Screening Of LncRNA-encoded Peptide CTCF-DT And Its Function And Mechanism In Esophageal Squamous Cell Carcinoma

ObjectiveThere is a fairly extensive ambiguous region between the currently annotated proteincoding genes and lncRNAs,and some lncRNAs in this region are capable of encoding functional short peptides associated with human disease.Although lncRNA is becoming comprehensively studied in cancer,the function and molecular mechanisms of lncRNAencoded short peptides in cancer development are still poorly understood.Esophageal cancer is the seventh most common cancer type in the world,and although some progress has been made in the treatment of esophageal cancer,there are few targeted therapies that have successfully achieved clinical translation in the Asian esophageal cancer patient cohort dominated by squamous cell carcinoma.Therefore,we intend to build a research platform for high-throughput screening of coding lncRNAs in esophageal squamous cell carcinoma based on the analysis process of multi-omics data,which is not only of great significance for understanding the regulation of lncRNA expression and the mechanism of esophageal cancer progression,but also provides new ideas for the clinical treatment of esophageal squamous cell carcinoma.MethodWe used magnetic beads with Poly(T)probes and RiboZero method to construct the expression profiles of mRNA and lncRNA in esophageal squamous cell cell lines.We then used CLIP-seq data of the translation initiation factor eIF3b combined with concurrently conducted iTRAQ protein profiling data to identify encoded lncRNAs.In combination with CAGE-seq(equivalent to genome-wide 5’-RACE),the intrinsic rules of its 5’ cap were analyzed to improve the existing lncRNA annotation rules.We firstly integrate and analyze the omics data of RNA-seq under different technical processing and the proteome data obtained by mass spectrometry at the research level of cell models,which can not only discover new coding lncRNAs,but also summarize their similarities and differences with known mRNA translation patterns.In order to verify the universality or tissue specificity of the conclusions,we also captured the above omics data in the human embryonic kidney 293 T cell and Hela cell models at the same time.We used expression plasmids and antibodies to verify the exogenous and endogenous expression of lncRNA CTCF-DT-201encoded polypeptide(CTCF-DT),respectively.The use of shRNA and antisense oligonucleotide(ASO)to knock down CTCF-DT detected malignant phenotypes such as proliferation,invasion and migration,and clonal formation of esophageal squamous cell carcinoma cells.Lentiviruses overexpressing lncRNA full-length and coding mutations were used to distinguish the functions of lncRNA CTCF-DT-201 and CTCF-DT polypeptides.The clinical significance of CTCF-DT was explored by using TCGA datasets,cDNA chips and tissue chips in esophageal squamous cell carcinoma patients.Immunoprecipitation/mass spectrometry(IP/MS)and RNA-seq were used to explore the interaction protein profiling and mechanism of CTCF-DT.The high-throughput drugscreening platform was used to identify the relationship between CTCF-DT expression level and the sensitivity of more than 380 anticancer drugs.Integrating GDSC and CCLE high-throughput data to analyze the relationship between CTCF-DT expression levels and the sensitivity of multiple anticancer drugs in esophageal squamous cell carcinoma cell lines.In vivo and in vitro experiments to explore the tumor inhibitory effect of ASO and carboplatin targeting CTCF-DT in the treatment of esophageal squamous cell carcinoma.ResultWe have built a platform for high-throughput screening of coding lncRNAs,that is,integrating and analyzing CLIP-seq data and iTRAQ mass spectrometry data of eIF3b target molecules,and using the Trans-Proteomic Pipeline(TPP)to enrich and screen encoded lncRNAs.We captured 7571 ncRNAs specifically bound to eIF3b,including 1142 lncRNAs.The TPP analysis indicated that 1280 potentially encoded ncRNAs that bind specifically to eIF3b.In the screening results,lncRNA CTCF-DT-201 encoded a 116amino acid polypeptide(CTCF-DT),which we successfully verified it with plasmids and antibodies.Knockdown CTCF-DT significantly inhibit multiple malignant phenotypes of esophageal cancer,such as proliferation,invasion migration,and clonalization.Overexpression of full-length lncRNA CTCF-DT-201 instead of translating lncRNA with initiation codon mutations can significantly promote proliferation of esophageal squamous cell carcinoma cells.RNA-seq and IP-MS results showed that CTCF-DT could directly bind to RhoA to maintain the level of RhoA-GTP and activate the downstream PI3K/NFκB signaling pathway,thereby promoting the malignant progression of esophageal cancer.Inhibition of CTCF-DT expression significantly downregulated the expression levels of pPI3K and p-IKBα.Survival analysis of three independent cohorts of esophageal squamous cell carcinoma patients showed that esophageal cancer patients with high expression of CTCF-DT had poorer survival,with HR risk values of 1.93,2.08 and 3.985,respectively.After knocking down CTCF-DT,the sensitivity of esophageal cancer cells to carboplatin was enhanced,and the IC50 of 48 hours of drug treatment was reduced from 100 μM to 30μM.The results of GDSC and CCLE data integration showed that the expression levels of CTCF-DT were positively correlated with the IC50s of eight PI3K signaling pathway inhibitors,namely AZD6482,PF-4708671,THR-101,CZC24832,Uprosertib,Ipatasertib,THR-103,THR-102 and MK-2206(r>0.2).In vivo and in vitro results showed that carboplatin combined with CTCF-DT targeting ASO could significantly inhibit PI3K/NFκB signaling pathway and inhibit the progression of esophageal squamous cell carcinoma.ConclusioneIF3b can successfully capture transcripts in the process of translation;Potentially coding ncRNAs have fewer transcription clusters(TCs)near the locus,resulting in lower transcription levels;Potentially encoded ncRNAs may initiate translation with nonclassical translation start codons,such as CTGs,but are less efficient.Peptides encoded by potentially coding ncRNAs have low mass spectral abundance and coverage.The polypeptide encoded by LncRNA CTCF-DT-201 can promote the proliferation,invasion and migration of esophageal squamous cell carcinoma.Esophageal cancer patients with high expression of CTCF-DT have poorer overall survival;The combined inhibition of CTCF-DT and carboplatin can obtain a significantly better tumor inhibition effect than that of monotherapy.CTCF-DT is a potential candidate molecule for esophageal cancer therapeutic targets and biomarkers.This study not only updates the existing understanding and database resources of ncRNA,which has high theoretical significance and scientific research application value,but also broadens the breadth and diversity of lncRNA in cancer development,and provides new potential targets for clinical translational research of esophageal squamous cell carcinoma.