As illustrated in Fig. 1, chr1q and chr8p were detected as the hotspot loci of copy number variation in the studied HCC samples. The obtained data will be discussed below.
Cytobands chr1q and chr8q were strongly associated with gene amplification in HCC samples
Of 24776 examined genes, 1024 (4.1%) were amplified in studied samples (linear copy number values cut-off ≥0.5). These genes were mapped on chr1p (1.3%; n=13), chr1q (86%; n=880) and chr8q (13%; n=131) (Fig. 2A). The highest scores were belonged to 34 genes including ADAM15, FLAD1, ZBTB7B, PYGO2, DCST2, LENEP, SHC1, PBXIP1, PMVK, DCST1, KCNN3, SLC50A1, DPM3, SCAMP3, FAM189B, MTX1, KRTCAP2, GBA, GBAP1, MUC1, THBS3, TRIM46, ASH1L, RUSC1, CLK2, HCN3, FDPS, PKLR, YY1AP1, MSTO1, GON4L, RIT1, SYT11 and KIAA0907. Most of these amplified genes were located on chr1q22 (67.6%; n=23), and the remaining genes were belonged to chr1q21.3 (32.4%; n=11) (Fig. 2A). These proteins were belonged to 15 signaling pathways which have been illustrated in Fig. 2B. Around 12.5 % of proteins were associated with Cholesterol biosynthesis. The amplification of 13 out of these genes were associated with tumor grade (Table 1). No association was found between gene amplification and other clinicopathologic parameters of HCC including tumor size, stage and metastasis. Although, all the 34 selected genes were amplified in HCC samples, only the expression of oncogene YY1AP1 (Yin Yang-1 AssociatedProtein 1) showed a strong correlation with their corresponding CNVs in 72% of studied samples (r>0.6). The obtained data from STRING Interaction Network showed that 25 proteins directly interacted with YY1AP1 gene among which NeuroG3 (transcription factor), SS18L2 (transcription coactivator), ZMYM4 (cell morphology and cytoskeletal organization), ZNF496 (transcription factor) and ZNF576 (presumably transcriptional regulation) are the top five proteins interacted with YY1AP1 gene (Fig. 3A). As illustrated in this figure, among the remaining 20 proteins, well-established oncogenes like, YY1 (Yin Yang 1), CCND1 (Cyclin D1), HDAC1 (Histone deacetylase 1) and tumor suppressors like VHL (Von Hippel-Lindau), MAD2L2 (Mitotic Arrest Deficient 2 Like 2) and CEBPA (CCAAT/enhancer-binding protein alpha) are observable.
Enrichment analysis through pathway common webserver showed that some of these 25 genes were belonged to the critical cellular pathway including Nucleolar Remodeling Complex (NoRC) which negatively regulates rRNA expression and regulation of cell cycle and DNA damage repair system like NER (Table2).
We also found that hsa-miR-375, hsa-miR-222-3p, are two miRNAs that target YY1AP1, therefore, are able to regulate the concentration of YY1AP1 protein in the cell (Table3).
Cytoband chr8p was strongly associated with gene deletion in HCC samples
Of 24776 examined genes, 172 (0.69%) were lost in studied samples (n=361) (with linear copy number values cut-off ≥0.5) which were mostly located on chr8p21-23. The most deletion scores were belonged to 17 genes including TNFRSF10B, RHOBTB2, PEBP4, TNFRSF10C, CHMP7, TNFRSF10A, ENTPD4, EGR3, BIN3, PDLIM2, R3HCC1, LOXL2, STC1, PIWIL2, SLC25A37, TNFRSF10D and CSMD1 that were considered for further analysis-. Among these, 16 genes were mapped on chr8p21.3 (94%) and - one gene chr8p23.2 (6%) (Fig. 4A). These genes are transcription factors and cytoskeletal proteins that act in apoptosis, EGF, FGF and P53 signaling pathways (Fig. 4B). Deletions of none of the mentioned genes were associated with tumor metastasis or grade. Although all of the mentioned genes were downregulated in studied cancerous tissues, the correlation with CNV was not significant expect for CHMP7 gene whose expression showed the moderate correlation (r=0.5) with corresponding CNV in 70% of studied samples. The obtained data from STRING Interaction Network showed that 10 proteins directly interacted with CHMP7 (Charged Multivesicular Body Protein 7) gene among which CHMP4A, CHMP5, CHMP2A, CHMP3 and ENSG00000249884 (RNF103-CHMP3 gene) are the top five proteins interacted with CHMP7 gene (Fig. 3B). Enrichment analysis through pathway common webserver showed that some of these genes were belonged to the vital cellular pathway including spindle organization, sister chromatid segregation, centrosome duplication, cytokinesis, Nucleus organization, Nuclear envelope reassembly and Vacuolar transport. (Table2).
We also found that hsa-miR-375, hsa-miR-222-3p, are two miRNAs that target CHMP7, therefore they are capable of regulating the concentration of CHMP7 protein in the cell (Table3).