The expression levels of eight CBX family members in patients with CESC
ONCOMINE, GEPIA and HPA were used to analyze the expression levels of the CBX family in CESC. In the ONCOMINE database, the mRNA expression of eight CBX family members in various tumors was analyzed and compared with normal tissues (Figure 1 and Table 1). Concerning CBX1, we found overexpression levels in the Dohun cervical statistics dataset (FC=3.762, p=1.72E-08). Regarding CBX2, low expression levels were found in the Luigi cervical statistics dataset (FC=-1.794, p=0.000145). Notably, we found that CBX3/5 mRNA is highly expressed in CESC tissues in multiple datasets. In the Yali dataset, for CBX3, compared to normal tissue, cervical squamous cell carcinoma (CSCC) tissues and high-grade cervical squamous intraepithelial neoplasias (HGCEIN) had fold changes of 1.911 (p=1.26E-08) and 1.576 (p=0.003), respectively. While Dohun found that CBX3 mRNA expression had a 2.701-fold increase in cervical cancer samples (p=2.37E-09), Luigi and Petra found a 1.812-fold and 2.202-fold increase in CBX1 mRNA expression in CESC tissues, respectively (p=0.00000164/p=0.0000113). Similarly, Yali observed a 2.166-fold/2.18-fold increase in CBX5 mRNA expression in HGCEIN/CSCC samples (p=0.000528/p=0.000545), while Dohun observed a 2.78-fold increase in CBX5 mRNA expression in cervical cancer samples (p=1.29E-08) and Luigi observed a 2.094-fold increase in CBX5 mRNA expression in CSCC samples (p=0.00000415). Importantly, Petra observed a -2.132-fold increase in CBX7 mRNA expression in CSCC samples (p=1.03E-12) [22-25].
As shown in Figure 2, compared to adjusted normal tissues, the GEPIA database indicated significant mRNA overexpression of CBX2/4/8 (Figure 2B, 2D and 2H) in CESC samples (p< 0.05). However, the expression levels of CBX6 (Figure 2F) and CBX7 (Figure 2G) were reduced in CESC tissues (p <0.05).We further confirmed the expression of CBX7 in cervical cancer via qRT-PCR.The results confirmed that CBX7 was lower expressed in cervical cancer tissues than benign cervical lesion tissues (p=0.0250)(Figure 2I) .
After inspecting the mRNA expression patterns of CBXs in CESC, we subsequently attempted to analyze the expression pattern of CBXs family proteins in CESC by HPA (Human Protein Atlas). As shown in Figure 3, the normal cervical tissues presented medium protein expression of CBX1/2/3/4/5, while medium and high protein expression was discovered in CESC tissues (Figure 3A-3E). Analogously, low protein expression of CBX6/8 was observed in normal cervical tissues, while medium and high protein expression of CBX6/8 was observed in CESC samples (Figure 3F and 3H). In contrast, we found that the CBX7 protein was medially expressed in healthy cervical tissues and was not detected in CESC tissues (Figure 3G).
The clinicopathological parameters associated with CESC patients were investigated using models of mRNA expression of eight members of the CBX family
Afterward, we analyzed the correlation between CBXs family mRNA expression levels and clinicopathological parameters in patients with CESC by GEPIA and UALCAN, containing patients’ cancer stages and tumor grades. In the Violin plot (Figure 4A), we found that the expression of CBX2/6/8 mRNA was correlated with the pathological stage of CESC patients.
Figure 4B shows the relationship of the mRNA expression level of distinct CBX family members with the tumor grades of CESC patients. The mRNA expression levels of CBX1/3/6 were related to tumor grade. When the tumor grade advanced, CBX1/3 tended to have a higher mRNA expression level, and CBX6 had the opposite trend. In tumor grade 4, we found the highest mRNA expression of CBX1/2/3. The highest mRNA expression of CBX4/5/8 was found in tumor grade 2, while the highest mRNA expression of CBX6/7 was found in normal tissues.
In summary, the results showed that the mRNA expression of several CBX family members was significantly correlated with clinicopathological parameters in patients with CESC.
The relationship between CBXs mRNA expression levels and prognosis in patients with CESC
Next, we analyzed the relationship between the CBXs mRNA expression levels and the prognosis of patients with CESC (Figure 5). Distinctly, CBX1/2/3/4/5/6/8 mRNA expression showed no correlation with prognosis in CESC patients (Figure 5A-5G), while higher mRNA expression of CBX7 (HR=0.43, 95% CI: 0.26-0.69, p=0.00042) was significantly correlated with favorable OS (overall survival) in CESC patients (Figure 5H). The results showed that the mRNA expression level of CBX7 could be used as an important biomarker to predict the OS of patients with CESC.
CBXs family gene mutations in the patients with CESC
We analyzed genetic changes in the patients using the cBioPortal tool and found mutations in the CBXS gene in 64 of 188 patients, with a mutation rate of 34%. In addition, the mutation rates of the CBX4, CBX1, CBX2, and CBX3 genes were 11%, 9%, 7%, and 7%, respectively (Figure 6).
Predicted functions and pathways in CBXs
After analyzing CBXs family gene mutations in patients with CESC, we analyzed neighboring genes associated with mutations in the CBXs family of genes and constructed a complete network. As shown in Figure 7A, Moreover, functions of 300 genes significantly associated with CBXs were analyzed and visualized with R project using the "clusterProfiler" and "org.Hs.eg.db" package.Under the condition of p.adj < 0.05 and qvalue < 0.2, the enrichment results included 11 MFs, 76 BPs, 15 CCs and 4 KEGG pathways.According to GO enrichment analysis, the molecular functions of CBXs were mainly in histone modification,peptidyl-lysine modification,covalent chromatin modification.The cellular components mainly consisted of histone methyltransferase complex,nuclear chromatin,nuclear speck.In addition, CBXs also
prominently affected the molecular functions,such as peptide-lysine-N-acetyltransferase activity,histone acetyltransferase activity,histone binding(Figure 7B).In KEGG analysis,3 pathways including mRNA surveillance pathway,Lysine degradation,Spliceosome were associated with the functions of CBXs(Figure 7C).