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It is the position of the American Dietetic Association that efforts to optimize nutritional status through individualized medical nutrition therapy, assurance of food and nutrition security, and nutrition education are essential to the total system of health care available to people with human immunodeficiency virus (HIV) infection throughout the continuum of care. Broad-based efforts to improve health care access and treatment have stabilized HIV prevalence levels in many parts of the world and led to longer survival for people living with HIV infection. Confounding clinical and social issues, such as medication interactions, comorbidities, wasting, lipodystrophy, food insecurity, aging, and other related conditions further complicate disease management. With greater understanding of the mechanisms of HIV disease and its impact on body function, development of new treatments, and wider ranges of populations affected, the management of chronic HIV infection continues to become more complex and demanding. Achievement of food and nutrition security and management of nutrition-related complications of HIV infection remain significant challenges for clients with HIV infection and health care professionals. Registered dietitians and dietetic technicians, registered, should integrate their efforts into the overall health care strategies to optimize their clinical and social influence for people living with HIV infection.

N6-Methyladenosine (m6A) modification has been implicated in many biological processes. It is important for the regulation of messenger RNA (mRNA) stability, splicing, and translation. However, its role in cancer has not been studied in detail. Here we investigated the biological role and underlying mechanism of m6A modification in hepatoblastoma (HB).

We used Reverse transcription quantitative real-time PCR (RT-qPCR) and Western blotting to determine the expression of m6A related factors. And we clarified the effects of these factors on HB cells using cell proliferation assay, colony formation, apoptotic assay. Then we investigated of methyltransferase-like 13 (METTL3) and its correlation with clinicopathological features and used xenograft experiment to check METTL3 effect in vivo. m6A-Seq was used to profiled m6A transcriptome-wide in hepatoblastoma tumor tissue and normal tissue. Finally, methylated RNA immunoprecipitation (MeRIP) assay, RNA remaining assay to perform the regulator mechanism of MEETL3 on the target CTNNB1 in HB.

In this research, we discovered that m6A modifications are increased in hepatoblastoma, and METTL3 is the main factor involved with aberrant m6A modification. We also profiled m6A across the whole transcriptome in hepatoblastoma tumor tissues and normal tissues. Our findings suggest that m6A is highly expressed in hepatoblastoma tumors. Also, m6A is enriched not only around the stop codon, but also around the coding sequence (CDS) region. Gene ontology analysis indicates that m6A mRNA methylation contributes significantly to regulate the Wnt/β-catenin pathway. Reduced m6A methylation can lead to a decrease in expression and stability of the CTNNB1.

Overall our findings suggest enhanced m6A mRNA methylation as an oncogenic mechanism in hepatoblastoma, METTL3 is significantly up-regulated in HB and promotes HB development. And identify CTNNB1 as a regulator of METTL3 guided m6A modification in HB.

In the present study we assessed levels of m6A, its key catalytic genes and profiled m6A transcriptome-wide in HB tissues. The level of m6A was increased in HB, and METTL3 was found to be the main factor involved in aberrant m6A modification. Furthermore, we also explored the target gene CTNNB1 and addressed the mechanism of methyltransferase METTL3 participates modulate CTNNB1 in HB.

To explore the potential role of m6A modification in HB, we first examined m6A levels in the tumor and normal tissues. The elevation in the m6A mRNA level was identified in tumor tissues (Fig. 1a) and HB cells (Additional file 1: Figure S1A). Additionally, we also evaluated the expression of m6A writers, erasers and readers in tumor and adjacent normal hepatic tissues by quantitative PCR coupled with reverse transcription (RT-qPCR) and Western blotting. We found that most of the tumors exhibit significantly up-regulated METTL3, WTAP, FTO and YTHDF2 levels when compared against adjacent normal tissues (Fig. 1b and c). In contrast, no significant difference was observed in the expression of METTL14, KIAA1429 and ALKBH5 between the tumor and normal tissues (Additional file 1: Figure S1B). Overall, these results confirmed that m6A modification was indeed up-regulated in tumor tissue.

To investigate the functional roles of METTL3, WTAP, FTO, YTHDF2 in HB. We established METTL3, WTAP, FTO, YTHDF2 knockdown HepG2 cells with two independent siRNA sequences. RT-qPCR and Western blotting were used to investigate the successful knockdown of METTL3, WTAP, FTO, YTHDF2 in HepG2 cells (Fig. 1d). The knockdown of METTL3, WTAP, FTO, YTHDF2 genes remarkably suppressed HB cell proliferation (Fig. 1e) and inhibited their colony-forming ability (Fig. 1f). Furthermore, annexin V-PI double staining assay revealed that METTL3, WTAP, FTO, YTHDF2 knockdown can also induce apoptosis (Fig. 1g). Considering the catalytic function of these genes and the up-regulated level of m6A modification. We finally choose METTL3 as the candidate molecule as a marker for the aberrant m6A modification in HB. It may act as an oncogene promoting HB proliferation and can also serve as a crucial active component of the m6A methyltransferase having a positive correlation with m6A levels.

m6A-Seq profiling of HB and identified CTNNB1 as a target of METTL3-mediated m6A modification. a Venn diagram of m6A modified genes in tumor and normal tissues. b The relationship between the number of genes and the peaks. c Top consensus motif identified DREME with m6A-seq in HB and normal tissues. d The normalized distribution of m6A peaks across the start codon, CDS, stop codon of mRNAs for normal and tumor m6A peaks. e Graphs of m6A peak distribution showing the proportion of total m6A peaks in the indicated regions in normal and tumor. f Comparison of the abundance of high level m6A peaks across the transcriptome of normal and tumor tissues. Genes with normal specific m6A peaks are highlighted in blue and genes with tumor-specific m6A peaks are highlighted in red. g Bar figure for the abundance of m6A modification percentage and gene expression. h Cumulative distribution curve for the gene expression changes between the tumor and normal tissues for up-regulated m6A methylation (Red) and down-regulated m6A methylation (Green). i The ratio of genes expression in normal (N) and tumor (T) tissues containing specific m6A peaks. Genes according to the peak position are divided into two groups (PeakStart and PeakStop). j KEGG pathway analysis of transcripts with increased m6A methylation and up-regulated mRNA expression in tumors against normal tissues. k Diagram of the Wnt/β-catenin pathway with genes affected by m6A marked by red. The diagram is based on KEGG annotations. l The m6A abundances on CTNNB1 mRNA transcripts in HB normal tissues and tumor tissues as detected by m6A-seq

In order to determine the effect of m6A in HB, we selected strongly expressed genes with up-regulated m6A methylation and identified the KEGG pathway. The KEGG pathway analyses were performed to organize and identify differentially activated biological processes between normal and tumor tissues. The significant genes in the tumor were related with viral carcinogenesis, proteoglycans in cancer, protein processing in endoplasmic reticulum and Wnt signaling pathway and so on. As HB is a Wnt/β-catenin-driven malignancy, we hypothesized that up-regulated m6A methylation might promote tumor growth through activation of the Wnt/β-catenin signaling pathway. Therefore, Wnt/β-catenin signaling pathway was chosen for further research (Fig. 4j). Indeed, several genes such as CTNNB1, CCND1 and NKD1 involved in the Wnt/β-catenin signaling pathway showed increased m6A methylation in tumor tissues with respect to normal (Fig. 4k). Figure 4l showed the m6A abundances of CTNNB1 were markedly increased HB tumor tissues. Overall these results are supportive of the fact that m6A has tendency of demonstrating a positive correlation with gene expression in large fraction of HB transcripts and activate m6A methylation activates the Wnt/β-catenin signaling pathway.

The discovery and mapping of m6A demethylases in mammalian systems suggests that m6A methylation of mRNA is a reversible and dynamic process with regulatory functions. Through a functional interplay between m6A methyltransferase and demethylases, the dynamic m6A modification is implicated in a variety of cellular processes, such as RNA splicing, protein translation, and stem cell renewal. Recently developed, antibody-based, high-throughput sequencing technology has allowed researchers to precisely map m6A sites to obtain further insights. Although, m6A is considered as the most prevalent alteration in human mRNA. Very few studies have focused upon the role of m6A modification in HB. In the present study we observed an increased tendency of m6A modification in HB, suggesting its potentially involvement in the malignancy. Genes METTL3, WTAP, FTO and YTHDF2 were up-regulated, and they all seem to play an essential role in promoting HB growth. Among the m6A related proteins, METTL3 and WTAP were chosen for further research as they were the m6A writers and have increased tendency of m6A modification. However, WTAP lacks methyltransferase domains and shows no catalytic activity of m6A modification. Collectively, it was found that METTL3 was the main factor for the aberrant m6A modification in HB.