Screening And Preliminary Validation Of Colorectal Cancer Associated Long Non-coding RNA Based On WGCNA

Objective Colorectal cancer(CRC)is the third highest incidence rate tumor in the world and one of the major causes of cancer related death.At present,the treatment effect of advanced CRC is still poor,and the lack of effective early diagnosis means is one of the main reasons for the poor treatment effect.Long Non-Coding RNA(lnc RNA)plays a critical role in the regulation of gene expression and the occurrence and development of tumors.However,the specific biological mechanism of lnc RNA in the occurrence and development of CRC remains to be further explored.Therefore,weighted gene co-expression network analysis(WGCNA)was adopted in this study.TCGA database was adopted to screen the lnc RNA possibly related to CRC,and q RT-PCR was used to verify its expression in CRC tissues and adjacent tissues and its relationship with clinical characteristics..It will provide a certain reference for the mechanism of CRC.Methods The RNA-seq data of CRC and normal colorectal tissues were downloaded from the TCGA database.Through the construction of WGCNA,gene modules related to CRC were divided,and hub lnc RNA in key modules were screened.Quantitave Real Time Polymerase Chain Reaction(q RT-PCR)was adopted to judge the possible CRC related lnc RNA screened from TCGA database,and analyze its expression in CRC tissues and adjacent tissues.Combined with the clinical information of CRC patients,the relationship between these lnc RNAs and clinical factors such as gender,age,tumor size,tumor location,degree of intestinal wall invasion and clinical stage of CRC patients was explored by statistical analysis.Receiver Operating Characteristic Curve(ROC)was utilized to probed the sensitivity and specificity of each lnc RNA in CRC.Results RNA-seq data of CRC and normal colorectal tissues were downloaded from TCGA database.The expression matrix was standardized and the Median Absolute Deviation(MAD)algorithm was applied to screen out 1627 lnc RNAs with relatively high absolute deviation.The WGCNA was constructed and finally divided into 8modules.Blue,green and yellow modules related to CRC were selected,and four hub lnc RNAs CDKN2B-AS1,CAPN10-DT,ADAMTS9-AS2 and MEF2C-AS1 were finally screened out.The q RT-PCR results showed that the relative expression levels of CDKN2B-AS1,MEF2C-AS1 and ADAMTS9-AS2 in CRC tissues were lower than those in adjacent tissues,and the difference was statistically significant(P<0.001).The relative expression of CAPN10-DT in CRC tissues was slightly lower than that in adjacent tissues,but the difference was not statistically significant(P=0.175).The expression differences of CDKN2B-AS1,CAPN10-DT,ADAMTS9-AS2 and MEF2C-AS1 in CRC patients with disparate clinical characteristics were analyzed.The results showed that the expression of CDKN2B-AS1 was significantly different in CRC patients with different ages(P<0.05).The expression of CDKN2B-AS1 in CRC patients under 60 years old was higher than that in CRC patients over 60 years old.There was no significant difference in CDKN2B-AS1 expression among CRC patients with different gender,tumor size,clinical stage,tumor location and degree of intestinal wall invasion.No correlation has been found between CAPN10-DT,ADAMTS9-AS2,and MEF2C-AS1 with clinical stage,tumor size,age,tumor site,sex,and degree of intestinal wall invasion in CRC patients.The results of ROC curve showed that the AUC of CDKN2B-AS1 was 0.94,the sensitivity was 0.98,the specificity was 0.85,and the Youden index was 0.83.The AUC of CAPN10-DT was 0.58,the sensitivity was0.85,the specificity was 0.38,and the Youden index was 0.23.The AUC of ADAMTS9-AS2 was 0.90,the sensitivity was 0.73,the specificity was 0.92,and the Youden index was 0.65.The AUC of MEF2C-AS1 was 0.79,the sensitivity was 0.56,the specificity was 0.90,and the Youden index was 0.46.Conclusions1.Four lnc RNAs that may be closely related to CRC were found by WGCNA analysis: CDKN2B-AS1,CAPN10-DT,ADAMTS9-AS2 and MEF2C-AS1.Among them,CAPN10-DT was up-regulated in CRC tissues,and CDKN2B-AS1,ADAMTS9-AS2 and MEF2C-AS1 were down-regulated in CRC tissues.2.The results of q RT-PCR confirmed that CDKN2B-AS1,ADAMTS9-AS2 and MEF2C-AS1 were down-regulated in CRC tissues,but there was no significant difference in the expression of CAPN10-DT between CRC tissues and adjacent tissues.3.The expression of CDKN2B-AS1 in CRC patients under 60 years old was higher than that in CRC patients over 60 years old.CAPN10-DT,ADAMTS9-AS2 and MEF2C-AS1 were not found to be related to clinical stage,tumor size,age,tumor location,gender and degree of intestinal wall invasion in CRC patients.The results of ROC analysis showed that CDKN2B-AS1,ADAMTS9-AS2 and MEF2C-AS1 had high predictive value in distinguishing CRC tissues from adjacent tissues.