Proteome Analyses Reveal S100A11,S100P And RBM25 As Potential Biomarkers In Colorectal Cancer

Background Colorectal cancer(CRC)is the third most commonmalignancyin the world withincreased incidence and mortality.Many patients subsequently develop lymph node metastases or liver metastases soon after diagnosis which drastically impacts their prognosis.Although some CRC patients have certain genetic background,most of them are still sporadic.Therefore,early diagnosis is very importantand specific CRC biomarkers are urgently needed to be found.DNA and RNA sequencing technologieshave been widely used in cancer research,diagnosis and treatment,but genetic information alone cannot predict protein activity in that proteins are often modified at post-transcriptional and post-translational levels.Comparing withgenome and transcriptome sequencing technologies,proteomics can provide information on protein level and shed light on disease prevention,diagnosis,and treatment of thediseases,and it has developed into a powerful tool for identifying diagnostic biomarkers and new therapeutic targets for diverse cancers.Objectives To screen potential molecular markers for diagnosis and prognosis of CRC by proteomics analysis;To preliminarily understand the function of potential molecular markers in the progression of CRC through the validation and subsequent functional experiments in CRC cell modes.Methods Non-metastatic tissues(NT)and metastatic tissues(MT)as well as their paired normal tissues(NN and MN)were performed proteomic analyses in 10 Chinese patients with CRC,and then the differentially expressed proteins(DEPs)between the groups were identified by mass spectrometry.The DEPs S100A11,S100 P and RBM25 were selected forimmunohistochemistry(IHC)analysesin paraffin-embedded colon cancer tissue microarray(TMA)and then validated in a larger cohortof 154 cases CRC TMA with corresponding statistical analyses,Western blot analyses in 20 cases of fresh CRC tissues were performed for further validation.The function of less studied RBM25 in CRC cell line HCT116 was investigated by sh RNA and si RNA knockdown techniques and subsequent CCK-8 and colony formation assays.Results Proteomic data mapped to approximately 83.76% of all annotated proteins with974 and 585 proteins being up-regulated and down-regulated,respectively.Assessing the inter-sample variation results indicated that CRC tissues are different from paired NTs in protein expression profile.DEPs mainly assigned tocytoplasm and nucleus,and showed that these DEPs were enrichedin several different pathways,biological processes,cellular components and molecularfunctions.The results of IHC validation of CRC tissue microarray showed that the expression of proteins including S100A11,S100 P,and RBM25 in both non-metastatic and metastatic CRC tissues were significantly up-regulated compared with normal tissues,and were also closely correlated with the degree of differentiation,respectively(P<0.05);Further Western blot analysis of fresh tissue samples also showed that the three proteins were significantly up-regulated in cancer tissues.The CCK-8 and colony formation assays by sh RNA or si RNA knockdown showed that RBM25 promoted cell growth and proliferation of CRC cell line HCT 116.Conclusion Our proteomic screening results reveal both previously identified CRC biomarkers along with novel candidates andprovide a ready resource of DEPs in CRC for further investigation.