Screening Of Differentially Expressed LncRNAs And Its Function In Patients With Colorectal Cancer

Recently,LncRNAs play important role in the occurrence and development of cancer,because of its unique characters and has become a new research hotspot.A number of researchers have found that many functional lncRNAs can directly or indirectly regulate the expression of oncogenes or tumor suppressor genes,suggesting that they can be used as a tool for tumor diagnosis and treatment,and open up a novel direction.Colorectal cancer?CRC?is one of most common human cancers,and the second cause of cancer death worldwide.However,the molecular mechanism driving their development and recurrence is still not fully known.The object of this study is to screen lncRNAs in CRC samples and normal colorectal samples,providing molecular mechanism for CRC diagnosis and therapy.Part One Screening of differentially expressed lncRNAs and mRNAs in colorectal cancer and bioinformatics analysisObjective: To explore the differences in the expression profiles of lncRNAs and mRNAs in colorectal cancer?CRC?tissues using next-generation sequencing?NGS?technology,to perform lncRNA-mRNA network analysis and differentially expressed genes by GO/KEGG enrichment analysis,and to predict new lncRNA,thereby providing experimental basis for diagnosis and treatment of colon cancer.Methods:1.CRC tissues and adjacent non-cancerous tissues from 10 patients with CRC were collected respectively.Following total RNA extraction and quantification,cDNA was hybridized and synthesized from RNA.The sequencing of the libraries was carried out using the Illumina HiSeq 3000 platform,and the original data was converted by statistical software for statistical analysis.Differential expression profiles of IncRNA and mRNA between the two tissues were obtained.2.In terms of differential genes,the sequencing results were analyzed using preliminary bioinformatics analysis,including KEGG pathway enrichment analysis and GO enrichment analysis,and the IncRNA-mRNA co-expression network was established.Results:A total of 3221 mRNAs?1606 up-regulated,and 1615 down-regulated?were differentially expressed in CRC tissues and adjacent non-cancerous tissues,according to sequencing of lncRNA and mRNA.Compared with the lncRNAs in non-tumorous tissues,1019 lncRNAs?512 up-regulated,and 507 down-regulated?were differentially expressed in CRC tissues.According to differential analysis of new lncRNA expression,109 new lncRNAs were identified in this assay,including 102 up-regulated and 7 down-regulated.GO analysis revealed that the mRNAs were mainly enriched in cellular components related to cell mitosis,such as spindles,chromosomes,and centromeres.This suggested that the effect of up-regulation or down-regulation of mRNAs on cell components might be related to mitosis.According to the results of pathway enrichment analyses and in combination with the number of enriched genes,the most potential networks for our differentially expressed lncRNAs and mRNAs included the cell cycle pathway,protein digestion and absorption pathway,cell adhesion molecules pathway and alcoholism pathway.Several classical pathways,including Wnt signaling pathway,the PI3K-Akt signaling pathway,the TGF-? signaling pathway,the MAPK signaling pathway and the P53 signaling pathway,were found in the CRC pathway diagram.Summary:Ten pairs of differentially expressed lncRNA and mRNA in CRC tissues and adjacent non-cancerous tissues were screened,the sequencing results were analyzed by bioinformatics,and the IncRNA-mRNA co-expression network was established.Part Two Verification of differentially expressed IncRNA and mRNA in CRC tissuesObjective: To investigate the expressions of 4 lncRNAs?FIRRE-201,SLCO4A1-AS1-202,LINC02163-201,and FEZF1-AS1-203?up-regulated in the expression profile,and 3 lncRNAs?PGM5-AS1-202,ADIPOQ-AS1-201,and SLC30A10-201?down-regulated in independent samples,and to explore the expressions of 3 up-regulated mRNAs?NM₀01062,NM₀02994,and NM₀01012964?,and 3 down-regulated mRNAs?NM₀05182,NM₀02594,and NM₀01080400?in independent samples.Methods:1.Expressions of 4 lncRNAs?FIRRE-201,SLCO4A1-AS1-202,LINC02163-201,and FEZF1-AS1-203?up-regulated in the expression profile,and 3 lncRNAs?PGM5-AS1-202,ADIPOQ-AS1-201,and SLC30A10-201?down-regulated in CRC tissues and non-cancerous tissues were detected by RT-qPCR.2.Expressions of 3 mRNAs?NM₀01062,NM₀02994,and NM₀01012964?up-regulated in the expression profile,and 3 down-regulated lncRNAs?NM₀05182,NM₀02594,and NM₀01080400?in CRC tissues and non-cancerous tissues were detected by RT-qPCR.Subsequently,the above-mentioned expressions were compared with sequencing results.Results:1.RT-qPCR assay demonstrated that the expressions of 4 lncRNAs?FIRRE-201,SLCO4A1-AS1-202,LINC02163-201,and FEZF1-AS1-203?up-regulated in the expression profile were consistent with sequencing results.2.RT-qPCR assay showed that the expressions of 3 lncRNAs?PGM5-AS1-202,ADIPOQ-AS1-201,and SLC30A10-201?down-regulated in the expression profile were in accordance with sequencing results.3.RT-qPCR assay revealed that the expressions of 3 mRNAs?NM₀01062,NM₀02994,and NM₀01012964?up-regulated in the expression profile accorded with sequencing results.4.RT-qPCR assay demonstrated that the expressions of 3 mRNAs?NM₀05182,NM₀02594,and NM₀01080400?down-regulated in the expression profile were in line with sequencing results.Summary:The expressions of IncRNA FIRRE-201,SLCO4A1-AS1-202,LINC02163-201,and FEZF1-AS1-203 were up-regulated in CRC tissues,whereas those of IncRNA PGM5-AS1-202,ADIPOQ-AS1-201,and SLC30A10-201 were down-regulated in CRC tissues.The expressions of mRNA NM₀01062,NM₀02994,and NM₀01012964 were up-regulated in CRC tissues,while those of mRNA NM₀05182,NM₀02594,and NM₀01080400 were down-regulated in CRC tissues.Part Three Knockdown of lncRNA FIRRE-201 or SLCO4A1-AS1-202 could inhibit proliferation,migration and metastasis of CRC cellsObjective: To investigate the effects of knockdown of lncRNA FIRRE-201 or SLCO4A1-AS1-202 on proliferation,migration,invasion and colony formation abilities of CRC cells.Methods:1.siRNA mediated knockdown of FIRRE-201 or SLCO4A1-AS1-202 expression in colon cancer cells HCT116 and SW480 cells was performed,which was subsequently verified by qPCR.2.MTS assay,Transwell and Matrigel assays,wound healing assay and colony formation assay were used to detect the effect of knockdown of lncRNA FIRRE-201 or SLCO4A1-AS1-202 on proliferation,migration,invasion and colony formation abilities of CRC cells.Results:1.MTS assay demonstrated that knockdown of FIRRE-201 or SLCO4A1-AS1-202 suppressed the proliferation of colon cancer cells HCT116 or SW480 in vitro?P<0.05?.2.Transwell assay showed that knockdown of FIRRE-201 or SLCO4A1-AS1-202 could suppress the migration of colon cancer cells HCT116 or SW480 in vitro?P<0.05?.Wound healing assay indicated that knockdown of lncRNA FIRRE-201 or SLCO4A1-AS1-202 could decrease migration abilities of HCT116 or SW480 cells in vitro?P<0.05?.3.Matrigel and Transwell assays showed that knockdown of lncRNA FIRRE-201 or SLCO4A1-AS1-202 could inhibit the invasion of colon cancer cells in vitro?P<0.05?.4.Colony formation assay demonstrated that knockdown of lncRNA FIRRE-201 or SLCO4A1-AS1-202 could suppress the colony formation abilities of colon cancer cells?P<0.05?.Summary:Knockdown of lncRNA FIRRE-201 or SLCO4A1-AS1-202 could inhibit the proliferation,migration,invasion and colony formation in vitro of CRC cells.Conclusions:1.Ten pairs of differentially expressed lncRNA and mRNA in CRC tissues and adjacent non-cancerous tissues were screened,the sequencing results were analyzed by bioinformatics,and thus the IncRNA-mRNA co-expression network was established.2.The expressions of IncRNA FIRRE-201,SLCO4A1-AS1-202,LINC02163-201,and FEZF1-AS1-203 were up-regulated in CRC tissues,whereas those of IncRNA PGM5-AS1-202,ADIPOQ-AS1-201,and SLC30A10-201 were down-regulated in CRC tissues.In addition,the expressions of mRNA NM₀01062,NM₀02994,and NM₀01012964 were up-regulated in CRC tissues,while those of mRNA NM₀05182,NM₀02594,and NM₀01080400 were down-regulated in CRC tissues.3.Knockdown of lncRNA FIRRE-201 or SLCO4A1-AS1-202 could inhibit invasion,proliferation,migration and colony formation ability of CRC cells.