1.The Molecular Mechanism Of LncRNA581 Promoting The Development Of Esophageal Squamous Cell Carcinoma 2.The Mechanism Of MiR-20b-5p That Promotes Lymph Node Metastasis Into Exosomes

According to the Cancer Incidence and Mortality Study in China(2016),published by the National Cancer Center in 2022,the incidence and mortality rate of esophageal squamous carcinoma ranked sixth and fifth,respectively,among all tumors.Due to the insidious onset of the disease,most patients are already in the middle-to-late stages with metastasis at the time of diagnosis,resulting in a poor overall outcome due to the lack of effective treatment options.Therefore,investigating the molecular mechanism of esophageal squamous carcinoma development and finding the predictive markers and therapeutic targets of esophageal squamous carcinoma metastasis has become an urgent problem in esophageal squamous carcinoma research.LncRNAs are a class of long non-coding RNAs greater than 200 nt in length with a wide variety and rich secondary structure,which can regulate gene expression at chromatin structure,transcriptional,and post-transcriptional levels through binding proteins,acting as ceRNAs adsorbing miRNAs,and even encoding small peptides,thereby regulating biological processes.Recent studies have shown that LncRNAs are dysregulated in a variety of tumors and are involved in the regulation of tumor proliferation,metastasis,immune escape,etc.Studying the molecular mechanisms of LncRNAs in tumor development can reveal the pathogenesis of tumors from a new perspective and provide new ideas for tumor diagnosis and treatment.During the master’s degree,the analysis of transcriptome sequencing data of esophageal squamous carcinoma from our group and the TCGA database revealed that the expression of LncRNA581 was elevated in esophageal squamous carcinoma.Functional assays showed that LncRNA581 promoted the malignant phenotype of esophageal squamous carcinoma cells,including proliferation,invasion,migration,and subcutaneous tumor formation.To investigate the mechanism of LncRNA581,RNA-Pulldown combined mass spectrometry analysis was used to screen the reciprocal proteins of LncRNA581,and the results showed that LncRNA581 could bind the cationic antimicrobial peptide HNP-1,which was verified by RIP assay.WB experiments showed that LncNRA581 could negatively regulate the expression of HNP-1.It has been reported that HNP-1 can promote M1-type polarization of macrophages.In this study,we found that overexpression of LncRNA581 in esophageal squamous cell carcinoma can inhibit the Ml-type polarization process of macrophages using conditional supernatants and co-culture experiments.During the Ph.D.period,the study focused on whether LncRNA581 can act as a ceRNA and whether it can encode small peptides that play a role in the development of esophageal squamous cell carcinoma.Tumor-associated macrophages are the main immune cell type for infiltration in multiple tumor microenvironments and consist of M1-type macrophages and M2-type macrophages.Among them,M1-type macrophages can mediate oncogenic functions as antigen-presenting cells,and M2-type macrophages can perform pro-cancer functions by promoting angiogenesis,etc.In the results of mass spectrometry,no protein associated with M2-type polarization was found among the proteins bound to LncRNA581,and a continuation investigation was conducted during the PhD period to investigate whether LncRNA can act as a ceRNA to regulate the M2-type polarization process in macrophages.CeRNA is a new mechanism of RNA interaction,which means that with the same miRNA binding sequence,LncRNA can competitively adsorb miRNA and then regulate the expression of its downstream target genes.The site prediction and dual luciferase reporter assay revealed that LncRNA5 81 has the same miR-1299 binding site as the 3’UTR of IL-33,and LncRNA581 can competitively bind miR-1299 with IL-33.qPCR and WB results showed that LncRNA581 can promote the expression of IL-33 by down-regulating miR IL-33 is a member of the IL-1 family,and it has been shown that IL-33 can further induce M2-type polarization of macrophages by binding to the ST2 receptor on the surface of macrophages.The overexpression of LncRNA581 in esophageal squamous carcinoma cells was found to promote the migration and M2 polarization of macrophages through conditioned medium and cell co-culture experiments.To investigate the specific mechanism,the secreted supernatants of macrophages were examined by cytokine microarray after co-culture with esophageal squamous cell carcinoma cells overexpressing LncRNA581.KEGG enrichment analysis of differentially secreted cytokines revealed that differentially secreted cytokines were mainly enriched in signaling pathways such as JAK/STAT and PI3K/AKT associated with M2-type polarization in macrophages,which was also verified in the subsequent WB experiments.In addition,PPAR-γ,a key regulator of M2-type polarization,and TGF-β3,a pro-angiogenic factor,were significantly upregulated in macrophages.Culturing vascular endothelial cells with this conditioned medium promoted the migration and tube-formation of vascular endothelial cells,which in turn promoted the metastasis of esophageal squamous cell carcinoma.Recent studies have shown that certain LncRNAs have short open reading frames on their sequences,which can regulate processes such as muscle contraction and tumor metabolism by encoding functional small peptides.To investigate whether LncRNA581 can encode small peptides,we evaluated whether LncRNA581 sequences have a short open reading frame using ORF finder and other websites.The results showed that the sequence of LncRNA581 contained two short open reading frames,Frame1 and Frame2.It was verified by Flag-Knock in the assay that only Frame2 was translated in esophageal squamous carcinoma cells,encoding the small peptide LncRNA581-pepN2.The expression of the small peptide was detected by WB in six groups of normal,paraneoplastic,and esophageal squamous carcinoma tissues.The results showed that the expression of this small peptide was upregulated in esophageal squamous cell carcinoma tissues compared with normal and paraneoplastic groups.Cell phenotyping assays showed that pepN2 promoted the malignant phenotype of esophageal squamous carcinoma.A preliminary mechanistic investigation using co-IP combined with mass spectrometry and WB revealed that pepN2 binds to and regulates the PRDX2 protein,a catalytic peroxide reductase that regulates intracellular levels of reactive oxygen species and thus promotes tumor proliferation,metastasis,and drug resistance.In this study,the ROS levels and cell viability of esophageal squamous carcinoma cells after hydrogen peroxide treatment were measured by flow cytometry and CCK8 after differential expression of the small peptide pepN2,The results showed that pepN2 reduced intracellular reactive oxygen species levels and enhanced the survival of esophageal squamous carcinoma cells under oxidative stress.In conclusion,LncRNA581,which is aberrantly up-regulated in esophageal squamous carcinoma,can on the one hand regulate tumor-associated macrophage polarization to M2 type in the microenvironment through HNP-1 and miR-1299/IL-33 axis,thus inducing angiogenesis and promoting esophageal squamous carcinoma metastasis;on the other hand,LncRNA581 can up-regulate peroxiredoxin reductase through encoding small peptide pepN2 PRDX2,which in turn improves the survival of esophageal squamous carcinoma cells under oxidative stress.Targeting LncRNA581 not only inhibits the growth of tumor cells but also inhibits the metastasis of esophageal squamous carcinoma by regulating the polarization of macrophages in the microenvironment,which may provide a new strategy for the treatment of esophageal squamous carcinoma.Esophageal squamous carcinoma is a common type of gastrointestinal malignancy in China,and the latest national cancer statistics show that its incidence and mortality rate are the sixth and fifth highest among all tumors,respectively.Due to its insidious onset and rapid progression,most of these patients are in the middle to late stage when diagnosed,accompanied by lymph node metastasis.The presence and number of lymph node metastasis is an important prognostic factor for esophageal squamous carcinoma.The survival rate of patients without lymph node metastasis in early stage can reach 85%-90%after treatment,while the 5-year survival rate of patients with metastasis is only 6%-15%,therefore,screening for diagnostic markers that accurately predict the occurrence of lymph node metastasis is important for early diagnosis and treatment of esophageal squamous carcinoma.Liquid biopsy is a non-invasive and simple test that can detect cancer by detecting the number of circulating tumor cells,circulating tumor DNA and tumor cell exosomes in blood,and has great application prospects in early screening and diagnosis,predicting treatment efficacy and prognosis,detecting recurrence,tumor heterogeneity and evolution.Among them,exosomes are derived from the secretion of living tumor cells and contain more parental cell information,and it is abundant and stable in blood,which has good prospects for application in liquid biopsy.Exosomes are a class of extracellular vesicles with a bilayer touch structure of 40-160 nm in diameter,which are secreted extracellularly by parental cells through plasma membrane invagination-forming early/late-stage sorting endosomes-further invagination to form multivesicular bodies,and exist stably in various body fluids,and can regulate recipient cells through direct fusion or ligand-receptor binding.During the assembly process,exosomes can wrap a series of proteins,nucleic acids and lipids,such as CD63,TSG101,miRNA,LncRNA,etc.Among the above substances,miRNAs are a class of small non-coding RNAs of 17-24 nt in length,which can regulate tumorigenesis and development by targeting downstream mRNAs and can exist stably in various types of body fluids.Therefore,in recent years,the use of abnormally expressed miRNAs in exosomes to evaluate tumorigenesis and progression has become a hot research direction in liquid biopsy.By sequencing exosomes in the plasma of patients with esophageal squamous carcinoma,our group found that miR-20b-5p was up-regulated in the plasma of patients with lymph node metastasis and could well predict the occurrence of lymph node metastasis(AUC=0.872,p<0.001),which provided a new basis for the evaluation of lymph node metastasis in patients with esophageal squamous carcinoma.To investigate the mechanism of miR-20b-5p entry into exosomes,we first detected the expression of miR-20b-5 by qPCR.Compared with immortalized epithelial cells,miR-20b-5p appeared differentially upregulated in esophageal squamous carcinoma cells.Exosomes were extracted by differential centrifugation and identified using projection electron microscopy,particle size analysis system and WB.qPCR showed that exosomes secreted by esophageal squamous carcinoma contained miR-20b-5p.Exosomes of different tissue or cellular origin were highly heterogeneous in size and contents,suggesting that exosomes have a high sorting mechanism for the substances they carry.It has been shown that exosomes can sort their contents by binding a variety of RNA-binding proteins,including the HnRNP family,nSMase2,and AGO2,among others.The mechanism of HnRNPA2Bl is the most clear:when miRNAs contain"GGAG" motifs,they can enter the exosome by binding to HnRNPA2B1.After sequence comparison,we found that miR-20b-5p contains the "GCAGGTAG" motif,and verified that it can bind to HnRNPA2B1 by RIP experiments.Therefore,we suggest that miR-20b-5p is wrapped by HnRNPA2B1 into exosomes and can be secreted into body fluids such as blood.In addition,experiments in nude mice foot pad injection model showed that miR-20b-5p in exosomes could promote lymph node metastasis of esophageal squamous carcinoma cells.In conclusion,miR-20b-5p in esophageal squamous carcinoma can be wrapped by HnRNPA2B1 into exosomes,enter blood with exosomes,and promote lymph node metastasis of esophageal squamous carcinoma cells,which provides a theoretical basis for using the expression of miR-20b-5p in serum exosomes to predict lymph node metastasis of esophageal squamous carcinoma.