The Function And Mechanistic Study Of IncRNAs And Transfer RNA Derived Small RNAs In Lung Adenocarcinoma

Chapter 1:LINC01290 suppresses lung adenocarcinoma stemness by binding to IGF2BP1 to regulate SOX-2 and NANOG expressionBackground and objectives:Lung adenocarcinoma(LUAD)is the most common type of lung cancer,with an incidence rate of 40%.Despite the fact that most patients receive comprehensive surgery-based multidisciplinary treatment,the 5year survival rate for lung adenocarcinoma remains below 15%.Lung adenocarcinoma,like many other solid tumors,has been discovered to be made up of a population of cancer cells that can self-renew and have undifferentiated stem cell features.Tumor recurrence and metastasis are thought to be caused by cancer stem cells.Exploring the essential regulatory molecules and signaling pathways that influence the sternness properties of CSCs is critical for designing effective treatment approaches to avoid tumor recurrence and invasive metastasis,as well as enhance patient survival.In recent years,with the development of high-throughput sequencing and bioinformatics,non-coding RNAs,which are viewed as transcriptional"noise",have attracted the attention of researchers.Accumulating studies have found that lncRNAs are involved in a variety of biological processes that affect the occurrence and development of various diseases such as cancer.However,the role of lncRNAs in lung adenocarcinoma stemness has not been fully elucidated.The aim of this study was to establish the characteristic lncRNA expression profile of lung adenocarcinoma CSCs;and investigated the function and mechanism of the lung adenocarcinoma stemness related lncRNA-linc01290.Methods:(1)The NANOG reporter system was used to sort lung cancer CSCs and non-CSCs,and ArrayStar LncRNA microarray hybridization was used to detect differentially expressed IncRNAs in lung adenocarcinoma CSCs;(2)Detected the expression levels of linc01290 in 97 lung adenocarcinoma patients’ tumors and adjacent lung tissues;gathered clinical data and assessing the connection between linc01290 expression levels and clinicopathological parameters;(3)Using bioinformatics tools such as phyloCSF software,UCSC database and RNAfold WebServer to analyze and predict the coding potential,secondary structure,conservation,etc.of linc01290;RACE experiment was performed to obtain the full-length sequence of linc01290;using stable overexpression of linc01290 and stable knockdown of linc01290 cell lines to conduct clone formation,spheroid formation,and subcutaneous tumor formation in nude mice assays to analyze the effects of overexpression or knockdown of linc01290 on cell proliferation and stemness in vivo and in vitro;(4)Fluorescence in situ hybridization(FISH)assay was used to detect the sublocalization of linc01290;Pull-down assay and RNA immunoprecipitation(RIP)assay were used to detect the interaction between linc1290 and IGF2BP1;different fragments of linc1290 were constructed based on the secondary structure of linc01290,and pulldown assay was conducted to analyze the key sequence of linc01290 binding to IGF2BP1;constructed IGF2BP1 protein deletion mutants based on IGF2BP1 protein structural domain,and RIP assay was used to analyze the key structural domain of IGF2BP1 binding to linc01290;using cell lines stably overexpressing different linc01290 fragments to carry out the clone formation,spheroid formation assay and subcutaneous tumor formation assay in nude mice to analyze the key function sequence of linc01290;the overall m6A content of cell lines overexpressing different linc01290 fragments was measured by colorimetric assay;the SOX-2 and NANOG m6 A levels of cell lines overexpressing different linc01290 fragments were measured by MazF assay;using UPF1 knockdown lung adenocarcinoma cell lines to analyze the degradation of linc1290 by nonsense-mediated mRNA decay(NMD)pathway.Results:(1)A total of 1,355 up-regulated lncRNAs and 273 down-regulated lncRNAs were screened in lung adenocarcinomas CSCs,among which linc01290 was down-regulated in CSCs and the expression level was negatively correlated with tumor growth and positively related to patient prognosis;(2)Overexpression of linc01290 inhibited its clonogenic ability,sphere formation ability,and subcutaneous tumorigenic ability in nude mice,whereas knockdown of linc1290 promoted its clonogenic ability,sphere formation ability,and subcutaneous tumorigenic ability in nude mice;(3)Linc01290 located in both nucleus and cytoplasm;linc01290 binded to m6A recognition protein IGF2BP1 KH3-4 structural domain via its exon 2 conserved region;conserved region of linc01290 exon 2 is the key region for its function,and its oncogenic effect is diminished by the absence of conserved region of exon 2;linc01290 suppresses the stemness characteristics of lung adenocarcinoma by affecting the m6A content of SOX-2 and NANOG through its exon 2 conserved region;(4)Linc01290 expression was down-regulated in lung adenocarcinoma,partly due to increased degradation of linc01290 as a result of over-activation of the nonsense-mediated mRNA decay(NMD)pathway in lung adenocarcinoma.Conclusions:(1)The lncRNA expression profile of lung adenocarcinoma CSCs was established for the first time,as well as the role and mechanism of linc01290 in maintaining the stemness characteristics of CSCs;(2)linc01290 impacts lung adenocarcinoma stemness by regulating SOX-2 and NANOG m6A levels through the interaction of its conserved region in exon 2 with the structural domain of m6A recognition protein IGF2BP1KH3-4;(3)Down-regulation of linc01290 in lung adenocarcinoma is partly due to overactivation of the NMD pathway in lung adenocarcinoma;(4)linc01290 is positively associated with prognosis and may serve as a potential diagnostic and therapeutic target for lung adenocarcinoma.Chapter 2:Expression profile of transfer RNA-derived small RNA in lung adenocarcinoma and the function and mechanism study of TRF-21RKP4P9L0 in lung adenocarcinomaBackground and objectives:With the advancement of high-throughput technology,some neglected transfer RNA(tRNA)-derived small RNAs(tsRNAs)are gradually discovered.They can participate in basic biological processes such as cell growth and differentiation,as well as play a role in the development of a variety of human diseases including neurological disorders,immune diseases and malignancies.Their potential as clinical biomarkers is progressively becoming recognized.This study aims to analyze the differentially expressed tsRNAs in blood and tissues of lung adenocarcinoma patients through public data;by constructing the expression profile of lung adenocarcinoma tsRNAs and establishing a tsRNA-mRNA regulatory network to find the tsRNAs that play key roles in lung adenocarcinoma;through machine learning methods to find the tsRNAs that can be used to predict lung adenocarcinoma thus providing new markers and targets for diagnosis and treatment of lung adenocarcinoma.Methods:(1)SRA database was used to download raw data of blood and tissue small RNA sequencing of lung adenocarcinoma and analyze differentially expressed tsRNAs;(2)tRFTar database was used to obtain differentially expressed tsRNA target mRNA,TCGA database was used to obtain differentially expressed mRNA in lung adenocarcinoma;cystoscope v3.8.2 and Gephi v0.9.2 were used to construct tsRNA and mRNA interaction network and look for hub genes;Metascape website was used to perform GO,KEGG,Wikipathway and Reactome enrichment analysis;(3)machine learning methods were used to identify tsRNAs that can be used for lung adenocarcinoma diagnosis;(4)cck8 and transwell experiments were used to explore the role and mechanism of tRF-21-RK9P4P9L0 in lung adenocarcinoma.Results:(1)expression level of tsRNAs were significantly different in plasma and tissues between normal people and lung adenocarcinoma patients,with more significant differences in plasma;(2)differential analysis identified a total of 155 tsRNAs with consistent expression in blood and tissues,of which 135 were up-regulated and 20 were down-regulated;(3)A tsRNA-mRNA regulatory network containing 155 tsRNAs and 406 mRNAs was constructed,and three tsRNAs(tRF-16-L85J3KE,tRF21-RK9P4PL0 and tRF-16-PSQP4PE)were the core genes in the network;enrichment analysis showed that the target genes of differential tsRNAs were enriched in multiple cancer-related pathways;(4)Lung adenocarcinoma prediction models were constructed using three tsRNAs(tRF-16-L85J3KE,tRF-21-RK9P4PL0 and tRF-16PSQP4PE),and the results suggested that combining three tsRNAs predicted better than single tsRNAs;(5)tRF-21-RK9P4PL0 was negatively correlated with the prognosis of lung adenocarcinoma;tRF-21-RK9P4PL0 promoted the proliferation,migration and invasion of A549 and H1299 through negative regulation of NOTCH1Conclusions:(1)The expression profile and tsRNA-mRNA regulatory network of transfer RNA-derived small RNAs in lung adenocarcinoma were first constructed,which is helpful for exploring the mechanism of lung adenocarcinoma carcinogenesis and finding new diagnostic markers for lung adenocarcinoma;(2)The diagnostic sensitivity of tsRNAs in blood is higher than that of tsRNAs in tissues;combining three tsRNAs is better than single tsRNAs for diagnosis;(3)tRF-21-RK9P4PL0 promotes proliferation,migration and invasion of lung adenocarcinoma cells through negative regulation of NOTCH1.