Multiomic Analysis Of Xuanwei Female Lung Adenocarcinoma And The Functional Mechanism Of DHX9 In P53 Mutant Lung Cancer

Objectives:Xuanwei lung cancer shows obvious gender and regional differences,and the incidence rate is higher in females.The pathological types are mainly adenocarcinoma,but its etiology and mechanism are poorly understood.This study aims to reveal the specific molecular characteristics of Xuanwei female lung adenocarcinoma from the multiomic level,so as to provide scientific basis and potential therapeutic targets for clinical diagnosis and treatment of Xuanwei female lung adenocarcinoma.Methods:(1)This study prospectively collected tumor tissues and adjacent normal tissue samples from 169 female patients diagnosed with lung adenocarcinoma in Xuanwei area.All of the 169 women had never smoked(less than 100 cigarettes in their lifetime),had no treatment and were diagnosed with primary lung adenocarcinoma for the first time.(2)Whole exome sequencing,transcriptome sequencing,label-free quantitative proteomics technology,and label-free quantitative phosphoproteomics technology were used to detect 135,136,102,and 102 tumor samples and 135,36,20,and 20 paired normal samples.Subsequently,multiomics analysis was performed.(3)DHX9 may be a potential therapeutic target for patients diagnosed with p53 mutant lung cancer screened by omics analysis.p53 wild-type lung cancer cell line A549 and p53 deletional lung cancer cell line Calu-1 were selected to construct p53 wild type and mutant lentivirus vectors,DHX9 interference and overexpression lentivirus vectors,and lentivirus were packed using packing cell lines to infect A549 and Calu-1 cells.Subsequently,purinomycin was used for screening.(4)q RT-PCR and western blot were used to detect the m RNA and protein expression levels of related genes.(5)The proliferative ability of lung cancer cells was detected by CCK-8 method;The migration ability of lung cancer cells was detected by scratch healing test.Transwell was used to detect the invasion and metastasis of lung cancer cells.Cell cycle and apoptosis of lung cancer were detected by flow cytometry assay.(6)Rescue experiment was used to analyze the regulation of p53 mutation on lung cancer development through DHX9/PCNA/CDK2.(7)The growth promotion effect of DHX9 on p53 mutant lung cancer was investigated in vivo by subcutaneous tumor formation in nude mice.(8)Immunohistochemistry and western blot were used to detect the expression of related proteins in nude mice.(9)TUNEL staining was used to detect apoptosis of tumor cells in nude mice.Results:(1)Whole-exome analysis identified 37149 somatic mutations,including 1797 insertion and deletion mutations,32972 missense mutations,2345 nonsense mutations and 35 nonstop mutations.Copy number analysis revealed 140,396 gene-level amplifications and 67605 gene-level deletions in 40 chromosome segments.The transcriptional profiles of 19182 genes were detected by m RNA-seq.Proteomic and phosphoproteomic analysis identified 1795 proteins and 476 phosphorylated proteins that were upregulated in tumor samples.(2)At the protein level,biological processes such as RNA metabolism,translation and intracellular protein transport are up-regulated,while at the phosphorylation level,biological processes such as chromatin organization,RNA metabolism and DNA metabolism are up-regulated.The protein levels of DHX9 and SF and the phosphorylation level of SF1＿S82 were significantly up-regulated in p53 mutant samples,which were associated with poor prognosis.DHX9 and SF1 may be potential therapeutic targets for p53-mutant lung cancer patients.(3)Compared with EGFR single mutation samples,Notch signaling pathway is significantly disturbed in TP53/EGFR double mutations samples,and down-regulated Notch signaling pathway may weaken the killing ability of effector monocytes.Compared with TP53/EGFR double mutations samples,there was extensive metabolic reprogramming in TP53 single mutation samples,including significant up-regulation of citric acid cycle and respiratory electron transport,metabolism of amino acids and their derivatives,lipid and pyrimidine metabolism,t RNA aminoacylation and other metabolic processes.(4)In the EGFR/RTK-RAS pathway,the double mutations of EGFR and RTK-RAS pathway genes endowed cancer cells with stronger glycolytic ability,and the glycolytic pathway and related enzymes were significantly up-regulated.(5)Through the construction of PPI network interfered by driver mutations,protein-protein interaction analysis was carried out,and multiple protein interactions affected by mutations were found,such as MAD1-MAD2,TPRN-PPP1 CA,etc.Enrichment analysis showed significant enrichment of biological processes such as mitotic cell cycle regulation,signaling by receptor tyrosine kinases,and cellular response to DNA damage stimulus.The MAD1 p.Arg558 His mutation greatly weakened the MAD1-MAD2 interaction,and may lead to the development of lung cancer by inhibiting the separation of sister chromatid during the metaphase to anaphase transition;The TPRN p.His550 Gln mutation also greatly weakened the TPRN-PPP1 CA interaction,which may lead to the dysfunction of serine/threonine specific protein phosphatase,causing the block of dephosphorylation,and ultimately leading to the progression of lung cancer.(6)In the cold tumor subtype,mannoglycosylation,cellular amino acid biosynthesis process,sulfide oxidation,glutamine family amino acid biosynthesis and so on are the most important biological pathways in cold tumor samples.(7)After transfection of A549 and Calu-1 cells with p53 wild-type and mutant lentivirus vectors and DHX9 interference and overexpression lentivirus vectors,DHX9 was successfully interfered and overexpressed tested by fluorescence microscopy and q PCR.After DHX9 knockdown in p53 mutant cells,compared with that in p53 wild-type knockdown group,the cell proliferation ability was significantly weakened,the number of tumor cells invading through matrigel was significantly reduced,the proportion of cell apoptosis was significantly increased and the levels of EMT and VEGF in lung cancer cells were inhibitted.After DHX9 overexpression,the healing ability of p53 mutant group was strengthened significantly compared to that of p53 wild group and the proportion of cells in G1 phase was reduced dramatically.(8)Overexpression or knockdown of DHX9 in p53 mutant A549 cells resulted in corresponding up-regulation or down-regulation of PCNA and CDK2 protein expression.When the overexpression of DHX9 was combined with the addition of PCNA inhibitor,it was found that the expression of CDK2 protein was also down-regulated,and the CCK8 assay showed that the proliferation ability of lung cancer cells was significantly weakened compared with before.(9)Compared with the p53 wild-type group,the tumor volume and tumor weight of nude mice after DHX9 overexpression in the p53 mutant group were significantly increased,E-cadherin was down-regulated,and the expressions of N-cadherin,Vimentin,VEGF,PCNA and CDK2 were up-regulated;the proportion of apoptosis in the tumor of nude mice was significantly increased after DHX9 knockdown in the p53 mutant group.Conclusions:(1)Multiomic analysis of Xuanwei female lung adenocarcinoma identified a large number of mutated genes,dysregulated proteins and downstream biological processes at the level of whole exome,transcriptome,proteome and phosphoproteome,respectively.These differences may have deeper mechanisms.(2)DHX9 and SF1 may be potential therapeutic targets for p53-mutant lung cancer patients.(3)Intervention of dysregulated Notch signaling pathway can be used as an adjuvant therapy for lung cancer patients with TP53/EGFR double mutations.For patients with double mutations of EGFR/RTK-RAS pathway genes,inhibiting the glycolysis pathway of tumor cells may achieve more significant therapeutic effects.(4)The development of inhibitors of the interaction between MAD1-MAD2 and TPRN-PPP1 CA may be an effective drug for the treatment of Xuanwei female lung adenocarcinoma.(5)Glutamine metabolism pathway may be a potential therapeutic target for cold tumor subtype of Xuanwei female lung adenocarcinoma.(6)DHX9 promoted the proliferation,migration,invasion and G1 phase of p53-mutant lung cancer cells and inhibits their apoptosis via up-regulation of PCNA/CDK2 axis,and may promote the progression of p53-mutant lung cancer by upregulating EMT-related proteins and promoting tumor angiogenesis.