Identification of DNA differentially methylated regions and differentially expressed genes in colon cancer tissues and cell lines
Five datasets (GSE17648, GSE25062, GSE29490, GSE47071, and GSE47592), including 272 tumoral samples and 151 normal controls, were downloaded to identify differentially methylated regions. The hypermethylated regions were indicated by generating a Venn diagram of five datasets. Altogether, two hundred and fifty-two common hypermethylated regions were defined (Fig. 2-A, Supplement 1).
Afterward, the common hypermethylated genes obtained from the GEO database were compared to SMART database hypermethylated genes through another Venn diagram. Two hundred two significant hypermethylated regions (genes) were obtained (Fig. 2-B, Supplement 2).
Ultimately, hypermethylated genes were compared to the significantly downregulated genes to identify the hypermethylated and downregulated genes simultaneously. HPSE2, SDC2, SPG20, RSPO2, ZNF667, SFRP2, CHST10, HAND2, NPY, ZNF677, FIGN, GPM6A, AMPH, D4S234E, ADHFE1, CNTN1, TRPC6, GRIK3, NRXN3, GFRA1, FLT4, JAM3, UCHL1, ATP8B2, MAL, CNR1, THBD, PHOX2A, EDNRB, KIF5A, NPR3, SOX17, NTRK3, VIPR2, CD34, GRASP, CDO1, INA, JAM2, RYR2, GAS7, PDE8B, SFRP1, and PRSS1 were significantly hypermethylated and downregulated in COAD samples (Fig. 2-C, Supplement 3). The HAND2 gene was selected in this study for further investigation because the potential role of HAND2 in CRC is not well understood.
The correlation of HAND2 methylation with its expression
Correlation analysis is a common method to examine the relationship between specific gene methylation and expression [16]. In this study, Pearson's correlation was calculated between the HAND2 methylation and expression. Interestingly, the aggregated (mean of all probes) Pearson's correlation (Pearson's correlation coefficient = -0.44, p = 6.6e-14 for COAD) conveyed that HAND2 significantly downregulated in CRC (288 colon cancer) and has a reverse correlation with the methylation status of CpG islands (Supplementary Fig. 1).
The HAND2 methylation and expression in CRC cell lines
The DepMap database was investigated to consolidate the correlation between HAND2 methylation and expression hypothesis. Interestingly, the data available on the DepMap database (https://depmap.org/) for cancerous cell lines conveyed that HAND2 is hypermethylated in CRC cell lines, simultaneously downregulated. Furthermore, the Pearson correlation coefficient test revealed a negative correlation (Pearson's correlation coefficient = -0.3035, p = 0.030) between the HADN2 expression and methylation (Supplement 4, Supplementary Fig. 3).
Identifying the HAND2 downstream genes, signaling pathways, and the interaction with other proteins
Using the ChIP-Atlas database, potential HAND2 targets were identified by an average score above 499 and the ± 1 kb distance from the transcription start site (TSS). The number of refined target genes was 104. Afterward, obtained genes were analyzed with ShinyGO for enrichment analysis (Fig. 3 and Supplement 5).
The final results of the biological process of HAND2 target genes conveyed that disruption in HAND2 expression could dysregulate ERK1 and ERK2 signaling pathways. Notably, the HAND2 downstream genes conveyed that HAND2 is a critical transcription factor for maintaining cell homeostasis. Interestingly, it was shown that HAND2 could directly bind to ERK and reduce the phosphorylation of ERK [17]. In this study, the results showed that HAND2 downstream genes could regulate ERK1 and ERK2 cascade.
On the other hand, by utilizing the String database, the interaction network of HAND2 revealed that it has numerous potential interactions with critical proteins, including ADSS, ELSPBP1, GATA4, HAND1, MEF2C, NFATC1, NKX2-5, TBX5, TCF3, and PHOX2A, which all of them are capable of binding DNA. Pathway enrichment analysis (KEGG) and functional enrichment analysis (GO) were applied to elucidate the biological functions of the putative interaction proteins related to HAND2. Enriched results were subjected to multiple testing adjustments with a threshold value FDR (q-value) less than 0.05. To better exhibit functional consequence, only the top twenty significant enriched GO terms are shown in Fig. 4. The BP enrichment analysis manifests that HAND2 misregulation could perturb Cardiac ventricle morphogenesis (FDR = 3.22E-10), Cardiac ventricle formation (FDR = 1.77E-09), and Cardiac chamber morphogenesis (FDR = 2.61E-09). The KEGG enrichment analysis reveals that the misregulation of HAND2 could impact the CGMP-PKG signaling pathway, Cellular senescence, and Signaling pathways regulating the pluripotency of stem cells.
The expression pattern of HAND2 antisense1 long non-coding RNA and its correlation with CpG island methylation
Previous investigations revealed that HAND2 has an antisense long non-coding RNA [18]. The expression data of 275 COAD and 308 normal samples, analyzed by the GEPIA2, revealed that the HAND2 and HAND2-AS1 were significantly downregulated in COAD samples compared to normal samples. The p-value cutoff was set to less than 0.01. (Fig. 5-A and Fig. 5-B) Afterward, the Pearson's correlation test revealed that the expression of HAND2 and its long non-coding RNA antisense, HAND2-AS1, are positively correlated (Pearson's correlation coefficient = 0.96, p < 0.001) (Fig. 5-C).
Another Pearson's correlation test revealed that the expression of HAND2-AS1 had a significant (Pearson's correlation coefficient = -0.41, p = 3.4e-13 for COAD) reverse correlation with the methylation status of CpG islands (mean of all probes) (Fig. 5-D, Supplementary Fig. 2). This evidence led to deduced that HAND2-AS1 could be under the control of DNA methylation, which hypermethylation of CpG islands affects the expression of HAND2-AS1. Aligning with our hypothesis, previous studies on ovarian carcinoma [19] and endometrioid endometrial carcinoma [20] conveyed that HAND2-AS1 expression could negatively correlate with its promoter CpG island methylation.