The ratio of apoptotic cells was increased by RAI2 under STS treatment in CRC cells. (mm), and represents the smallest diameter (mm). Mice were sacrificed on the 22nd day, and tumor weights were measured. All procedures were approved by the Animal Ethics Committee of the Chinese PLA General Hospital. Statistical analysis The RNA sequencing (RNA-Seq) data for RAI2 gene expression in the dataset of CRC and normal tissues were downloaded from Genotype-Tissue Expression (GTEx) database (https://www.gtexportal.org/home/datasets) and the Cancer Genome Atlas (TCGA) (http://xena.ucsc.edu/, 08/16/2016), respectively. Statistical analysis was performed using SPSS 17.0 software (SPSS, Chicago, IL). Chi-square or Fishers exact tests were used to evaluate the relationship between methylation status and clinical pathological characteristics. The two-tailed independent samples test was applied to determine YH239-EE the statistical significance of the differences between the two experimental groups. Survival rates were calculated by the Kaplan-Meier method, and the differences in survival curves were evaluated using the log-rank test. Cox proportional hazards models were fit to determine independent associations of RAI2 methylation with 5-year OS and 5-year relapse-free survival (RFS) outcomes. Two-sided tests were used to determine the significance, and valuevalues are obtained from the chi-squared test Statistically significant *P?0.05, **P?0.01, ***P?0.001 Table 2 Analysis of RAI2 methylation status with OS or RFS in colorectal cancer patients by Cox regression analysis
Variables
OS
RFS
Univariate analysis
Multivariate analysis
Univariate analysis
Multivariate analysis
HR (95% CI)
P
HR (95% CI)
P
HR YH239-EE (95% CI)
kalinin-140kDa colspan=”1″> P
HR (95% CI)
P
RAI2 methylation0.4810.004**0.4050.002*0.5040.008**0.5120.022*?M vs U(0.290C0.796)(0.226C0.726)(0.305C0.833)(0.288C0.907)Age (years)1.9130.0580.4600.027*1.1870.5670.7900.440??50 vs 50(0.979C3.737)(0.231C0.915)(0.659C2.137)(0.434C1.437)Gender1.0130.9561.9690.018*1.0290.9071.5760.101?Female vs male(0.630C1.631)(1.121C3.455)(0.635C1.668)(0.916C2.714)Tumor location0.8640.5921.6610.1060.9860.961.5640.176?Distal colon or rectum vs proximal colon(0.505C1.476)(0.898C3.070)(0.563C1.727)(0.818C2.989)Tumor size0.8540.5191.2130.4800.7590.2761.3520.299??5 vs 5?cm(0.527C1.381)(0.710C2.072)(0.461C1.248)(0.765C2.388)Differentiation0.3960.000***0.4600.002**0.3690.000***0.4490.001**?Low vs high/ middle(0.247C0.634)(0.283C0.748)(0.229C0.597)(0.274C0.735)TNM stage0.2620.000***0.0890.000***0.2370.000***0.0690.006**?III/IV vs I/II(0.125C0.546)(0.027C0.294)(0.113C0.496)(0.010C0.461)Pathologic N stage0.4180.006**3.9850.008**0.2830.000***4.5050.105?N1C2 vs N0(0.224C0.778)(1.439C11.034)(0.140C0.570)(0.732C27.731)Intravascular cancerous embolus0.5970.0930.6720.2200.9960.9921.0950.812?Yes vs no(0.327C1.090)(0.357C1.267)(0.476C2.084)(0.517C2.320) Open in a separate window *P?0.05, **P?0.01, ***P?0.001 To explore the regulation of RAI2 expression in primary colorectal cancer, RAI2 expression YH239-EE was evaluated by immunohistochemistry (IHC) in 32 cases of matched colorectal cancer and adjacent tissue samples. The expression of RAI2 was YH239-EE reduced significantly in cancer tissue compared to the adjacent normal tissue (P?0.01, Fig.?2c, d). Among the 19 cases of cancer samples that had loss of/reduced expression of RAI2, 12 cases were methylated (63.15%). In contrast, in 13 cases of cancer tissue samples that expressed RAI2, only 3 cases were methylated (23.1%). Loss/reduction of RAI2 expression was significantly associated with promoter region methylation in CRC (P?0.05, Fig.?2d, bottom panel). These results suggest that the expression of RAI2 is regulated by promoter region methylation in human CRC. To further validate our results, RAI2 mRNA expression and promoter region methylation data were extracted from Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) (http://xena.ucsc.edu/) databases. RAI2 expression data were obtained from RNA sequencing (RNA-Seq) in 383 cases of CRC samples and 50 cases of adjacent colorectal tissue samples. The levels of RAI2 expression were significantly lower in CRC samples compared to adjacent normal colorectal mucosa samples (P?0.0001, Fig.?2e), while no association was found between RAI2 mRNA expression and 5-year OS (n?=?333, P?=?0.3168, Fig.?2f) or 5-year RFS (n?=?341, P?=?0.0951, Fig.?2f) in this cohort. Methylation of RAI2 was analyzed by Illumina Infinium Human Methylation 450 (HM450) based on the methylation status of 16 CpGs in the promoter region. Available data were obtained from 373 cases of colorectal cancer samples for both RAI2 expression YH239-EE and methylation. The expression of RAI2 was inversely associated with promoter region methylation (P?0.05; Fig.?2g). These data further support our results. Thus, methylation of RAI2 may serve as a detection and poor prognostic marker in CRC. Restoration of RAI2 expression suppresses cell proliferation and induces cell apoptosis in CRC Colony formation assays were performed to evaluate the effect of RAI2 on clonogenicity. The colony numbers were 233??11 versus 164??6 in RKO cells (P?0.001) and 155??6 versus 85??5.