ABSTRACT
Objective:To investigate the expression, regulation, potential mechanism and clinical significance of microRNA(miRNA)-129-1 in colon cancer.Methods:The changes of expression and methylation of miRNA-129-1 were analyzed from the methylation, mRNA expression and miRNA expression data of colon cancer in the cancer genome atlas (TCGA) database. The target genes of miRNA-129-1 were predicted from miRwalk 2.0 and TargetScan database. DAVID 6.7 online software was used for gene oncology and Kyoto encyclopedia of genes and genomes enrichment analysis. STRING database was used for protein-protein interaction analysis. TCGA data were applied again to analyze the differential expression and prognosis of key target genes of miRNA-129-1. Paired t test and independent sample t test were used for statistical analysis. The receiver operating characteristic curve (ROC) was used to evaluate the diagnostic value of miRNA-129-1 gene methylation in colon cancer. Kaplan-Meier method and log-rank test were used to analyze the effects of miRNA-129-1 expression on survival. Results:The sequence of miRNA-129-1 among different species was conserved. After all colon cancer samples, and control samples of TCGA database were analyzed, the results showed that compared with those of control samples, the expression of miRNA-129-1 decreased in cancer samples (0.98±0.81 vs. 5.74±0.59), and the methylation levels of cg04524088, cg04840800, cg11364290, cg20734982 and cg24044186 locus of miRNA-129-1 significantly decreased (0.321±0.130 vs. 0.563±0.051, 0.432±0.123 vs. 0.624±0.064, 0.475±0.153 vs. 0.768±0.033, 0.659±0.180 vs. 0.816±0.037 and 0.862±0.096 vs. 0.916±0.019, respectively) in colon cancer tissues, and the differences were all statistically significant ( t=14.95, 11.36, 9.39, 11.74, 5.32 and 3.47, all P<0.01). The results of ROC analysis showed that the methylation levels of the above five locus of miRNA-129-1 gene had high diagnostic efficiency in colon cancer (area under curve=0.946, 0.915, 0.950, 0.758 and 0.667, all P<0.01). The results of survival analysis indicated that low expression of miRNA-129-1 was associated with poor prognosis (hazard ratio ( HR)=0.55, P=0.018). The results of bioinformatics analysis demonstrated that the target genes of miRNA-129-1 were enriched in serine / threonine kinase receptor, mitogen-activated protein kinase and other functional gene clusters closely related to tumor, and there was a complex interaction network among the target genes proteins. The high expression of ephrin type-B receptor2 ( EPHB2) gene, a potential key target gene of miRNA-129-1, was associated with the short overall survival and disease-free survival time ( HR=1.9 and 1.6, both P<0.01). Conclusions:The expression and methylation of miRNA-129-1 play an important regulatory role in the development and development of colon cancer. The methylation of miRNA-129-1 has potential value in the diagnosis of colon cancer, and miRNA-129-1 is an influencing factor for the prognosis of patients with colon cancer. EPHB2 may be a potential key target gene of miRNA-129-1.
ABSTRACT
Objective To evaluate the role of circular RNA protein arginine methyltransferase 5 (circPRMT5) in the genesis and progression of colorectal cancer.Methods From January 2013 to December 2017,96 patients with colorectal cancer who underwent radical resection in Department of General Surgery,Jiading District Central Hospital Affiliated to Shanghai Medical College of Health were collected.The expression of circPRMT5 in colorectal cancer tissues was examined by real-time polymerase chain reaction (RT-PCR).The correlation between circPRMT5 expression level and age,gender,tumor size,tumor location,pathological differentiation,TNM stage,lymph node metastasis of patients with colorectal cancer was analyzed.The SW620 and LOVO cells were divided into control group,circPRMT5-lenti group and circPRMT5-shRNA-lenti group according to different interventions.The effects of circPRMT5 expression level on viability,apoptosis,mitochondrial membrane potential and migration of SW620 and LOVO cells were detected.The influence of circPRMT5 expression level on E-cadherin,Slug,N-cadherin and vimentin was determined by Western blotting method.The potential target miRNA of circPRMT5 was predicted by Starbase V2.0.Student's t test,analysis of variance and chi-square test were performed for statistical analysis.Results The results of RT-PCR showed that the expression of circPRMT5 in colorectal cancer tissues was higher than that of adjacent cancer tissues (2.167 ± 0.345 vs.1.103 ± 0.144),and the difference was statistically significant (t =26.847,P ＜ 0.01).The circPRMT5 expression level was positively correlated with tumor size,TNM stage,lymph node metastasis and distant metastasis (x2 =6.010,10.971,5.321 and 6.272,all P ＜0.05).The upregulation of circPRMT5 expression could promote proliferation and migration of SW620 and LOVO cells.The circPRMT5 downregulation could inhibit cell proliferation,induce apoptosis and decrease mitochondrial membrane potential.The results of Western blotting indicated that,compared with those of control group,the expression of Slug,N-cadherin and vimentin increased in circPRMT5-1enti group (1.023 ±0.038 vs.2.105 ±0.042,1.051 ±0.309 vs.2.277 ± 0.111,1.055 ± 0.040 vs.2.002 ± 0.537,respectively),however the expression of E-cadherin decreased (2.074 ± 0.214 vs.0.627 ± 0.023),and the differences were statistically significant (t =31.817,22.065,14.536 and 9.148,all P ＜ 0.01).Compared with the control group,the expression of Slug,N-cadherin and vimentin decreased in circPRMT5-shRNA-lenti group (1.023 ± 0.038 vs.0.585 ± 0.023,1.051 ± 0.309 vs.0.616 ± 0.043,1.055 ±0.040 vs.0.537 ±0.022),while the expression of E-cadherin increased (2.074 ± 0.214 vs.2.756 ± 0.148),and the differences were statistically significant (t =-13.795,-14.252,-11.794 and-13.116,all P ＜ 0.05).A total of 21 miRNAs might have potential binding sites with circPRMT5 predicted by Starbase V2.0 software.The expression of miRNA4735-3p,miRNA202-3p,miRNA326,let-7i-5p and miRNA4500 was negatively correlated with circPRMT5 expression in both SW620 and LOVO cells confirmed by RT-PCR.Conclusion CircPRM75 is an important oneogenic gene in the genesis and progress of colorectal cancer,and may have certain potential application prospect in the research and development for colorectal cancer.