Study On The Association And Mechanism Of Abnormal Nuclear Acids Modification In Hepatocellular Carcinoma And Type 2 Diabetes Mellitus

Objective Liver is an important organ of glucose and lipid metabolism. Type 2 diabetes mellitus (T2DM) is one of the most common metabolic disease. The abnormal of nuclear acid modifications is tightly related to the development of cancer and T2DM, and 5-methylcytosine (5mC) is the most abundant and common DNA methylation. The finding of 5-hydroxymethylcytosine (5hmC), an intermediate product of DNA demethylation, provided a vital clue for the research of DNA epigenetic in many cancers. Previous reports demonstrated that the overexpression of fat mass-and obesity-associated (FTO) protein is related to T2DM and obesity. Recent researches found that FTO is a RNA demethylase, but its function as a RNA demethylase in human diseases was rarely reported. Here, we firstly explored the association of 5hmC with the development of Hepatocellular carcinoma (HCC), and then we investigated the associations of RNA methylation with both T2DM patients and diabetic animal model. We try to find out potential biomarkers that can be used for the earlier diagnosis of HCC and T2DM, which also can be provided some new ideas for the treatment of these diseases in the future.Material and Methods The formalin-fixed, paraffin-embedded (FFPE) tissue samples from the tumor and tumor-adjacent of HCC patients were collected for DNA epigenetic study. Whole blood samples from T2DM patients and healthy individuals as well as the streptozocin (STZ) diabetic and non-diabetic model Male Sprague Dawley (SD) rats were collected for RNA epigenetic study. The capillary hydrophilic-interaction liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry (cHILIC-ESI-qTOF-MS) method was established to detect the contents of 5mC and 5hmC in genomic DNA from HCC FFPE tissues, while the liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method was performed to detect the N6-methyladenosine (m6A) content in RNA from the whole blood samples. Real-time quantitative PCR (RT-qPCR) was used to examine the mitochondrial DNA (mtDNA) contents from HCC FFPE tissues and the mRNA expression levels of FTO, alkB homolog 5 (ALKBH5), methyltransferase like 3 (METTL3), methyltransferase like 14 (METTL14) and Wilms tumor 1 associated protein (WTAP) genes in peripheral blood samples. High-resolution melting (HRM) analysis and DNA sequencing were used to detect the four common FTO single-nucleotide polymorphisms. SPSS software package was used to analysis all the data in these studies.Conclusions For the DNA epigenetic study, we found that 5hmC content is lower in HCC tumor tissues than tumor-adjacent tissues, and 5hmC content highly correlated with tumor stages and tumor size. The marked depletion of 5hmC may have profound effects on epigenetic regulation in HCC and could be a potential biomarker for the early detection and prognosis of HCC. In addition, mtDNA content of HCC tumor tissues was significant negatively correlated with 5mC content, but not 5hmC. The linkages among mtDNA content,5mC and 5hmC in the genomic DNA of HCC tumor tissues will provide an important new insight on the pathogenesis of HCC. For the RNA epigenetic study, our results showed that the mRNA expression level of FTO was significantly higher in T2DM patients and diabetic rats than that of the controls and was associated with the risk of T2DM. And the m6A content were correlated with FTO mRNA expression. These results suggest that the increased mRNA expression of FTO could be responsible for the reduction of m6 A in T2DM, which may further increase the risk of complications of T2DM. The increased FTO mRNA expression level might be investigated further as a novel potential biomarker of T2DM. Moreover, the mRNA expression levels of METTL3, METTL14 and WTAP genes were significantly higher in T2DM patients than healthy controls, and there are similar results in the rat model experiments. METTL14 mRNA level was negatively correlated with m6A contents. Interestingly, the METTL3, METTL14 and WTAP mRNA levels were correlated with both FTO and ALKBH5 expressions. METTL14 seems play a central role in the methyltransferase complex arising suggestions for crystallographic studies.