Characteristics of the study population
A total of 507 CRC patients (209 colon cancer cases and 298 rectal cancer cases) and 497 cancer-free controls were involved in our study. The baseline characteristics and lifestyle factors are shown in Table 1. There was no significant difference in age between the cases and controls, but the proportion of males was higher in the case group (64.89% vs. 57.95%). CRC patients were more likely to have a lower education level and lower body mass index (P<0.05). CRC patients also reported higher percentages of family history of cancer and history of appendicitis in comparison with controls (P=0.034, P<0.001 respectively). However, no significant differences were found between the case and control groups with respect to tobacco smoking, alcohol drinking, or tea drinking.
mRNA expression analysis of miR-143, miR-145, and KRAS
We extracted published microarray data from GEO datasets GSE38389 and compared the mRNA expression of miR-143, miR-145 between rectal cancer tissue and adjacent normal mucosa. As shown in Figure 1, miR-143 expression was under-expressed (log2-fold difference<-1) 26 out of 66 matched pairs of rectal tumor samples and normal samples (P value for paired t test <0.001). MiR-145 showed the same trend, with decreased expression(log2-fold difference<-1) in 28 out of 66 pairs of samples (P value for paired t test <0.001). (Figure1, 2)
We performed a statistical comparison of KRAS expression from multiple colorectal cancer studies published in Oncomine database. Eight independent microarray studies comprising a total of 639 colorectal cancer and 202 normal colorectal mucosa samples were evaluated from meta-analysis data by Oncomine. Meta-analysis identified that KRAS mRNA was under-expressed no matter in colon cancer or rectal cancer(P<0.001, P=0.016 respectively).
Polymorphisms of miR-143, miR-145, and KRAS 3′ UTR and CRC risk
KRAS rs712 and rs1137196, miR-143 rs41291957, and miR-145 rs74693964 and rs80026971 were genotyped in this study. The genotype distribution of the five SNPs in the control group all conformed to Hardy–Weinberg equilibrium; their associations with the risk of CRC are presented in Table 2. As shown in the table, subjects with the heterozygous genotype CT of rs74693964 were more than twice as likely to have CRC as subjects with the wild genotype CC [adjusted odds ratio (OR)=2.414, 95% CI:1.385–4.206]. MiR-145 rs74693964 was associated with a significantly increased risk of CRC. However, KRAS rs712 and rs1137196, miR-143 rs41291957, and miR-145 rs80026971 showed no association with the risk of CRC. In the subgroup analysis, rs41291957 and rs74693964 were found to be associated with an increased risk of rectal cancer but not colon cancer (rs41291957 GA vs. AA: adjusted OR=1.367, 95% CI: 1.005–1.860; rs74693964 CT vs. CC: adjusted OR=2.820, 95% CI: 1.547–5.140) (Tables 2, 3).
When stratified by smoking status, we found that the genotype distributions of miR-143 rs41291957 among non-smokers differed significantly between cases and controls. Compared with the GG genotype, those carrying heterozygous genotype GA had a nearly 40% increased risk for developing CRC (adjusted OR=1.397, 95% CI: 1.007–1.936). In non-smokers, miR-145 rs74693964 remained a significant risk factor for CRC among subjects carrying the CT genotype (adjusted OR =3.086, 95% CI: 1.468–6.484). Interaction analyses of the two SNPs and tobacco smoking were conducted using a multiplicative model; neither interaction effect showed statistical significance (Table 4).
Although miR-143 and miR-145 located close to each other on 5q33, our analysis showed no linkage disequilibrium between them. To evaluate the potential cumulative effects of miR-143 and miR-145, we defined at-risk genotypes as those with OR values greater than 1 under a dominant model of rs41291957 and rs74693964. We compared the distributions of the number of at-risk genotypes between cases and controls. CRC risk increased with the number of at-risk genotypes (Ptrend=0.003). When split by cancer type, individuals harboring two at-risk genotypes had an increased risk of rectal cancer relative to those with none (OR=3.738, 95% CI: 1.725–8.101) (Table 5).
Polymorphisms of miR-143, miR-145, and KRAS 3′ UTR and CRC survival
We collected and evaluated the overall survival time of 222 CRC cases with Hangzhou household registration from the Cancer Registration System and Death Surveillance System of Hangzhou Center for Disease Control and Prevention. Of the 222 cases recruited between May 2014 and May 2015, a total of 34 had died of CRC by Jan 1 2019. Associations between polymorphisms of miR-143, miR-145, and the KRAS 3′ UTR and CRC survival were explored. First, we used the Kaplan–Meier method to compare overall survival among different genotypes of selected SNPs. Then, adjusted HRs were obtained by Cox regression analysis for further confirmation of the relationships between the genotypes and CRC survival. The results showed that the mutant homozygote TT of rs712 was associated with decreased survival time in CRC (log-rank P=0.044). Compared with the reference genotype GG of rs712, the CRC cases with TT genotype had a significant increase in number of deaths (adjusted HR=4.328, 95% CI: 1.236–15.147). The polymorphisms of miR-143 and miR-145 did not show any statistical association with prognosis of CRC cases (Table 6).