaction network analysis showed that comparing the interactions between UVA group and UVA+metformin group proteins,ISY1-RAB43,BCL-2,SNX22,etc.were significantly upregulated,while DAG1,MAP1S,GABARAPL1,etc.were significantly downregulated.The nodes of UBC,ATM,MKI67,etc.were larger,indicating that the above proteins play a key role in the inhibition of UVA induced photoaging by metformin.11.Functional annotation and enrichment analysis of differentially expressed genes obtained.GO enrichment analysis is evaluated and analyzed from three aspects:biological processes,cell composition,and molecular function.Comparing the UVA+metformin group with the UVA group,a series of genes involved in biological processes such as fiber assembly pressure regulation,light stimulation response,sterol response,cholesterol response,and regulation of T cell response were significantly downregulated,while the basic cellular composition,including basic structural components within the cell,extracellular matrix,cell membrane,DNA binding and transcriptional repair,MAPK kinase activity,was significantly upregulated,This indicates that metformin can significantly inhibit the damage of UVA to human foreskin fibroblasts,promote the integrity repair of cell structure,and reduce the stimulation of cells to UVA.KEGG signaling pathway analysis found that multiple signaling pathways,including neuroactive ligand receptor interactions,MAPK signaling pathway,Jak STAT signaling pathway,Wnt signaling pathway,and Ras signaling pathway,are associated with metformin inhibiting UVA induced skin photoaging.Conclusion:1.The successful preparation of the photoaging model of the C.Elegans effectively solves the shortcomings of traditional animal photoaging models,such as long preparation time and high economic cost,and provides a new experimental method for studying photoaging.2.Transcriptome analyzed the differential genes and expression of metformin resistance to UVA irradiation induced photoaging of human foreskin fibroblasts,predicted the role of MAPK signaling pathway,Wnt signaling pathway,etc.in this,and pointed out the direction for targeted skin photoaging treatment and drug development.