| Objective To explore the PRDI-BF1 and RIZ homology domain containing 12(PRDM12),Forkhead Box E1(FOXE1),Beta-1,3-Glucuronyltransferase 2(B3GAT2),Vimentin(VIM),Secreted frizzled-related protein 2(SFRP2)gene promoter methylation expression,analyze its sensitivity and specificity in the detection of colorectal cancer,and To explore its application value in clinical auxiliary diagnosis of colorectal cancer.Method This study was divided into two parts.The first part Detection of PRDM12,FOXE1,B3GAT2,VIM,SFRP2 gene promoter methylation levels in colorectal cancer tissues and normal tissues adjacent to cancer,preliminary evaluation of the potential of these genes as early screening markers for colorectal cancer.The paraffin section specimens of 31 patients with colorectal cancer who underwent surgical resection were selected from the First Affiliated Hospital of Naval Military Medical University.Pyrosequencing was used to detect PRDM12,FOXE1,B3GAT2,VIM,SFRP2-1,SFRP2-2 gene promoter methylation status in cancer tissues and normal tissues adjacent to cancer.The second part According to the results of the first part of the study,occult blood test and PRDM12,FOXE1,B3GAT2,VIM gene promoter methylation detection were performed on the stool of healthy people,colorectal cancer and adenomatous polyps patients,and then statistical analysis of single-gene detection and multi-gene combined detection Diagnostic performance,further study the potential and clinical application value of these gene methylation tests as early screening markers for colorectal cancer.A total of248 research subjects were selected in this part: 105 healthy patients,78 colorectal cancer patients,and 65 adenomatous polyps patients.This study was approved by the Medical Ethics Committee of Changhai Hospital,and all patients gave informed consent.The research team(Xiamen Sencidox)developed a self-made CRC 4 gene methylation detection kit(fluorescent PCR method)to detect gene methylation status.Statistical analysis Analysis was performed via SPSS 20.0 statistical software.The measurement data of non-normal distribution is expressed as median(quartile),and the measurement data of normal distribution is expressed as?x±S.The count data use case number(rate or composition ratio),The χ~2test was used for comparison between groups,When the expected count is less than 5,the continuous correction ? 2 test is used.The difference was statistically significant at P<0.05,and P<0.01 was considered to be significant.Results The first part The methylation index of PRDM12,FOXE1,B3GAT2,VIM,SFRP2-1 and SFRP2-2gene promoters in the cancer tissues of 31 patients were significantly higher than the corresponding normal tissues adjacent to cancer,and the differences were statistically significant(P<0.001).The positive rates of PRDM12,FOXE1,B3GAT2,VIM,SFRP2-1,SFRP2-2 gene hypermethylation in cancer tissues were 87.10%(27/31),90.32%(28/31),80.64%(25/31),77.42 %(24/31),74.20%(23/31),64.52%(20/31).The detection rates of hypermethylation of colorectal cancer patients with PRDM12,FOXE1,B3GAT2,VIM,SFRP2-1,SFRP2-2 gene In 18 cases of early stage(TNM Ⅰ ~ Ⅱ)were 88.90%(16/18),94.45%(17/18),83.33%(15/18),77.78%(14/18),83.33%(15/18)),61.11%(11/18).The detection rates of PRDM12,FOXE1,B3GAT2,VIM,SFRP2-1 and SFRP2-2gene hypermethylation in 13 patients with advanced(TNM Ⅲ~Ⅳ)colorectal cancer were84.62%(11/13)and 84.62%(11/13),76.92%(10/13),76.92%(10/13),61.54%(8/13),69.23%(9/13).The second Part1.Comparison of the positive rate of PRDM12,FOXE1,B3GAT2,VIM gene methylation detection in stool of colorectal cancer group,adenomatous polyp group and healthy group:The positive rates of fecal PRDM12,FOXE1,B3GAT2 and VIM gene promoter methylation tests in the colorectal cancer group were 82.05%,67.94%,52.56%,and57.70%,and the positive rates in the adenomatous polyp group were 36.92%,33.84%,27.69%,30.77%,the positive rates in the healthy group were 1.90%,0.95%,0,4.76%.There were significant statistical differences in the methylation positive rate of each gene in the three groups(P<0.001).The sensitivity of PRDM12 gene methylation detection in the diagnosis of colorectal cancer and adenomatous polyps was 82.05% and 36.92%,and the specificity was 98.10%.The sensitivity of FOXE1 gene methylation detection in the diagnosis of colorectal cancer and adenomatous polyps was 67.64% and 33.84%,and the specificity was 99.05%.The sensitivity of B3GAT2 gene methylation detection in the diagnosis of colorectal cancer and adenomatous polyps was 52.56% and 27.69%,and the specificity was 100.0%.The sensitivity of VIM gene methylation detection in the diagnosis of colorectal cancer and adenomatous polyps was 57.70% and 30.77%,and the specificity was 95.24%.PRDM12 gene has better sensitivity and specificity than the other three genes in diagnosing colorectal cancer and adenomatous polyps.2.Comparison of the positive rate of methylation detection in the stool PRDM12,FOXE1,B3GAT2 and VIM gene serial test of two in the colorectal cancer group,adenomatous polyp group and healthy group:Stool PRDM12+FOXE1,PRDM12+B3GAT2,PRDM12+VIM,FOXE1+B3GAT2,FOXE1+VIM,B3GAT2+VIM gene methylation test positive rates in colorectal cancer group were: 62.82%,41.02%,51.28%,41.02%,47.43%,37.18%,the positive rates of methylation in the adenomatous polyp group were 23.07%,18.46%,16.92%,21.54%,20.00%,21.54%,and the positive rates of methylation in the healthy group were : 0,0,1.90%,0,0.95%,0,the positive rate of methylation of each gene combination in the three groups was significantly different(P<0.001).The sensitivity of PRDM12+FOXE1 gene combination methylation detection in the diagnosis of colorectal cancer and adenomatous polyps was 62.82% and 23.07%,and the specificity was 100.0%.The sensitivity of PRDM12+B3GAT2 gene combination methylation detection in the diagnosis of colorectal cancer and adenomatous polyps was 41.02% and 18.46%,and the specificity was 100.0%. The sensitivity of PRDM12+VIM gene combination methylation detection in the diagnosis of colorectal cancer and adenomatous polyps was 51.28% and 16.92%,and the specificity was 98.10%.The sensitivity of FOXE1+B3GAT2 gene combination methylation detection in the diagnosis of colorectal cancer and adenomatous polyps was 41.02% and 21.54%,and the specificity was 100%.The sensitivity of FOXE1+VIM gene combination methylation detection in the diagnosis of colorectal cancer and adenomatous polyps was47.43% and 20.00%,and the specificity was 99.05%.The sensitivity of B3GAT2+VIM gene combination methylation detection in the diagnosis of colorectal cancer and adenomatous polyps was 37.18% and 21.54%,and the specificity was 100%.The PRDM12+FOXE1 gene combination has better sensitivity and specificity in the diagnosis of colorectal cancer and adenomatous polyps than the other five gene combinations.3.Comparison of the positive rate of methylation detection in the stool PRDM12,FOXE1,B3GAT2 and VIM gene parallel test of two in the colorectal cancer group,adenomatous polyp group and healthy group:Stool PRDM12/FOXE1,PRDM12/B3GAT2,PRDM12/VIM,FOXE1/B3GAT2,FOXE1/VIM,B3GAT2/VIM gene methylation test positive rates in colorectal cancer group were:88.46%,91.02%,92.30%,80.76%,85.89%,75.64%,the positive rates of methylation in the adenomatous polyp group were 47.69%,46.15%,49.23%,40.00%,44.62%,36.92%,and the positive rates of methylation in the healthy group were respectively:2.86%,1.90%,4.76%,0.95%,4.76%,4.76%,the methylation positive rate of each gene combination in the three groups was significantly different(P<0.001).The sensitivity of PRDM12/FOXE1 gene combination methylation detection for the diagnosis of colorectal cancer and adenomatous polyps was 88.46% and 47.69%,and the specificity was 97.14%.The sensitivity of PRDM12/B3GAT2 gene combination methylation detection in the diagnosis of colorectal cancer and adenomatous polyps was 91.02% and 46.15%,and the specificity was 98.10%.The sensitivity of PRDM12/VIM gene combination methylation detection in the diagnosis of colorectal cancer and adenomatous polyps was 92.30% and 49.23%,and the specificity was 95.24%.The sensitivity of FOXE1/B3GAT2 gene combination methylation detection for the diagnosis of colorectal cancer and adenomatous polyps was80.76% and 40.00%,and the specificity was 99.05%.The sensitivity of FOXE1/VIM gene combination methylation detection for the diagnosis of colorectal cancer and adenomatous polyps was 85.89% and 44.62%,and the specificity was 95.24%.The sensitivity of B3GAT2/VIM gene combination methylation detection in the diagnosis of colorectal cancer and adenomatous polyps was 75.64% and 36.92%,and the specificity was 95.24%.The PRDM12/VIM gene combination has better sensitivity and specificity than the other five gene combinations in the diagnosis of colorectal cancer and adenomatous polyps.4.Comparison of the positive rate of methylation detection in the stool PRDM12,FOXE1,B3GAT2 and VIM gene serial test of three in the colorectal cancer group,adenomatous polyp group and healthy group:Stool PRDM12+FOXE1+B3GAT2,PRDM12+FOXE1+VIM,PRDM12+B3GAT2+VIM,FOXE1+B3GAT2+VIM gene methylation test positive rates in colorectal cancer group were: 37.18%,39.74%,35.89%,33.33%,The positive rates of methylation in the adenomatous polyp group were 18.46%,16.92%,15.38%,16.92%,and the positive rates of methylation in the healthy group were: 0,0.95%,0,0,There was a statistically significant difference in the methylation positive rate of each gene combination in the three groups(P<0.001).The sensitivity of PRDM12+FOXE1+B3GAT2 gene combination methylation detection in the diagnosis of colorectal cancer and adenomatous polyps was37.18% and 18.46%,and the specificity was 97.14%.The sensitivity of PRDM12+FOXE1+VIM gene combination methylation detection in the diagnosis of colorectal cancer and adenomatous polyps was 39.74% and 16.92%,and the specificity was 99.05%.The sensitivity of PRDM12+B3GAT2+VIM gene combination methylation detection in the diagnosis of colorectal cancer and adenomatous polyps was 35.89% and15.38%,and the specificity was 100.0%.The sensitivity of FOXE1+B3GAT2+VIM gene combination methylation test for the diagnosis of colorectal cancer and adenomatous polyps is 33.33% and 16.92%,respectively,and the specificity is 100.0%.The sensitivity of any three-gene serial test to diagnose diseases is relatively low. 5.Comparison of the positive rate of methylation detection in the stool PRDM12、FOXE1、B3GAT2 and VIM gene parallel test of three in the colorectal cancer group,adenomatous polyp group and healthy group:Stool PRDM12/FOXE1/B3GAT2,PRDM12/FOXE1/VIM,PRDM12/B3GAT2/VIM,FOXE1/B3GAT2/VIM gene methylation test positive rates in colorectal cancer group were:92.30%,91.01%,92.30%,87.18%,The positive rates of methylation in the adenomatous polyp group were 53.84%,56.92%,50.76% and 46.15%,and the positive rates of methylation in the healthy group were 2.86%,7.62%,6.67%,and 5.70%.There was a statistically significant difference in the methylation positive rate of each gene combination in the three groups(P<0.001).The sensitivity of PRDM12/FOXE1/B3GAT2 gene combination methylation detection in the diagnosis of colorectal cancer and adenomatous polyps was 92.30% and 53.84%,and the specificity was 97.14%.The sensitivity of PRDM12/FOXE1/VIM gene combination methylation detection in the diagnosis of colorectal cancer and adenomatous polyps was 91.01% and 56.92%,and the specificity was 92.38%.The sensitivity of PRDM12/B3GAT2/VIM gene combination methylation detection in the diagnosis of colorectal cancer and adenomatous polyps was 92.30% and50.76%,and the specificity was 95.34%.The sensitivity of the FOXE1/B3GAT2/VIM gene combination methylation test for the diagnosis of colorectal cancer and adenomatous polyps is 87.18% and 46.15%,and the specificity is 94.30%.Among these combinations,the sensitivity and specificity of the PRDM12/FOXE1/B3GAT2 combination test is the best.6.Comparison of the positive rate of methylation detection in fecal PRDM12,FOXE1,B3GAT2,VIM four genes in serial and parallel tests in colorectal cancer group,adenomatous polyp group and healthy group:The positive rates of fecal PRDM12+FOXE1+B3GAT2+VIM,PRDM12/FOXE1/B3GAT2/VIM gene methylation tests in the colorectal cancer group were 30.76% and 93.58%,and the methylation positive rates in the adenomatous polyps group were 15.38% and 58.46%.In the healthy group,the methylation positive rates were 0 and 7.62%.There was a statistically significant difference in the methylation positive rate of each gene combination in the three groups(P<0.001).The sensitivity of the PRDM12+FOXE1+B3GAT2+VIM gene combination methylation test for the diagnosis of colorectal cancer and adenomatous polyps is 30.76% and 15.38%,and the specificity is 100.0%.The sensitivity of the PRDM12/FOXE1/B3GAT2/VIM gene combination methylation test for the diagnosis of colorectal cancer and adenomatous polyps was 93.58 and58.46%,and the specificity was 92.38%.When the four genes are tested in parallel,the sensitivity and specificity of the diagnosis of CRC and adenomatous polyps are the best.Conclusion The results of the study show that PRDM12,FOXE1,B3GAT2,VIM,SFRP2-1,SFRP2-2 gene promoters have significant abnormal methylation expression in colorectal cancer tissues,and the methylation index of gene promoters is significantly higher than that normal tissues adjacent to cancer,the difference between them was statistically significant(P<0.001).The methylation levels of PRDM12 and FOXE1 genes in colorectal cancer tissues are higher than those of the other four genes.It is judged that the two genes have the potential of molecular markers for early screening and auxiliary diagnosis colorectal cancer.Then in healthy people,colorectal cancer and adenomatous polyps patients stool PRDM12 、 FOXE1 、 B3GAT2 、 VIM gene methylation detection,the distribution of the positive rate of methylation of each gene has a significant statistical difference(P<0.001),Analyze the diagnostic ability of each gene and its combination test.The sensitivity,specificity,positive predictive value and negative predictive value of PRDM12 single gene and PRDM12,FOXE1,B3GAT2,VIM four genes combined parallel detection are 90% greater than that of clinical outcome.Auxiliary diagnosis of colorectal cancer has certain clinical application value. |
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