| Background and object:Colorectal cancer is a tumorous disease with a very high incidence and mortality in China and even the world.Although great progress has been made in its diagnosis and treatment for colorectal cancer,most patients has progressed to middle and advanced stages,such distant metastases.Therefore,the discovery of early diagnosis and prognostic markers of colorectal cancer has become more and more important for patients.The purpose of this study was to screen for abnormally expressed lncRNA between colorectal cancer tissues and normal tissues,and to explore lncRNAs that affect the survival prognosis of colorectal cancer patients and the function of colorectal cancer cell lines.This study includes two parts:In the first part,we obtain differentially expressed lncRNA through bioinformatics,screen out the target lncRNA,analyze the relationship between the lncRNA and survival prognosis,and its potential biological functions and involved signaling pathways;In the second part,we regulated the expression of target lncRNA to observe its effect on the proliferation,invasion and migration of colorectal cancer cell lines(Hctll6,HT29,SW480,SW620).Part 1 Abnormal expression,survival prognosis,and potential biological functions of lncRNA in colorectal cancer.Materials and Methods1.Download the HTSeq-FPKM transcript of TCGA database colorectal cancer and organize data the data in Perl language;2.Differential analysis of lncRNA transcripts between 647 colorectal cancer tissues and 51 normal tissue using edgeR package by R language 3.6.1;3.The linc01977 obtained by screening the differentially expressed lncRNAs was analyzed by Wilcoxon rank sum test(unpaired)and Wilcoxon sign-rank test(paired),its correlation with clinical stage,specificity and sensitivity analysis of tumor tissues relative to normal tissues;4.The median value of linc01977 in all tumor patients was divided into high and low expression groups,and log-rank test of Kaplan-Meier method was used for survival analysis;5.Correlation analysis between mRNAs and linc01977 in the all HTSeq-FPKM samples was performed(correlation coefficient>0.4),furthermore,GO(gene ontology)analysis and KEGG(Kyoto Encyclopedia of Genes and Genomes)signal pathway enrichment also was performed.In addition,PPI network analysis(minimum required interaction score-high confidence=0.700)was performed on these related mRNAs to screen out the top 10 hub mRNAs.Results1.Analysis of differential expression of lncRNA transcripts between colorectal cancer tissues and normal tissues revealed 1229 differentially expressed lncRNAs,of which 278 were up-regulated and 951 were down-regulated;2.Wilcoxon rank sum test and Wilcoxon sign rank test results showed that the linc01977 expression in tumor tissues was significantly higher than that in normal tissues(p<0.001),and its expression in mid-advanced colorectal cancer patients(Ⅲ+Ⅳ)were significantly higher than early(Ⅰ+Ⅱ)(p<0.001);The specificity and sensitivity of linc01977 expression in tumor tissue relative to normal tissue are 0.711 and 0.893,respectively,and the AUC value is 0.86;3.Colorectal cancer patients with high expression of linc01977 exhibit shorter overall survival(OS)and disease-free survival(DFS)than those with low expression of linc01977.4.According to the screening criteria,696 mRNAs related to linc01977 were obtained.The GO analysis showed that the molecular functions(MF)involved in these mRNAs include:transferase activity,methyltransferase activity,and S-adenosylmethionine.Acid-dependent methyltransferase activity,N-methyltransferase activity,protein lysine N-methyltransferase activity,lysine N-methyltransferase activity;KEGG signaling pathway enrichment analysis revealed tumors involved Signal-related pathways include:mTOR signaling pathway,VEGF signaling pathway,Notch signaling pathway,NF-kappa B signaling pathway,and choline metabolism information pathways in cancer.The related genes were analyzed by PPI network.According to high confidence.Finally,253 mRNAs were obtained to form 473 relationships with each other.The top 10 hub mRNAs(NAT10,DDX27,DDX56,RRP12,BYSL,RRS1,RBM19,NOL6,WDR74,ODF2)were found by MCC method using Cytoscape version 3.6.3 and plug-in cytoHubb;In addition,we also found that two common tumor proliferation markers PCNA and KI67 had a certain correlation with linc01977Part 2 Effect of regulating lncRNA-linc01977 expression on the proliferation,invasion and migration of colorectal cancer cell lines(Hctll6,HT29,SW480,SW620).Materials and Methods1.RT-PCR detection of lncRNA-linc01977 in colorectal cancer cell lines Hctll6,HT29,SW480,SW620 and normal colorectal epithelial mucosal epithelium FHC expression;2.Transfect linc01977-siRNA with cell lines(SW480,HT29)that express higher linc01977 and verify the transfection efficiency;3.CCK-8 assay,DAPI and EDU staining and colony formation assay were used to detect the effects of down-regulating linc01977 on the proliferation of SW480 and HT29 cells;4.The Wound Healing and Transwell assay were used to detect the effect of down-regulating linc011977 on the invasion and migration ability of SW480 and HT29 cells.Results1.RT-PCR showed that the relative expression of linc01977 in human colorectal cancer cell lines(Hctll6,SW620,SW480,HT29)was significantly higher than that of human normal colonic epithelial cells(FHC)[(1.23±0.10)&(1.83±0.06)&(3.04±0.07)&(3.87±0.14)vs(0.98±0.13)],which was obvious with SW480 and HT29.2.The relative expression levels of linc01977-siRNA1 group,linc01977-siRNA2 group,linc01977-siRNA3 group and NC-si group in SW480 cells were(0.35±0.17),(0.21±0.06),(0.37±0.08),and(1.01±0.11).Compared with the NC-si group,the first three groups have statistically significant differences(p<0.05).The relative expression levels of linc01977-siRNA1 group,linc01977-siRNA2 group,linc01977-siRNA3 group and NC-si group in HT29 cells were respectively(0.19.±0.03),(0.37±0.04),(0.35±0.08),and(1.11±0.03),the first three groups have statistically significant differences(p<0.05).The results showed that linc01977 siRNA effectively down-regulated the expression of linc01977 in the two cell lines.3.CCK-8 assay showed that the proliferation ability of transfecting linc01977-siRNA2 SW480 and HT29 cells at 24h,48h,and 72h were significantly lower than that of the NC-si group,and the difference was statistically significant(p<0.05).In addition,DAPI and EDU staining and colony formation assay also showed that the proliferation ability of transfecting linc01977-siRNA2 SW480 and HT29 cells at 24h were significantly lower than that of the NC-si group,the difference was statistically significant(p<0.05),these suggested that down-regulation of linc01977 could significantly inhibit the proliferation of colorectal cancer cell lines(SW480,HT29).4.The Wound Healing and Transwell assay showed that the migration and invasion ability of transfecting linc01977-siRNA2 SW480 cells and HT29 cells were reduced compared with NC-si control group(p<0.05).ConclusionsLinc01977 may promote the proliferation,invasion and migration of colorectal cancer cells through multiple tumor-related signal pathways and tumor-related mRNA,and play an oncogene role as a prognostic factor for patients with colorectal cancer,which provides a theoretical basis for the occurrence and development of colorectal cancer. |
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