Expression And Clinical Significance Of FEN1 And RFC2 In Lung Squamous Cell Carcinoma Tissues

Objective:Flap-endonuclease-1(FEN 1)is a divalent metal ion-dependent multifunctional nuclease with a molecular weight of about 42 kDa.It is a member of the Rad2 structure-spec ific nuclease family.The multiple nuclease activities of FEN1 enable it to participate in a variety of DNA metabolic pathways,such as Okazaki fragment maturation,stalle d replication fork rescue,telomere maintenance,long base excision repair etc.FEN1 plays a role in DNA replication and modification in normal tissues and is essential f or normal cell proliferation.Several studies have shown that FEN1 is strongly presen t in cervical cancer,prostate cancer,breast cancer and stomach cancer,liver cancer a nd other tumor tissues,and is closely related to prognosis.However,the role of FEN 1 in LUSC is unclear.Replication factor C subunit 2(RFC2)is a member of the fa mily of replication factor RFC(Replication factor C).RFC family members are main ly involved in telomere maintenance,nuclear DNA replication,cell division cycle etc.And it can be used as a key factor to initiate checkpoints in the downstream of D NA damage sites,and signal transduction is carried out by binding to cell cycle chec kpoint proteins to participate in the process of DNA excision and mismatch repair.In addition,RFC2 is the only RFC subunit that can independently unload PCNA(proli ferating cell nuclear antigen)and affect DNA polymerase activity.It has been confir med to be involved in the development of various tumors.RFC complex can also ac t as an activator of FEN1 together with PCNA,playing a synergistic role in cell cyc le,DNA replication and other processes.The purpose of this study was to explore the expression of FEN1 and RFC2 in LUSC,and the relationship between their expressi on and clinicopathological features and prognosis of patients with LUSC.At the sam e time,the possible gene pathways involved in FEN1 and RFC2 and the immune inf iltration of FEN1 and RFC2 were studied.Research Methods:The first part:The RNA-seq data set was extracted based on the UCSC Xena database for pan-cancer analysis,and the expression levels of FEN 1 and RFC2 in LUSC were analyzed.According to the obtained clinicopathological data,the relationships between FEN1 and RFC2 and the age,gender,clinical stages,TNM stage,radiotherapy and chemotherapy of patients with LUSC were analyzed.The pathological tissue wax blocks of 80 patients with LUSC who underwent surgical resection in the Department of Thoracic Surgery,the Affiliated Hospital of Anhui Medical University from January 2015 to June 2022 were selected to make tissue microarrays.Immunohistochemistry and qRT-PCR were used to detect and verify the expression of FEN1 and RFC2 in LUSC tissues and adjacent tissues,and to analyze their relationship with clinicopathological features and prognosis.The second part:Based on GO and KEGG databases,R language were used for GO,KEGG and GSEA enrichment analysis.Based on the hallmark database,R language was used for enrichment analysis with GSVA algorithm.The third part:The ssGSEA algorithm was used to compare the infiltration of 28 immune cells between the high and low expression groups of FEN1 and RFC2 in LUSC.Spearman correlation was used to analyze the correlation between 28 immune cells and the expression of FEN1 and RFC2.Research results:The first part:1.Through the analysis of UCSC Xena LUSC data set,it is concluded that FEN1 an d RFC2 are highly expressed in various malignant tumor tissues,and the differences are statistically significant(P<0.05);The expression levels of FEN1 and RFC2 in LU SC were higher than those in LUAD(P<0.05),and the expression levels in LUSC w ere also higher than those in normal tissues(P<0.05);The expression of FEN1 have relation with N stage(P=0.022),M stage(P=0.014)and clinical stage(P=0.013),but not with gender(P=0.7),age(P=0.47),T stage(P=0.4)and radiotherapy(P=0.4);RFC2 expression was correlated with age(P=0.034),T stage(P=0.045)and radiotherapy(P=0.012),but not with gender(P=0.66),N stage(P=0.29),M stage(P=0.95)and clinic al stage(P=0.24).2.The results of IHC confirmed that FEN1 and RFC2 were highly expressed in LUS C tissues compared with adjacent tissues,and the difference was statistically significa nt(P<0.05);The expression of FEN1 protein in LUSC patients in the aspect of diffe rent TNM stages(P<0.01)was statistically disparate;The high expression rate of RF C2 protein in LUSC patients with different TNM stage(P<0.01)and pleural invasion(P=0.016)was statistically significant;There were no significant difference in the hi gh expression rates of FEN1 and RFC2 protein in LUSC and adjacent tissues with d ifferent age,gender,body mass index,smoking,tumor diameter,pleural invasion,lym ph node metastasis and differentiation(P>0.05).The OS of LUSC patients in FEN1high expression group was lower than that in FEN1 low expression group(P<0.0001),OS of patients with LUSC in the RFC2 high expression group was lower than tha t in the RFC2 low expression group,and the difference was statistically significant(P<0.0001),The OS of patients with LUSC with that both FEN1 and RFC2 are highe r expression was lower than that of patients with both lower expression(P<0.0001);through univariate analysis,FEN1 expression,RFC2 expression and TNM stage were related to the prognosis of LUSC patients,and the differences were statistically signif icant(P<0.05);Multivariate analysis showed that FEN1 expression,RFC2 expression and TNM stage were related to the prognosis of LUSC patients(P<0.05).3.The expression of FEN1 and RFC2 mRNA in LUSC tissues was significantly higher than that in adjacent tissues(P<0.01);The relative expressions of FEN1 and RFC2 were correlated and there was a significant positive correlation(P<0.01).The second part:1.The results of GO and KEGG enrichment analysis showed that FEN1 was mainly enriched in DNA replication,nuclear division,organ fission function pathway,catalytic activity on DNA,DNA polymerase and ATP synthase activity,oocyte meiosis,Fanconi anemia pathway,cell senescence and other pathways;RFC enrichment results were DNA replication,nuclear division,sister chromatid separation,histocompatibility complex Ⅱ(MHC-Ⅱ),extracellular matrix component activity,DNA catalytic activity,human T cell leukemia virus 1 infection,phagocytic vacuoles,cell adhesion molecules,complement and coagulation cascades,etc.2.GSEA enriched FEN1 was significantly enriched in the GO data set in the five pathways of DNA strand elongation involved in DNA replication,double strand break repair via breakinduced replication,positive regulation of protein localization to telomere establishment,protein localization to nucleosome,and protein localization to telomere regulation.In the KEGG data set,the following five pathways were enriched in DNA replication,panconi anemia pathway,homologous recombination,mismatch repair,nuclear cytoplasmic transport;the GO dataset showed that RFC2 was associated with establishment of protein localization to telomere,DNA double-strand break repair via break-induced replication,DNA strand elongation involved in DNA replication,regulation of establishment of protein localization to chromosome,regulation of esta blishment of protein localization to telomer.The KEGG database showed that RFC2 was associated with Base excision repair,DNA replication,chromosome homologous recombination,mismatch repair,Steroid biosynthesis.3.The results of GSVA enrichment showed that 10 pathways such as G2/M checkpoint,E2F target,MYC target V2,unfolded protein response pathway and MYC target V1 etc were upregulated when FEN1 was highly expressed;11 pathways including E2F target,G2/M checkpoint,DNA repair,MYC target V1,mammalian target of rapamycin(mTOR)signaling pathway etc were up-regulated when RFC2 was highly expressed.The third part:1.The results of immune infiltration analysis showed that the expression of FEN 1 and RFC2 was negatively correlated with the infiltration of immune cells in the microenvironment of LUSC.2.In the FEN1 high expression group and the low expression group,the levels of 25 immune cells were different,with statistical significance(P<0.05);The levels of the remaining 23 immune cells including mast cells,central memory T cells,eosinophils,plasmacytoid dendritic cells,etc were higher in the FEN 1 low expression group than in the FEN1 high expression group.The levels of memory B cells and Th2 cell in the FEN1 high expression group were higher than those in the FEN 1 low expression group,and the differences were statistically significant(P<0.05);When the expression of RFC2 was different,the infiltration level of 26 kinds of immune cells was different,with statistical significance(P<0.05).The rest included 25 kinds of immune cells,such as plasmacytoid dendritic cells,central memory T cells and mast cells.The infiltration level of CD56+natural killer cells in RFC2 high expression group was higher than that in RFC2 low expression group(P<0.05).3.Memory B cells and helper T cell 2(Th2)were positively correlated with FEN1 expression,and the remaining 23 immune cells were negatively correlated with FEN1 expression.The most relevant cells were mast cells(P=0.000);Memory B cells were positively correlated with RFC2 expression,and the remaining 25 immune cells were negatively correlated with RFC2 expression.Among them,plasmacytoid dendritic cells had the strongest correlation with RFC2 expression(P=0.000).Conclusion:1.FEN1 and RFC2 are highly expressed in LUSC tissues compared with adjacent normal tissues,The high expression levels of FEN1 and RFC2 are associated with multiple clinicopathological parameters.2.FEN1 expression is positively correlated with RFC2 expression,and the expression levels of FEN1 and RFC2 are significantly correlated with the poor prognosis of LUSC,and may be new tumor markers and potential therapeutic targets for LUSC.3.FEN1 and RFC2 may be involved in multiple signaling pathways related to LUSC,the results of enrichment analysis are similar,the both are significantly related to important growth and metabolic processes of tumor cells such as mitosis,cell cycle and DNA mismatch repair.4.The expression levels of FEN1 and RFC2 are negatively correlated with immune infiltration,among which FEN1 is most correlated with mast cells,and RFC2 is most correlated with plasmacytoid dendritic cells.