Fecal Metabolomics,mitochondrial DNA Genome And SNRPA Bioinformatics Analysis In Colorectal Cancer Patients

Colorectal cancer(CRC),as one of the most common malignant tumors,increased nearly 2 million new cases and 1 million deaths in 2020,accounting for approximately one in ten new cancer cases and deaths.At present,most CRC patients have appeared metastasis when diagnosed due to the limitation of available targets for clinical diagnosis,therefore,explore the new targets can provide great value and significance.In order to explore potential molecular therapeutic targets and provide new thoughts for CRC,we have identified specific metabolites between CRC and the normal groups through fecal metabolomics analysis,correlation and metabolic pathway enrichment analysis were also performed.We have explored mitochondrial DNA mutations,haplogroups classification and phylogenetic relationships between tumor and normal tissues through mitochondrial genome analysis,suggesting that specific haplogroups might be associated with the risk of CRC.In addition,according to our previous proteomics data,we analyzed the expression of U1 small nuclear ribonucleoprotein A(SNRPA)and the correlation with pathological data in CRC.We identified downstream differential expressed genes and KEGG enrichment pathways by RNA-seq of HCT116 cells with or without SNRPA si RNA knockdown,revealing that SNRPA might be a key gene for the progression of CRC.In this study,fecal samples were collected from 10 CRC patients(WC1-WC10)and10 healthy volunteers(HC1-HC10)at the Department of Gastrointestinal Surgery of the First Affiliated Hospital of Anhui Medical University for Liquid Chromatography Mass Spectrometer(LC-MS).The results showed significant differences in fecal metabolites between WC and HC groups.In total,we found 700 differentially expressed metabolites,of which 322 metabolites were up-regulated in WC group,including isoleucine and isopalmitic acid but 378 metabolites,including phosphatidylinositol and glutamate,were down-regulated.Then,we further identified 66 specific metabolites were different enrichment between HC and WC groups,which divided into Ethers,Amines,Eicosanoids and Bile acids and derivatives.Pearson correlation analysis showed significant correlations among the four subclass metabolites.Through metabolic pathways enrichment analysis of differentially expressed metabolites,we found the main enrichment pathways were amino acids and fatty acids biosynthesis,lactose metabolism and choline metabolism in cancer,and the biosynthesis of valine,leucine and isoleucine was the most significant enrichment pathway.By this fecal metabolomics analysis of CRC,the specifically metabolites were identified,especially Amines,Eicosanoids,Bile acids and derivatives might be potential biomarkers for the occurrence and development of CRC,and can also provide basic information for the early CRC screening and diagnosis.Mitochondrial genome(mt DNA)was sequenced in 20 cancer tissues and the corresponding adjacent non-cancerous tissues collected from 10 CRC patients,and sequence alignment,haplogroups classification and phylogenetic analysis were also performed.The results showed that the length of 20 mt DNA sequences were ranged from16 558-16 675 bp,and 2 to 286 bp mt DNA mutations were observed between each pair of samples,the mutation rate was 0.012% to 1.175%.The total number of mutation sites were 748,and the PCGs region observed the most mutation sites(399),followed by CR region(252)and r RNA region(89),while t RNA region showed the least mutation sites(8).However,the mutation rate in CR region(1.13%)was significantly higher than PCGs region(0.19%).Amongst the 20 mt DNA sequences screened,5 haplogroups were identified and classified into predicted haplogroups A,B,C,F and M,and haplogroups B and F accounted for the most.The mt DNA sequences were divided into East Asian,Southeast Asian and North Asian types,and 80% sequences showed East Asian and Southeast Asian haplogroups.Through this mt DNA sequences analysis,haplogroups B and F might be potential risk factors for CRC,and East Asian and Southeast Asian types might be more susceptible to CRC.In addition,we detected SNRPA expression by immunohistochemical(IHC)experiments in TMA tissue microarray from 196 CRC patients,and Western blotting(WB)experiments were also conducted in tumor tissues and normal adjacent tissues from 30 CRC patients.RNA sequencing was used to screen the downstream signaling pathways and target genes by SNRPA knockdown in HCT116 cells.The results showed that 74 upregulated genes and 57 down-regulated genes were obtained in the two knockdown groups,and the differentially expressed genes were enriched into 5 and 7 significantly different pathways by KEGG enrichment analysis.Glycosphingolipid biosynthesis-lacto and neolacto series/ globo and isoglobo series were the common enrichment pathway.The expression of SNRPA and related bioinformatics analysis showed that SNRPA was related to the progression of CRC,and maybe a potential biomarker of CRC.SNRPA might promote the occurrence and development of CRC by specific signaling pathways and downstream target molecules.In all,this study provides basic data for subsequent studies on the molecular mechanism of SNRPA in CRC.