Screening And Preliminary Validation Of Long Non-coding Associated With Colorectal Cancer Based On GEO Database

ObjectiveThe incidence rate of colorectal cancer（CRC）is increasing year by year.It has become one of the major public health problems threatening human health.At present,the treatment effect of advanced CRC is still poor,and the lack of effective early diagnosis is one of the main reasons for the poor treatment effect.Long non-coding RNA（lnc RNA）regulates genes expression and plays an important role in the development of tumors,but its role in the development of CRC remains to be further studied.Therefore,in this study,we used the public database（GEO）to screen lnc RNA that may be associated with pathogenesis of CRC and experimentally validate their expression in CRC and the relationship with clinical features,so as to provide some reference for the follow-up study of function and mechanism.MethodsDownload the CRC related RNA sequence data in the GEO database,select the qualified data set according to the inclusion and exclusion criteria,preliminarily process the data to obtain lnc RNA with differential expression in cancer tissues and adjacent non-cancerous tissues.The target genes of lnc RNA were predicted by bioinformatics.Analyze the function,pathway enrichment and protein interaction network of lnc RNA targeted m RNA,and predict whether they may participate in the occurrence and development of CRC.q RT-PCR was used to validate the CRC-related lnc RNA selected by GEO,analyze their expression in CRC cancer tissues and adjacent non-cancerous tissues,and investigate the relationship between CRC-related lnc RNA and clinical factors such as gender,age,the size of tumor,depth of intestinal wall invasion,lymphatic metastasis and clinical stage by statistical analysis in combination with the clinical data of CRC patients,and analyze the sensitivity and specificity of each lnc RNA in CRC by receiver operating characteristic curve（ROC）.ResultsThe datasets were obtained by GEO database screening: GSE18105 and GSE126092,of which 97 and 322 differentially expressed lnc RNA,respectively.Among these differentially expressed lnc RNA,the four lnc RNA with the greatest fold change in differential expression were: up-regulated: CRNDE and ZFAS1;down-regulated: DPP10-AS1 and MBNL1-AS1.According to the lnc RNA target gene prediction website,it was predicted that these four lnc RNA may interact with 12 mi RNA.Further prediction revealed that 12 mi RNA may interact with 181 m RNA.Functional enrichment analysis of target genes revealed that CRNDE,ZFAS1,DPP10-AS1 and MBNL1-AS1 may be involved in tumor processes and regulate biological processes and pathways.The results of real time PCR revealed that the relative expression of CRNDE in CRC tissues was higher than that in adjacent non-cancerous tissues,and the difference was statistically significant;the relative expression of DPP10-AS1 and MBNL1-AS1 in CRC tissues was lower than that in adjacent non-cancerous tissues,and the difference was statistically significant;the ZFAS1 in CRC tissues was slightly higher,but the difference was not statistically significant（P=0.162）.The expression level of CRNDE was correlated with lymphatic metastasis,TNM stage and degree of intestinal wall invasion in CRC patients,regardless of age,the size of tumor,gender and tumor location;the expression level of DPP10-AS1 was correlated with the size of tumor and degree of intestinal wall invasion in CRC patients;and the expression level of MBNL1-AS1 was correlated with clinical stage and lymphatic metastasis in CRC patients.The area under the ROC curve AUC of CRNDE was 0.786,sensitivity 71.1%,specificity 77.8%,and Youden index 0.623;the area under the ROC curve AUC of DPP10-AS1 was 0.863,sensitivity 95.6%,specificity 66.7%,and Youden index 0.623;the area under the ROC curve AUC of MBNL1-AS1 was 0.883,sensitivity 91.1%,specificity 75.6%,and Youden index 0.827;and the area under the ROC curve AUC of ZFAS1 was 0.624,sensitivity 66.7%,specificity 64.4%,and Youden index 0.311.Conclusions1.Four lnc RNA that may be closely related to CRC were mined by bioinformatics:CRNDE,DPP10-AS1,MBNL1-AS1 and ZFAS1,of which CRNDE and ZFAS1 were up-regulated genes and MBNL1-AS1 and DPP10-AS1 were down-regulated genes in CRC tissues.The results of target gene pathway analysis indicated that they may be involved in the process of tumor development and regulate biological processes and pathways.2.CRNDE was up-regulated in CRC tissues;DPP10-AS1 and MBNL1-AS1 were down-regulated in CRC tissues.CRNDE expression levels were associated with lymphatic metastasis,TNM stage and degree of intestinal wall invasion in CRC patients;DPP10-AS1 expression levels were correlated with the size of tumor and degree of intestinal wall invasion in CRC patients;and MBNL1-AS1 expression levels were related to clinical stage and lymphatic metastasis.CRNDE,DPP10-AS1 and MBNL1-AS1 have some reference value as diagnostic effects of tumor markers.