*3.1. RUNX1 Hypermethylation Is Inversely Associated with Its Expression*

Data reported previously were used to identify differentially methylated CpGs in *RUNX1* gene in tumor and matched normal tissues from 42 NSCLC patients. The Wilcoxon rank-sum test was applied because the distribution of β-values obtained from tumor tissues using a 450 K array was negatively skewed and did not follow a normal distribution (Shapiro-Wilk test, *p* < 0.05). Three CpGs with a *p*-value less than or equal to 1.03 <sup>×</sup> 10−<sup>7</sup> (Bonferroni significance threshold) were identified from the 450K array: three CpGs (cg11498607, cg04228935, cg05000748) at the CpG island of *RUNX1* showed hypermethylation in tumor tissues compared with normal tissues (Figure 1A). The methylation levels of the three CpGs did not vary significantly with histology (Figure 1B). Altered methylation of three CpGs was not significantly correlated with a patient's age (Figure 1C).The methylation levels were not also associated with smoking status (Figure 1D) and recurrence (Figure 1E). However, the methylation levels were found to be higher in the poorly differentiated type of NSCLC than in the well differentiated type (Figure S1). The *RUNX1* mRNA levels were analyzed using the HT-12 array to determine the association between methylation changes and changes in *RUNX1* gene expression. The methylation levels of individual CpGs were negatively associated with the mRNA levels of *RUNX1* (*p* < 0.05; Figure 1F).

*J. Clin. Med.* **2020**, *9*, 1694

**Figure 1.** Relationship between methylation and mRNA levels of runt-related transcription factor 1 (*RUNX1)* in 42 lung tumor and matched normal tissues. (**A**) Methylation levels of three CpGs at the CpG island of *RUNX1* gene were compared between the tumor and matched normal tissues obtained from 42 NSCLC patients. Y-axis indicates β-values. (**B**) Methylation levels of the three CpGs were compared according to histologic subtypes. (**C**) Correlations between the patient's age and the methylation levels of the three CpGs were analyzed in 42 tumor tissues. Spearman's correlation coefficient was used to calculate *p*-values. Magenta color indicates *p* < 0.05. (**D**) Methylation levels of three CpGs at a CpG island of *RUNX1* were compared in never-smokers, former smokers, and current smokers. Y-axis indicates sample identification numbers. Methylation levels are represented using gradient-based colors from green (0%–20%) to yellow (21%–50%) to red (51%–100%). (**E**) The association between recurrence and the methylation levels at three CpGs were analyzed in 42 NSCLCs. (**F**) The correlation between methylation levels of three CpGs and the mRNA expression of *RUNX1* was analyzed in 42 tumor tissues from patients with NSCLC. Y-axis indicates the log2 fold change (= log2(tumor/normal)) between tumor and matched normal tissues. X-axis indicates β-values in tumor tissues.
