Deficiency of serum vitamin B12 and ferritin is recognized in cancer patients
Out of 200 patients screened and offered baseline hemoglobin, serum ferritin, serum folate, and vitamin B12 estimation, 40 patients in each type of cancer were finally enrolled in the study. As shown in table 2, the prevalence of anemia was almost 80% in patients with HCC, since the persons who showed deficient levels of hemoglobin were 32 out of 40. These patients also showed deficient levels of ferritin, vitamin B12, transferrin saturation, and increasing levels of homocysteine. In patients with BC, 75% of patients have severe anemia with deficient levels of ferritin, vitamin B12, transferrin saturation, and increasing levels of homocysteine. The prevalence of anemia in patients with LC was 70% of patients accompanied by deficient levels of ferritin, vitamin B12, and transferrin saturation, and high levels of homocysteine. Likewise, 85% of patients with CC were anemic with deficient levels of ferritin, vitamin B12, and transferrin saturation, and high levels of homocysteine. Unlikely, more than 80% of cancer patients showed normal levels of serum folate, while a maximum of 20% of cancer patients showed low levels of serum folate. These data indicate that the cancer patients with HCC, BC, LC, or CC represented deficient levels of hemoglobin, serum ferritin, vitamin B12, and subsequently low saturation levels and accumulated levels of homocysteine.
The relative expression of DNMTs is associated with deficiency of vitamin B12/ferritin in cancer patients
To assess the correlation between DNA methylation and vitamin B12/ferritin deficiency in cancer, the relative gene expression of DNMT1, DNMT2, DNMT3a, and DNMT3b in obtained blood samples from patients with HCC, BC, LC, and CC were compared to blood samples derived from healthy individuals using qRT-PCR. Our results revealed a significantly increased expression level of DNMT1, DNMT3a, and DNMT3b, but not DNMT2 that connected with the low level of vitamin B12 and ferritin, while their expression significantly reduced in patients represented normal levels of vitamin B12 and ferritin (Figure 1A, B, C, and D). Notably, the calculated P values for the expression level of DNMT1 and DNMT3b were lower than 0.01 in samples derived from patients with HCC, BC, LC, and CC. Whereas the calculated P values for the expression level of DNMT3b were about 0.05 (Tables 3-6). These findings indicate the possible connection between DNMTs expression, particularly DNMT1, DNMT3a, and DNMT3b, and levels of vitamin B12/ferritin in patients with the indicated cancer diseases.
Deficiency of vitamin B12 and ferritin associated with low expression level of MS, TET1, and TET3 genes in cancer patients
To achieve the correlation between low levels of vitamin B12/ferritin and gene expression of other essential cofactors involved in DNA methylation in cancer patients, the relative gene expression of MS, TET1, TET2, and TET3 was evaluated by qRT-PCR in obtained blood samples. Interestingly, the results showed a significant reduction in the expression level of indicated genes; expect TET2 expression, associated with the deficiency of vitamin B12/ferritin in patients with HCC, BC, LC, and CC (Figure 2A, B, C, and D). Importantly, the calculated P values for MS, TET1, and TET3 expression level were lower than 0.01 in obtained samples from patients with low level of vitamin B12/ferritin compared with samples derived from patients with normal level of vitamin B12/ferritin and healthy individuals (Tables 7, 8, and 10). Whereas the calculated P values for TET2 expression level showed insignificant differences between patients and healthy individuals (Table 10). These data reveal that the deficiency of serum vitamin B12 and ferritin in patients with cancer may contribute in the regulation of MS, TET1, and TET3 genes expression and subsequently controlling the DNA methylation process.
Patients with deficient vitamin B12 and ferritin revealed a methylation activity particularly in TET1 and TET3 promoter region
DNA methylation at CpG dinucleotides is a major epigenetic marker leading to gene silencing when located in the promoter region of a certain gene [27]. Noteworthy, the identified restriction site for HapII enzyme is (CCGG) with an independent sensitivity to cleave the methylated CpG [28, 29]. Based on this, we incubated the purified genomic DNA with HapII to compare and analyze the differences in cleaved fragments in cancer patients that are associated with the level of vitamin B12 and ferritin. Interestingly, agarose gel electrophoresis of HapII-cleaved fragments showed an alteration in the number of cleaved fragments in patients with a normal level of vitamin B12/ferritin and healthy on one side and patients with a low level of vitamin B12/ferritin on the other side. As shown in figures 3A and B, almost 7 cleaved fragments were found in patients with the normal level of vitamin B12/ferritin, while only 2 cleaved fragments were detected in patients with the low level of vitamin B12/ferritin. These findings clearly indicate that the HapII resection enzyme has failed to digest the genomic DNA isolated from cancer patients with a deficient level of vitamin B12/ferritin due to the potential DNA methylation activity on its binding site. Furthermore, to identify the potential methylated region, the low-intensity band cleaved from genomic DNA purified from patients with the normal level of vitamin B12/ferritin (approximately 100bp) was sequenced and aligned with deferent sequences of various related genes. Fortunately, the sequence and alignment analysis showed a consensus sequence of about 80 nucleotides within the promoter region of TET1 and TET3, started at the location 318/CG and 385/CG, respectively (Figure 3C and D). These findings strongly suggest that TET1 and TET3 were downregulated in cancer patients in a vitamin B12/ferritin dependent manner due to the methylation potential in their promoter regions.
Bisulfite-converted DNA revealed a significant CpG methylated motif in TET1 and TET3 genes associated with vitamin B12/ferritin level in cancer patients
Bisulfite DNA sequencing was recognized as an accurate method to detect DNA methylation-based on the conversion of genomic DNA using sodium bisulfite [30]. In this way, the methylated cytosine is converted into uracil and recognized in PCR amplification as thymine [31]. Therefore, we designed specific oligoniclutids for both TET1 and TET3 methylated regions including common forward primers and two reverse primers for each TET gene. The first reverse primer contains the complementary sequences of the normal motif CG named wild-type reverse primer (R-W) at the 3- end of the primer. While the second reverse primer contains the complementary sequences of converted cytosine in CpG motif and named methylated reverse primer (R-M) at the 5- end of the primer, to make the amplified fragment shorter than the amplified wild-type fragment, and contained adenine nucleotide instead of guanidine nucleotide. These triple primers were used to amplify the specific fragments from bisulfate-converted DNA isolated from cancer patients with a vary level of vitamin B12 and ferritin. Interestingly, the agarose gel electrophoresis of PCR products showed only one specific band with a molecular size of about 175bp when used the triple specific primers for TET1 and genomic DNA isolated from cancer patients with the normal level of vitamin B12/ferritin (Figure 4A). Whereas, two specific bands with a molecular size of 175bp and 165bp were detected using the genomic DNA isolated from cancer patient with a low level of vitamin B12 and ferritin (Figure 4B). Likewise, the specific triple primers for TET3 amplified only the wild-type fragment when using the genomic DNA isolated from cancer patients with a low level of vitamin B12/ferritin. Meanwhile, the same primers amplified both of the fragments with the molecular size of 175bp and 165bp using the obtained DNA from cancer patients with a low level of vitamin B12 and ferritin (Figure 4C and D). These findings reveal the methylation potential in the promoter region of TET1 and TET3 associated with deficiency of vitamin B12 and ferritin in cancer patients like HCC, BC, LC, and CC.