| Objective: Colorectal cancer is the third largest cancer in the world after breast and lung cancer,and in 2020 statistics,colorectal cancer had the second highest mortality rate of all cancers,at 940 000.Among them,the incidence of colorectal cancer in China has reached the second place,with about 550,000 new cases per year,and the mortality rate is the fifth.According to cancer data released in 2018,the incidence of colorectal cancer has been declining from 1976 to 2014.The occurrence and progression of colorectal cancer is inseparable from the interstitial transformation of epithelial cells,and the resulting cell migration and invasion is one of the main causes of colorectal cancer death.Epithelial-mesenchymal transition(EMT)refers to the process by which epithelial cells undergo a series of programmed changes to eventually form mesenchymal cells.This leads to metastasis and invasion of colorectal cancer,which in turn leads to the death of colorectal cancer.At present,it has been found that there are many signal pathways that can promote the occurrence of EMT,such as Wnt/β-catenin signaling pathway,TGF-β signaling pathway,RTK signaling pathway,Notch signaling pathway,etc.The Wnt/β-catenin signaling pathway can promote the EMT process in many cancers.In colorectal cancer,the Wnt/β-catenin signaling pathway has been shown to activate the EMT pathway,and the Wnt pathway plays a role by promoting β-catenin nucleation to activate TCF/LEF,which is closely related to the occurrence and development of EMT in colorectal cancer.Long noncoding RNA(lncRNA)has a nucleotide length greater than 200,unlike m RNA,lncRNA is an RNA transcript that lacks an obvious open reading frame,does not encode proteins or peptides,but can participate in the development of many cancers.LASTR,also known as LINC02657,can promote the occurrence of cancer in stomach,breast and lung cancer.In this study,we demonstrated that lncRNA LASTR promotes the proliferation and metastasis of colorectal cancer,and that LASTR promotes the entry of β-catenin protein into the nucleus by regulating CRIP1,and the β-catenin protein entering the nucleus strengthens the binding of TCF/LEF binding elements in the nucleus to promote the occurrence of colorectal cancer.In addition,we have verified in clinical specimens that the molecule is significantly higher in tissues from colorectal cancer patients than normal colorectal tissue.The research results of this experiment will provide new options for early diagnosis and treatment of colorectal cancer.Methods:1.TCGA database colorectal cancer data analysis and colorectal cancer cell line expression difference verification(1)The data of 514 colorectal cancers downloaded were screened for differential genes,and the screening criteria were |log2FC|>2,FDR<0.01;(2)Univariate COX regression(HR>1,P<0.05)was performed on the screened differential genes and multivariate COX regression analysis to further screen for differential genes;(3)Tumor sample and normal sample expression analysis and survival analysis of target gene;(4)The expression of LASTR was detected by quantitative real time PCR(q RT-PCR)in common bowel cancer cell lines(HCT116,HT29,Caco-2,SW480,SW620)and normal cell line HIEC.2.Evaluation of the biological function of LASTR in colorectal cancer cells(1)HCT116 and HT29 were selected as follow-up research objects,and overexpression plasmids and knockdown models of LASTR were constructed in HCT116 and HT29;(2)CCK8 and cell plate cloning experiments verified whether LASTR had any effect on CRC cell proliferation;(3)Transwell and cell scratch experiments verified whether LASTR had an effect on CRC cell migration;(4)Flow apoptosis detection experiments verified whether LASTR had an effect on the apoptosis level of CRC cells.3.LASTR promotes the mechanism of EMT process in CRC cells(1)WB experiments detected the changes of EMT-related indexes(Desmin,E-cad,Snail)after knockdown and overexpression of HCT116 and HT29 cell lines;(2)transcriptome gene sequencing for the LASTR knockdown group,GO analysis and KEGG pathway pathway analysis on the sequencing data;(3)Bioinformation analysis CRIP1 was evaluated in tumor tissue and normal tissue expression in TCGA database,and CRIP1 expression detection was carried out in PCR experiment and WB experiment for knockdown LASTR group;(4)WB experiments were performed on cells after knockdown and overexpression of HCT116 and HT29 cell lines,and changes in Wnt pathway index β-catenin and downstream index c-myc were observed;(5)Set up the recovery experiment,WB experiment verified the relationship between LASTR activating Wnt/β-catenin pathway by regulating CRIP1 to promote the occurrence of CRC cell EMT;(6)In situ hybridization was used to detect the expression level of LASTR in paraffin sections of clinical tissues;Results:1.Biocredit analysis and differential expression results(1)717 differential lncRNAs(594 up-regulated and 123 down-regulated)were screened for colorectal cancer,and 7 genes(P<0.05)were further screened by COX regression analysis;(2)During the analysis of clinical samples,it was observed that LASTR had certain differences between CRC tissues and normal tissues,and the survival time of the high-expression LASTR group was shorter than that of the low-expression group(P<0.05);(3)It was verified that the expression of LASTR in bowel cancer was higher than that of normal intestinal epithelial cells(P<0.05);2.The results of cell function experiments showed that LASTR overexpression can promote CRC cell proliferation,migration and inhibit apoptosis,and LASTR knockdown can inhibit cancer cell proliferation,migration and promote apoptosis(P <0.05).3.LASTR promotes the EMT process of CRC cells by regulating the activation of Wnt/β-catenin by regulating CRIP1(1)WB results showed that LASTR could promote the occurrence of EMT process,and the expression of E-cad in epithelial indexes decreased in HCT116 and HT29 after overexpression of LASTR,the expression of interstitial indexes Desmin and Snail increased,the expression of E-cad increased after knockdown,and the expression of Desmin and Snail decreased(P<0.05);(2)The transcriptome sequencing results showed that the differential genes of knocking out LASTR could be enriched into the Wnt signaling pathway,and LASTR could be related to the development and progression of cancer(P<0.05);(3)The sequencing results showed that the level of CRIP1 decreased after LASTR knockdown,and PCR and WB experiments proved that the expression of CRIP1 after LASTR knockdown decreased(P<0.05);(4)LASTR can promote the occurrence of EMT process in colorectal cancer through Wnt/β-catenin,and the expression of Wnt pathway indexes β-catenin and downstream c-myc indexes increases and decreases after overexpression and knockdown LASTR(P<0.05);(5)WB recovery experiments proved that LASTR promoted the occurrence of EMT in CRC cells by regulating the Wnt/β-catenin pathway by regulating CRIP1(P<0.05);(6)The expression of LASTR in CRC tissues was higher than that in adjacent tissues(P<0.05).Conclusion: LASTR activates the Wnt/β-catenin signaling pathway by regulating CRIP1,thereby promoting EMT in colorectal cancer. |
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