MRTO4 Enhances Glycolysis to Facilitate HCC Progression by Inhibiting ALDOB.

BACKGROUND MRT4 Homolog, Ribosome Maturation Factor (MRTO4) is often upregulated in cancer cells. However, its impact in hepatocellular carcinoma (HCC) is less well understood. Herein, we explored the prognostic and energy metabolism reprogramming role of MRTO4 in HCC. MATERIAL AND METHODS Clinical data were obtained from The Cancer Genome Atlas (TCGA), and the expression of MRTO4 in clinical samples was analyzed. The association between different variables and overall survival (OS) was studied, as well as their potential as independent prognostic factors, using Cox regression analysis. We constructed a nomogram including clinical pathological variables and MRTO4 expression to provide a predictive model for prognosis. Heatmaps, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed the relationship between energy metabolism pathways and MRTO4. We used classic molecular biology research methods, including RT-qPCR, Western blotting, CCK8, TUNEL, Clone formation, Transwell assay, ELISA, and immunohistochemistry, to study the role of MRTO4 in promoting the progression of HCC through glycolysis regulation. RESULTS Our study showed that MRTO4 is an independent prognostic risk factor for HCC and that MRTO4 accelerates glycolysis of HCC cells, promotes proliferation and invasion, and suppresses apoptosis of HCC cells. The underlying mechanism involves MRTO4 promoting glycolysis and accelerating HCC by inhibiting ALDOB. CONCLUSIONS Our study revealed a novel mechanism by which MRTO4 promotes glycolysis and accelerates HCC progression, and suggests that inhibiting MRTO4 could be a potential therapeutic strategy for HCC.

Exosomal IGFBP2 derived from highly metastatic promotes hepatocellular carcinoma metastasis by inducing epithelial mesenchymal transition.

Liver cancer metastasis is the main cause of death in liver cancer patients. Exosomes, which are small vesicles released by cancer cells, play a crucial role in the metastasis of cancer. The aim of this study was to investigate the effect of exosomes derived from high metastatic potential liver cancer cells acting as cell to cell communication on liver cancer metastasis. Bioinformatics analysis was used to obtain the differential expression of exosomal mRNAs from the plasma of both liver cancer patients and healthy volunteers. Transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and protein blot were employed to characterize the exosomes. The molecular mechanisms and were explored by conducting CCK8, Transwell, Tunel, RTqPCR, western blot, and immunofluorescence staining. We examined IGFBP2 special expression in the plasma exosomes of both liver cancer patients and healthy volunteers, and its presence was associated with a poor prognosis in liver cancer patients. Furthermore, we observed that exosomes from highly metastatic liver cancer cells (MHCC97H) contained high levels of IGFBP2 and could enhance the metastatic potential of less aggressive liver cancer cells (Hep3B). Additionally, we discovered that IGFBP2 in MHCC97H-derived exosomes activated ERK signaling pathway, which triggered epithelial-mesenchymal transition (EMT) in Hep3B cells. Our study underscores the significance of exosomal IGFBP2 from highly metastatic liver cancer cells as a driver of metastasis in less invasive liver cancer cells. This suggests that targeting IGFBP2 in exosomes could be a promising strategy for the treatment and prognosis of liver cancer patients.

Genome-Wide mRNA-Seq Profiling Reveals that LEF1 and SMAD3 Regulate Epithelial-Mesenchymal Transition Through the Hippo Signaling Pathway During Palatal Fusion.

BACKGROUND: Epithelial-mesenchymal transition (EMT) of the medial edge epithelium (MEE) occurs through fusion of the palatal shelves and is a crucial step in palatogenesis. The key genes, however, and the related signaling pathway of EMT are not yet fully understood. Therefore, the aim of this study was to reveal the key genes and the related signaling pathway of EMT during palatal fusion. MATERIALS AND METHODS: C57BL/6J mice at embryonic gestation day 14.5 (E14.5; n = 6) were used to establish the cleft palate model for mRNA-Seq (HiSeq X Ten). The Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed for functional annotations of the differentially expressed genes. Quantitative polymerase chain reaction (qPCR) assays were used to validate the RNAseq data. RESULTS: A total of 936 differentially expressed genes, including 558 upregulated and 378 downregulated genes were identified in cases versus controls, respectively. Among these genes, the GO analysis showed that Lymphoid Enhancer-Binding Factor 1 (LEF1) and SMAD Family Member 3 (SMAD3) significantly enriched biological processes, which were EMT related. The KEGG analysis showed that these genes regulated EMT through the Hippo signaling pathway. LEF1 and SMAD3 were downregulated, and the qPCR results corroborated the RNA-seq data. CONCLUSIONS: These results demonstrate that LEF1 and SMAD3 inhibits EMT at the MEE through the Hippo signaling pathway; and that this could contribute to cleft palate formation in embryonic palatal fusion at E 14.5.

A novel lncRNA-mediated trans-regulatory mechanism in the development of cleft palate in mouse.

BACKGROUND: Increasing evidence indicates that long non-coding RNAs (lncRNAs) play crucial regulatory roles in epithelial-mesenchymal transition (EMT). However, the regulatory mechanisms during EMT of the medial edge epithelium (MEE) remain elusive. The aim of this work is to reveal a novel lncRNA-regulated dysfunction of EMT involved in the development of cleft palate (CP). METHODS: C57BL/6 J mice at embryonic gestation day 14.5 (n = 6, 3 case samples vs. 3 control samples) were used to establish the CP model for lncRNA-mRNA co-expression profile analysis after high-throughput sequencing. Functional predictions for the differentially expressed lncRNA-mRNA co-expression with transcription factor (TF)-target gene relationship Gene Ontology/Kyoto Encyclopedia of Genes and Genomes pathway (GO/KEGG) analyses identified the regulatory "lncRNA-TF-target gene" trans model. RESULTS: A total of 583 differentially expressed lncRNAs and 703 differentially expressed mRNAs were identified. The results of trans analysis revealed that some TFs (LEF1, SMAD4, and FOXD3) regulate lncRNAs and gene expression. Finally, we identified the NONMMUT034790.2-LEF1-SMAD7 co-expression trans-regulatory network that might be associated with CP. CONCLUSIONS: Our results revealed that NONMMUT034790.2 might be a novel epigenetic biomarker in CP. The integration of lncRNA modulators into trans-regulatory networks will further enhance our understanding of lncRNA functions and regulatory mechanisms during palatal fusion in ATRA-induced mouse CP.

MYO18B promotes hepatocellular carcinoma progression by activating PI3K/AKT/mTOR signaling pathway.

BACKGROUND: MYO18B has been identified as a novel tumor suppressor gene in several cancers. However, its specific roles in the progression of hepatocellular carcinoma (HCC) has not been well defined. METHODS: We firstly identified the expression and prognostic values of MYO18B in HCC using TCGA cohort and our clinical data. Then, MYO18B knockdown by RNA inference was implemented to investigate the effects of MYO18B on HCC cells. Quantitative RT-PCR and Western blot were used to determine gene and protein expression levels. CCK-8 and colony formation assays were performed to examine cell proliferation capacity. Wound healing and transwell assays were used to evaluate the migration and invasion of HepG2 cells. RESULTS: MYO18B was overexpressed and correlated with poor prognosis in HCC. MYO18B expression was an independent risk factor for overall survival. Knockdown of MYO18B significantly inhibited the proliferation, migration and invasion of HepG2 cells. Meanwhile, MYO18B knockdown could effectively suppress the phosphorylation of PI3K, AKT, mTOR and P70S6K, suggesting that MYO18B might promote HCC progression by targeting PI3K/AKT/mTOR signaling pathway. CONCLUSIONS: MYO18B promoted tumor growth and migration via the activation of PI3K/AKT/mTOR signaling pathway. MYO18B might be a promising target for clinical intervention of HCC.

Correlation between HDAC4 enhancer DNA methylation and mRNA expression during palatal fusion induced by all-trans retinoic acid.

Epithelial-mesenchymal transformation of the medial edge epithelium is the most crucial process in embryonic palatal fusion. This study aimed to explore the relationship and potential mechanism between enhancer DNA methylation and mRNA expression of histone deacetylase 4 (HDAC4) during palatal fusion induced by maternal exposure to all-trans retinoic acid (ATRA). Pregnant mice were administered ATRA (70 mg/kg) by gavage at embryonic gestation day 10.5 (E10.5) to establish a cleft palate (CP) model in C57BL/6J mice. Control groups were given an equivalent volume of corn oil. Pregnant mice were dissected at E14.5 (n = 6) to obtain embryonic palates. HDAC4 enhancer DNA methylation data were obtained from a previous MethylRAD-seq. Methylation-specific polymerase chain reaction (MSP) and real-time quantitative PCR were used to quantify enhancer methylation and the mRNA expression level of HDAC4. Enhancer DNA methylation at a non-CpG site within the HDAC4 gene was hyper-methylated at E14.5 (P: 0.011, log2 FC:1.67). The MSP results indicated a similar trend, in agreement with the MethylRAD-seq results. The change in the HDAC4 expression level was negatively correlated with its enhancer DNA methylation level, at the non-CpG site, during palatal fusion induced by ATRA. Enhancer DNA methylation of HDAC4 might play an important regulatory role during palatogenesis, especially in embryonic palatal fusion at E 14.5, and may facilitate the development of novel epigenetic biomarkers in the treatment of CP.

Altered serum copper homeostasis suggests higher oxidative stress and lower antioxidant capability in patients with chronic hepatitis B.

Copper homeostasis can be altered by inflammation. This study aimed to investigate the alteration of serum copper homeostasis and to explore its clinical significance in patients with chronic hepatitis B (CHB).Thirty-two patients with CHB and 10 aged- and sex-matched healthy controls were recruited. Analyses included serum levels of total copper (TCu), copper ions (Cu), small molecule copper (SMC), ceruloplasmin (CP), Cu/Zn superoxide dismutase 1 (SOD1), urinary copper, and the activities of serum CP and SOD1.The serum TCu and urinary copper levels in patients with CHB were significantly higher than the controls (P = .04 and .003), while the serum Cu was lower than the controls (P = .0002). CP and SOD1 activities in the serum were significantly lower in patients with CHB compared to controls (P = .005) despite higher serum concentrations. In addition, serum alanine aminotransferase inversely correlated with serum CP activity (P = .0318, r = -0.4065).Serum copper homeostasis was altered in this cohort of patients with CHB. The results suggest increased oxidative stress and impaired antioxidant capability in patients with CHB, in addition to necroinflammation. These results may provide novel insights into the diagnosis and treatment of patients with CHB.

Genome-Wide DNA Methylation Analysis During Palatal Fusion Reveals the Potential Mechanism of Enhancer Methylation Regulating Epithelial Mesenchyme Transformation.

Epithelial mesenchyme transformation (EMT) of the medial edge epithelium (MEE) is the crucial process during palatal fusion. This work is aimed to elucidate the enhancer regulatory mechanism by genome-wide DNA methylation analysis of EMT during palatal fusion. Over 800 million clean reads, 325 million enzyme reads, and 234 million mapping reads were generated. The mapping rate was 68.85-74.32%, which included differentially methylated 17299 CCGG sites and 2363 CCWGG sites (p < 0.05, log2FC >1). Methylated sites in intron and intergenic regions were more compared to other regions of all DNA elements. GO and KEGG analysis indicated that differential methylation sites related to embryonic palatal fusion genes (HDAC4, TCF7L2, and PDGFRB) at the enhancer were located on CCWGG region of non-CpG islands. In addition, the results showed that the enhancer for HDAC4 was hypermethylated, whereas the enhancers for TCF7L2 and PDGFRB were hypomethylated. The methylation status of enhancer regions of HDAC4, PDGFRB, and TCF7L2, involved in the regulation of the EMT during palatal fusion, may enlighten the development of novel epigenetic biomarkers in the treatment of cleft palate.

[Association between single nucleotide polymorphisms of v-maf musculoaponeurotic fibrosarcoma oncogene homolog B gene and non-syndromic cleft lip with or without cleft palate].

OBJECTIVE: To reveal the association between the single nucleotide polymorphism (SNP) of v-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) gene rs17820943 locus and non-syndromic cleft lip with or without cleft palate (NSCL/P) in the southern Chinese Han population. METHODS: Genotyping of MAFB gene rs17820943 polymorphism was carried out in 300 patients with NSCL/P, 354 normal controls, and an additional 168 case-parent trios with matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry. Then based on the genotyping results, both a case-control association study and a case-parent trio association study were performed. RESULTS: Significant differences were found in the allele and genotype frequencies of rs17820943 locus between case and control groups (Pallele = 0.001 and Pgenotype = 0.002, respectively). To be specific, the odds radio (OR) values and 95% confidence interval (95% CI) of allele T (frequencies of cases:controls = 0.358:0.448) and genotype TT (frequencies of cases:controls = 0.110:0.195) were ORT = 0.69 (95% CI: 0.55-0.86) and OR(TT) = 0.43 (95% CI: 0.26-0.70), respectively. Subsequent case-parent trio analysis also indicated an association between MAFB rs17820943 variant and the risk of NSCL/P (ORT(T vs. C) = 0.55, 95% CI: 0.41-0.75, P value of transmission disequilibrium test was 0.000). CONCLUSION: Polymorphism of MAFB gene rs17820943 locus is associated with NSCL/P in the southern Chinese Han population; MAFB rs17820943 variant may be a susceptible gene of NSCL/P.

Association between single-nucleotide polymorphisms on chromosome 1p22 and 20q12 and nonsyndromic cleft lip with or without cleft palate: new data in Han Chinese and meta-analysis.

BACKGROUND: Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a common congenital malformation associated with genetic and environmental risk factors. A recent genome-wide association study identified two novel susceptibility loci on chromosomes 1p22 and 20q12; however, conflicting results, especially for 1p22, have been reported in Han Chinese population. The aims of this study were to replicate this association with risk of NSCL/P in the southern Han Chinese population and to discern the effect of these loci by a meta-analysis. METHODS: To this end, 305 patients with NSCL/P, 356 phenotypically normal controls, and an additional 176 case-parent trios were recruited. Four of the previously associated single nucleotide polymorphisms (SNPs) were genotyped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Furthermore, two published datasets were combined with the present results to determine the precise roles of the loci. RESULTS: SNPs (rs6072081, rs13041247, and rs6102085) on 20q12 were found to be strongly associated with NSCL/P (Bonferroni-corrected and χ(2) test; p values < 0.05). Subsequent analysis of the case-parent trio provided similar results. However, neither the association study nor the trio analysis supported a causative role for SNP rs560426 on 1p22 in NSCL/P susceptibility. Stratified meta- analysis combining Chinese samples supported our findings. CONCLUSIONS: This cross-validation study confirmed the previous findings that SNPs in 20q12 are associated with NSCL/P in Han Chinese population. We further conclude that rs560426 on 1p22 might not have a major influence on susceptibility to NSCL/P in southern Han Chinese, but future studies with other Han Chinese populations are needed.

PVRL1 as a candidate gene for nonsyndromic cleft lip with or without cleft palate: no evidence for the involvement of common or rare variants in southern Han Chinese patients.

The poliovirus receptor related-1 (PVRL1) gene encodes nectin-1, a cell-cell adhesion molecule (OMIM #600644), and is mutated in the cleft lip with or without cleft palate/ectodermal dysplasia-1 syndrome (CLPED1, OMIM #225000). In addition, PVRL1 mutations have been associated with nonsyndromic cleft lip with or without a cleft palate (NSCL/P) in studies of multiethnic samples. To investigate the possible involvement of this gene in southern Han Chinese NSCL/P patients, we performed (i) a case-control association study, and (ii) a resequencing study. A set of 470 patients with NSCL/P and 693 controls were recruited, and a total of 45 tagging single-nucleotide polymorphisms (SNPs) were genotyped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In the resequencing study, the coding regions of the PVRL1 α isoform were direct sequenced in 45 trios from multiply affected families. One (rs7128327) of the 45 tested SNPs showed a trend toward statistical significance in the genotypic-level chi-square test (p = 0.009567). However, this result did not withstand correction for multiple testing. Likewise, sliding window haplotype analyses consisting of two, three, or four SNPs failed to detect any positive association. Resequencing analysis also failed to identify any novel rare sequence variants. In conclusion, the present study provided no support for the hypothesis that common or rare variants in PVRL1 play a significant role in NSCL/P development in the southern Han Chinese population. This is the first study that has used tagging SNPs covering all the coding and noncoding regions to search for common NSCL/P-associated mutations of PVRL1.

[Studies on mother-to-infant transmission of hepatitis C virus (HCV) and hepatitis G virus (HGV) co-infection].

OBJECTIVE: To explore mother-to-infant transmission of hepatitis C virus (HCV) and hepatitis G virus (HGV) co-infection and the influence factors. METHODS: Antihepatitis C virus (anti-HCV) and anti-hepatitis G virus (anti-HGV) antibodies were detected by third generation enzyme linked immunosorbent assay (ELISA). HCV RNA and HGV RNA were detected by fluorogenic quantitative-PCR (FQ-PCR). RESULTS: Totally 4506 common pregnant women were tested positive of serum anti-HCV. In these women, 878 were detected of serum anti-HGV, and 10 of them were found with both HCV RNA and HGV RNA positivities. In their 11 infants, two were positive for HCV RNA, and two were positive for HGV RNA. In these 4 infected infants, three were delivered by birth canal, one was delivered by cesarean section. All four were fed by breast-feeding. Three mother's ALTs were abnormally high before delivery. CONCLUSIONS: Hepatitis C and G virus co-infection does not increase the rate of mother-to-infant transmission. Birth canal delivery, breast-feeding and high alanine aminotransferase before delivery are high risk factors for mother-to-infant transmission of HCV and HGV co-infection.