The Biological Function And Mechanism Of Long Non-coding RNA LINC02474 In Colorectal Cancer

Colorectal cancer(CRC),which comprises colon cancer and rectal cancer,is the third most malignant tumor worldwide.Its fatality rate ranks up to the second,seriously threatening human health.In the past ten years,the incidence and fatality of CRC is a continuous upward trend and going younger.Metastasis is the crucial feature of CRC,also the leading cause of high incidence and fatality.Tumor regulators play an important role during this process.Exploring effective targets for CRC metastasis,and revealing their biological function and mechanism to block tumor progression as early as possible is the main issue in reducing CRC incidence and fatality rate and improve prognosis.With the development of high-throughput sequencing technology,new markers are spring up.Among these,the role of long non-coding RNA(lncRNA)in tumorigenesis has attracted much public attention.lncRNAs can function in serval aspects,such as epigenetic modification,transcriptional regulation,and protein translation,as well as many core biological processes consist of tumor proliferation,metastasis,and apoptosis.As we all known,the onset of CRC involves a complex process,including multi-factor,multi-gene,and multi-step participation.It is the combined result of genetic and environmental factors,as one of the participates,lncRNAs power critical regulatory roles.Objective:This study mainly explores the biological functions of LINC02474 on CRC cells,and its potential mechanism to pave novel ways to find new biomarkers for CRC.Methods:1.Acquiring the transcriptome data of tumor and compared adjacent tissues in CRC patients from the TCGA database.Then,the R 3.5.2 software and the "DESeq2"package were used to obtain differentially expressed lncRNAs and highly expressed molecule,LINC02474.2.qRT-PCR identified the expression characteristics of LINC02474 among CRC tissues and adjacent tissues,CRC cells and normal epithelial cells,as well as CRC serum and healthy people serum.The Kaplan-Meier survival analysis and ROC curve were used to evaluate patients’ prognosis and the diagnostic efficacy of LINC02474 expression in patients.In addition,the distribution features of clinicopathological parameters for CRC patients was assessed by the Kolmogorov-Smirnov test.And the distribution characteristic of LINC02474 expression between pathological parameters of two groups was detected by Mann-Whitney U test,while multiple groups by Kruskal-Wallis one-way ANOVA test.3.Using the lentiviral packaging vector to construct cell lines in which LINC02474 were stably knocked down or overexpressed.The effects of LINC02474 on CRC metastasis was explored by nude mice experiments and transwell assays.Apoptosis of CRC cells and surface biomarkers of CRC serum exosomes were identified by flow cytometry and Western blot.RNA FISH was used for subcellular localization of LINC02474.Electron microscopy was used to detect morphological features of exosomes.And NTA was used to confirm the size distribution of exosomes.4.RNA-seq,differential gene analysis,functional enrichment analysis and other bioinformatics methods were used to reveal the potential biological processes,signal pathways as well as target genes related to LINC02474.Dual-luciferase report experiment and Actinomycin D assay were used to explore the effect of LINC02474 on the transcription of Granzyme B(GZMB)and their potential regulation.Results:1.A total of 698 cases of CRC-related samples were downloaded from the TCGA database with RNA-seq data types,including 647 cases of tumor tissues and 51 adjacent tissues.It was found that LINC02474 was highly expressed in CRC tissues from the differential lncRNAs profiles(p<0.05).And the area under ROC curve(AUC)was 0.7915,95%confidence interval(CI)was 0.7495-0.8335.Besides,the high expression of LINC02474 in CRC tissues associated with a trend of shorter survival time(p=0.065).2.qRT-PCR results from 80 pairs of CRC tissues and corresponding adjacent tissues,6 CRC cell lines and 1 normal cell line,and 128 CRC serum samples and 128 healthy serum samples all identified that LINC02474 was highly expressed in CRC(p<0.05).At the same time,the Kolmogorov-Smirnov test showed that the clinicopathological parameters of CRC patients representing a non-normal distribution;the Mann-Whitney U test and Kruskal-Wallis one-way ANOVA test analyzed the relationship between LINC02474 and the clinicopathological parameters of CRC patients,finding that the expression of serumal LINC02474 in colon region(2.721(0.6449-4.801))was higher than the rectal region(1.030(0.1847-2.955)).3.In constructed DLD-1,LOVO and HCT116 cell lines which LINC02474 was stably knocked down.The transwell experiment found that all 3 cell lines showing weakened migration and invasion ability.In contrast,flow cytometry showed increased apoptosis after LINC02474 was downregulated(p<0.05).4.In vivo animal experiments found that after nudes were injected with DLD-1 cells in which LINC02474 was stably knocked down through the tail vein,fewer metastatic cancer nodules in the lungs than the control groups(p<0.05).5.SW1116 and SW480 cell lines were constructed in which LINC02474 was stably overexpressed.It was shown that migration and invasion ability was enhanced and apoptosis was reduced after LINC02474 was upregulated(p<0.05).6.RNA-seq showed that GZMB might be the target gene of LINC02474.GZMB expression was upregulated in cells in which LI-NC02474 was knocked down.And the downregulated metastatic ability caused by LINC02474 deletion could be reversed,after GZMB was deleted in cells in which LINC02474 was downregulated.7.The luciferase activity of the GZMB promoter in groups of which LINC0274 stably knocked down was more robust than their control groups,which indicated that LINC02474 might exert its regulation on CRC metastasis and apoptosis by mediating the transcriptional inhibition to GZMB.However,LINC02474 had no apparent effect on the mRNA stability of GZMB.Conclusion:1.In this study,a novel lncRNA LINC02474 was selected from CRC IncRNAs profiles,it represented high expression in CRC tissues,cells and serum samples.2.This study found that LINC02474,as an oncogene,can promote the invasion and migration of CRC while inhibiting cell apoptosis.3.This study initially revealed that LINC02474 could play its regulatory roles on CRC metastasis and apoptosis by exerting transcriptional suppression on GZMB.