Based On 4D-DIA Proteomics And GC-MS/UPLC-QE-MS Non-Targeted Metabonomics To Explore The Mechanism Of Transformation From Actinic Keratosis To Cutaneous Squamous Cell Carcinoma

Objective:In order to explore the potential molecular mechanism of the progression from Actinic Keratosis(AK)to Cutaneous Squamous Cell Carcinoma(CSCC),we used 4D-data independent acquisition(4D-DIA)proteomics combined with gas chromatography-mass spectrometry / ultra-high performance liquid chromatography-mass spectrometry(GCMS/UPLC-QE-MS)dual-platform non-targeted metabonomics to identify and analyze the differences of proteins and metabolites in the skin of SKH-1 genotypic mice.Thus,we screened out the key differential proteins and metabolites related to the development of UVB-induced AK to CSCC.Combined protein-metabolite analysis was carried out to explore the potential molecular mechanism of the transformation from AK to CSCC.Method:SKH-1 genotype mice were irradiated by UVB for 22 weeks,and the histopathological results of skin lesions were used as the inclusion and exclusion criteria.6 normal mouse skin tissues,6 AK skin tissues and 6 CSCC skin tissues were identified by 4D-DIA proteomics and GC-MS/UPLC-QE-MS metabonomics,and the differential proteins and metabolites significantly related to the disease progression were obtained through screening and bioinformatics analysis.Then,through the joint analysis of protein and metabolite,the results were explored from the expression correlation and metabolic pathway correlation,and the effect on disease progression was analyzed.Result:According to 4D-DIA proteomic results,46 common differential proteins(9 upregulated proteins and 37 down-regulated proteins)were identified by Venn analysis.After integrating the results of GC-MS and UPLC-QE-MS,22 differential metabolites were identified by metabonomics(14 up-regulated metabolites and 8 down-regulated metabolites).After the combined analysis of proteomics and metabolomics results,5differential proteins were found(Ectonucleoside triphosphate diphosphohydrolase 2(Entpd2),Monoglyceride lipase(Mgll),Aldehyde dehydrogenase 3 family member A1(Aldh3a1),Gamma-butyrobetaine dioxygenase(Bbox1)and Calcium-activated chloride channel regulator 3A-1(Clca3a1))and 3 differential metabolites(Adenosine,Aminoadipic Acid and Carnosine)were the most closely related to the occurrence of disease.Then the relevant network diagram was obtained through the KGML analysis.The related metabolic pathways involved were purine metabolism,lysine degradation,histidine metabolism,betaalanine metabolism,regulation of lipolysis in adipocytes and renin secretion.Conclusion:We take the differential metabolites as the key to analyze the tumor promoting effect of high concentration adenosine and low concentration carnosine levels in AK and CSCC.And explore the relevant pathways and protein expression changes.It is found that the persistent low expression of protein Entpd2 in purine metabolic pathway may be the main reason for the extracellular production of high concentration of adenosine in tumor tissues,and the persistent low expression of Entpd2 reveals the enhancement of the invasiveness of AK and CSCC tissues in a certain sense.It is reasonable to believe that Entpd2 is a potential biomarker of the transition from AK to CSCC.At the same time,the changes of Mgll in regulation of lipolysis in adipocytes pathway and renin secretion pathway also promote the high concentration of adenosine accumulation.The low expression of Aldh3a1 in lysine degradation pathway and beta-alanine metabolism pathway is related to the low concentration of carnosine.This study can provide certain molecular biological theoretical basis for further elucidating the mechanism of UVB-induced AK to CSCC transformation.