Mechanisms and clinical landscape of N6-methyladenosine (m6A) RNA modification in gastrointestinal tract cancers.

Epigenetics encompasses reversible and heritable chemical modifications of non-nuclear DNA sequences, including DNA and RNA methylation, histone modifications, non-coding RNA modifications, and chromatin rearrangements. In addition to well-studied DNA and histone methylation, RNA methylation has emerged as a hot topic in biological sciences over the past decade. N6-methyladenosine (m6A) is the most common and abundant modification in eukaryotic mRNA, affecting all RNA stages, including transcription, translation, and degradation. Advances in high-throughput sequencing technologies made it feasible to identify the chemical basis and biological functions of m6A RNA. Dysregulation of m6A levels and associated modifying proteins can both inhibit and promote cancer, highlighting the importance of the tumor microenvironment in diverse biological processes. Gastrointestinal tract cancers, including gastric, colorectal, and pancreatic cancers, are among the most common and deadly malignancies in humans. Growing evidence suggests a close association between m6A levels and the progression of gastrointestinal tumors. Global m6A modification levels are substantially modified in gastrointestinal tumor tissues and cell lines compared to healthy tissues and cells, possibly influencing various biological behaviors such as tumor cell proliferation, invasion, metastasis, and drug resistance. Exploring the diagnostic and therapeutic potential of m6A-related proteins is critical from a clinical standpoint. Developing more specific and effective m6A modulators offers new options for treating these tumors and deeper insights into gastrointestinal tract cancers.

miR-3682-3p Activated by c-Myc Aggravates the Migration and Stemness in Hepatocellular Carcinoma Cells by Regulating PTEN/PI3K/AKT/ß-Catenin Signaling.

BACKGROUND: Hepatocellular carcinoma (HCC) is a leading cancer worldwide. miRNA has been linked to cancer processes. We want to figure out what the underlying mechanism and functions of miR-3682-3p are in HCC. METHODS: Thirty pairs of tumor tissues and adjacent tissues were obtained from HCC patients. mRNA and protein expressions were detected by quantitative real-time PCR and Western blot, respectively. The migration and invasion were measured using transwell or wound-healing assays. Dual luciferase and ChIP assays were utilized to detect gene interactions. RESULTS: miR-3682-3p was highly expressed in HCC tissues and cell lines. Silencing of miR-3682-3p inhibited cell migration and invasion, increased E-cadherin expression, and decreased N-cadherin, vimentin, and snail expressions, as well as the SOX2, OCT4, and Bmi1 expression, thereby restraining EMT and stemness of HCC in vitro. miR-3682-3p was positively activated by c-Myc and could directly target PTEN to activate PI3K/AKT/ß-catenin pathway. In addition, inhibition of PTEN weakened the anti-migration and anti-stemness effects of miR-3682-3p downregulation in HCC cells. CONCLUSION: miR-3682-3p promoted HCC migration and stemness through PTEN/PI3K/AKT/ß-catenin signaling, implying that miR-3682-3p might be a promising target for HCC clinical treatment.

Expression, Prognostic Value, and Functional Mechanism of Polarity-Related Genes in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a common malignant tumor with high mortality and poor prognoses around the world. Within-cell polarity is crucial to cell development and function maintenance, and some studies have found that it is closely related to cancer initiation, metastasis, and prognosis. The aim of our research was to find polarity-related biomarkers which improve the treatment and prognosis of HCC. For the knowledge-driven analysis, 189 polarity-related genes (PRGs) were retrieved and curated manually from the molecular signatures database and reviews. Meanwhile, in the data-driven part, genomic datasets and clinical records of HCC was obtained from the cancer genome atlas database. The potential candidates were considered in the respect to differential expression, mutation rate, and prognostic value. Sixty-one PRGs that passed the knowledge and data-driven screening were applied for function analysis and mechanism deduction. Elastic net model combing least absolute shrinkage and selection operator and ridge regression analysis refined the input into a 12-PRG risk model, and its pharmaceutical potency was evaluated. These findings demonstrated that the integration of multi-omics of PRGs can help us in untangling the liver cancer pathogenesis as well as illustrate the underlying mechanisms and therapeutic targets.

Five-lipoxygenase-activating protein-mediated CYLD attenuation is a candidate driver in hepatic malignant lesion.

Hepatocellular carcinoma (HCC) is an inflammation-associated cancer. However, the lipid pro-inflammatory mediators have only been seldom investigated in HCC pathogenesis. Cylindromatosis (CYLD) attenuation is involved in hepatocarcinogenesis. Here, we aimed to evaluate the significance of hepatic lipid pro-inflammatory metabolites of arachidonate-affected CYLD expression via the 5-lipoxygenase (5-LO) pathway. Resection liver tissues from HCC patients or donors were evaluated for the correlation of 5-LO/cysteinyl leukotrienes (CysLTs) signaling to the expression of CYLD. The impact of functional components in 5-LO/CysLTs cascade on survival of HCC patients was subsequently assessed. Both livers from canines, a preponderant animal for cancer research, and genetic-modified human HCC cells treated with hepatocarcinogen aristolochic acid I (AAI) were further used to reveal the possible relevance between 5-LO pathway activation and CYLD suppression. Five-LO-activating protein (FLAP), an essential partner of 5-LO, was significantly overexpressed and was parallel to CYLD depression, CD34 neovascular localization, and high Ki-67 expression in the resection tissues from HCC patients. Importantly, high hepatic FLAP transcription markedly shortened the median survival time of HCC patients after surgical resection. In the livers of AAI-treated canines, FLAP overexpression was parallel to enhanced CysLTs contents and the simultaneous attenuation of CYLD. Moreover, knock-in FLAP significantly diminished the expression of CYLD in AAI-treated human HCC cells. In summary, the hepatic FLAP/CysLTs axis is a crucial suppressor of CYLD in HCC pathogenesis, which highlights a novel mechanism in hepatocarcinogenesis and progression. FLAP therefore can be explored for the early HCC detection and a target of anti-HCC therapy.

Human menstrual blood-derived stem cells as immunoregulatory therapy in COVID-19: A case report and review of the literature.

BACKGROUND: The coronavirus disease 2019 (COVID-19) caused by novel coronavirus 2019 in December 2019 has spread all around the globe and has caused a pandemic. There is still no current effective guidance on the clinical management of COVID-19. Mesenchymal stem cell therapy has been shown to be one of the therapeutic approaches to alleviate pneumonia and symptoms through their immunomo-dulatory effect in COVID-19 patients. CASE SUMMARY: We describe the first confirmed case of COVID-19 in Hangzhou to explore the role of human menstrual blood-derived stem cells (MenSCs) in the treatment of COVID-19. Moreover, we review the immunomodulation effect including non-specific and specific immune functions of MenSCs for the therapy of COVID-19. CONCLUSION: MenSCs can be helpful to find a promising therapeutic approach for COVID-19.

A Quality Assurance Scheme for Prenatal Detection of Aneuploidy Based on Developing Chimeric Quality-Control Cells.

BACKGROUND: The shortage of quality-control materials caused by non-renewable utilization of rare disease samples is the key factor to limit the quality control of prenatal molecular diagnosis. This study aimed to prepare aneuploid amniocyte lines for the development of quality control cells for fluorescence in situ hybridization (FISH)-mediated detection of aneuploidy. METHODS: Recombinant SV40LTag-pcDNA3.1(-) vectors were transfected into 47,XY,+18 amniotic fluid cells with the use of liposomes. After culturing, these cells were mixed with primary amniocytes with the karyotype 46,XY to prepare four groups of chimeric quality control cells comprising recombinant cells with the karyotypes 47,XY,+18 and primary cells with 46,XY, with theoretical ratios of 47,XY,+18 cells at 5%, 10%, 20%, and 40%. Subsequently, the chimeric quality control cells were tested as clinical samples by three technicians to examine their feasibility for use as internal quality controls (IQC) for FISH detection. RESULTS: After being immortalized by the SV40 large T antigen gene (SV40LT), these aneuploid amniocytes can be cultured indefinitely to prepare chimeric quality control cells. The actual ratio of the 47,XY,+18 cells was identified by FISH to be 1.5 ± 1.1%, 10.3 ± 1.0%, 19.9 ± 0.4%, and 40.8 ± 0.3%, respectively, and the fluorescence signals of chromosomes 13, 18, 21, X, and Y in these cells were consistent with that of the primary cells. CONCLUSIONS: The present study may resolve the shortage of quality control cells in the prenatal detection of chromosomal aneuploidy and may provide a foundation for IQC-based detection in FISH.

Assessment of biological functions for C3A cells interacting with adverse environments of liver failure plasma.

BACKGROUND: For its better differentiated hepatocyte phenotype, C3A cell line has been utilized in bioartificial liver system. However, up to now, there are only a few of studies working at the metabolic alternations of C3A cells under the culture conditions with liver failure plasma, which mainly focus on carbohydrate metabolism, total protein synthesis and ureagenesis. In this study, we investigated the effects of acute liver failure plasma on the growth and biological functions of C3A cells, especially on CYP450 enzymes. METHODS: C3A cells were treated with fresh DMEM medium containing 10% FBS, fresh DMEM medium containing 10% normal plasma and acute liver failure plasma, respectively. After incubation, the C3A cells were assessed for cell viabilities, lactate dehydrogenase leakage, gene transcription, protein levels, albumin secretion, ammonia metabolism and CYP450 enzyme activities. RESULTS: Cell viabilities decreased 15%, and lactate dehydrogenase leakage had 1.3-fold elevation in acute liver failure plasma group. Gene transcription exhibited up-regulation, down-regulation or stability for different hepatic genes. In contrast, protein expression levels for several CYP450 enzymes kept constant, while the CYP450 enzyme activities decreased or remained stable. Albumin secretion reduced about 48%, and ammonia accumulation increased approximately 41%. CONCLUSIONS: C3A cells cultured with acute liver failure plasma showed mild inhibition of cell viabilities, reduction of albumin secretion, and increase of ammonia accumulation. Furthermore, CYP450 enzymes demonstrated various alterations on gene transcription, protein expression and enzyme activities.

Efficient generation of functional hepatocyte-like cells from mouse liver progenitor cells via indirect co-culture with immortalized human hepatic stellate cells.

BACKGROUND: Differentiation of liver progenitor cells (LPCs) to functional hepatocytes holds great potential to develop new strategies for hepatocyte transplantation and the screening of drug-induced cytotoxicity. However, reports on the efficient and convenient hepatic differentiation of LPCs to hepatocytes are few. The present study aims to investigate the possibility of generating functional hepatocytes from LPCs in an indirect co-culture system. METHODS: Mouse LPCs were co-cultured in Transwell plates with an immortalized human hepatic stellate cell line (HSC-Li) we previously established. The morphology, expression of hepatic markers, and functions of mouse LPC-derived cells were monitored and compared with those of conventionally cultured LPCs. RESULTS: Co-culturing with HSC-Li cells induced differentiation of mouse LPCs into functional hepatocyte-like cells. The differentiated cells were morphologically transformed into hepatocyte-like cells 3 days after co-culture initiation. In addition, the differentiated cells expressed liver-specific genes and possessed hepatic functions, including glycogen storage, low-density lipoprotein uptake, albumin secretion, urea synthesis, and cytochrome P450 1A2 enzymatic activity. CONCLUSIONS: Our method, which employs indirect co-culture with HSC-Li cells, can efficiently induce the differentiation of LPCs into functional hepatocytes. This finding suggests that this co-culture system can be a useful method for the efficient generation of functional hepatocytes from LPCs.

Melatonin enhances plant growth and abiotic stress tolerance in soybean plants.

Melatonin is a well-known agent that plays multiple roles in animals. Its possible function in plants is less clear. In the present study, we tested the effect of melatonin (N-acetyl-5-methoxytryptamine) on soybean growth and development. Coating seeds with melatonin significantly promoted soybean growth as judged from leaf size and plant height. This enhancement was also observed in soybean production and their fatty acid content. Melatonin increased pod number and seed number, but not 100-seed weight. Melatonin also improved soybean tolerance to salt and drought stresses. Transcriptome analysis revealed that salt stress inhibited expressions of genes related to binding, oxidoreductase activity/process, and secondary metabolic processes. Melatonin up-regulated expressions of the genes inhibited by salt stress, and hence alleviated the inhibitory effects of salt stress on gene expressions. Further detailed analysis of the affected pathways documents that melatonin probably achieved its promotional roles in soybean through enhancement of genes involved in cell division, photosynthesis, carbohydrate metabolism, fatty acid biosynthesis, and ascorbate metabolism. Our results demonstrate that melatonin has significant potential for improvement of soybean growth and seed production. Further study should uncover more about the molecular mechanisms of melatonin's function in soybeans and other crops.

Identification and characterization of the soybean IPK1 ortholog of a low phytic acid mutant reveals an exon-excluding splice-site mutation.

Phytic acid (myo-inositol 1, 2, 3, 4, 5, 6 hexakisphosphate) is an important constituent of soybean meal. Since phytic acid and its mineral salts (phytates) are almost indigestible for monogastrics, their abundance in grain food/feed causes nutritional and environmental problems; interest in breeding low phytic acid has therefore increased considerably. Based on gene mapping and the characteristics of inositol polyphosphates profile in the seeds of a soybean mutant line Gm-lpa-ZC-2, the soybean ortholog of inositol 1,3,4,5,6 pentakisphosphate (InsP(5)) 2-kinase (IPK1), which transforms InsP(5) into phytic acid, was first hypothesized as the candidate gene responsible for the low phytic acid alteration in Gm-lpa-ZC-2. One IPK1 ortholog (Glyma14g07880, GmIPK1) was then identified in the mapped region on chromosome 14. Sequencing revealed a G → A point mutation in the genomic DNA sequence and the exclusion of the entire fifth exon in the cDNA sequence of GmIPK1 in Gm-lpa-ZC-2 compared with its wild-type progenitor Zhechun No. 3. The excluded exon encodes 37 amino acids that spread across two conserved IPK1 motifs. Furthermore, complete co-segregation of low phytic acid phenotype with the G → A mutation was observed in the F(2) population of ZC-lpa x Zhexiandou No. 4 (a wild-type cultivar). Put together, the G → A point mutation affected the pre-mRNA splicing and resulted in the exclusion of the fifth exon of GmIPK1 which is expected to disrupt the GmIPK1 functionality, leading to low phytic acid level in Gm-lpa-ZC-2. Gm-lpa-ZC-2, would be a good germplasm source in low phytic acid soybean breeding.

[In vitro studies on the growth and proliferation characteristics of rat bone mesenchymal stem cells].

OBJECTIVE: This study was to investigate the growth and proliferation characteristics of rat bone mesenchymal stem cells (BMSCs) isolated by the method of whole bone marrow culture and to explore the effect of cell inoculation density and incubation period on cell proliferation, with an aim to provide multipotential seed cells for preventing from degenerative disease. METHODS: Bone mesenchymal stem cells were isolated by the method of whole bone marrow culture and then cultured in vitro. The cell morphologic features were observed by inverted microscope. The cell surface antigens were identified by flow cytometry. The effect of cell inoculation density and culture period on cell growth and proliferation was explored by analyzing the characteristics of a ten-day cell growth curve in 96-well plates. RESULTS: Flow cytometry results showed the detection rates for CD29, CD34 and CD45 were 97.68% (7607/7788), 7.93% (661/8340) and 2.76% (215/7788) respectively, which was consistent with the expression characteristics of BMSCs surface antigens. BMSCs became uniform after three cell passages, existing in a typical shape of whirlpool or radial colony. The senescent cells started to appear at 7(th) passage, and more senescent cells were found at 10(th) passage. The growth curve for moderate inoculation density was typically S-shaped. Lag phase was found during the first two days, and logarithm growth phase was in the following three days. Plateau phase started from the 6(th) day and cell numbers decreased slightly from the 8(th) day. CONCLUSION: The whole bone marrow culture is an effective way to obtain BMSCs. A moderate inoculation density was more advantageous to cell proliferation, by which more seed cells could be obtained.

[Effects of drynaria total flavonoid on osteogenic differentiation of bone marrow mesenchymal stem cells at different glucose concentrations: experiment with rats].

OBJECTIVE: To study the effects of drynaria total flavonoid on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) at different glucose concentrations. METHODS: BMSCs of SD rats were isolated, cultivated in vitro, and divided into 6 groups to be induced to differentiate into osteoblasts under different conditions: (1) low glucose control group, (2) high glucose control group, (3) low glucose classical induction group (sodium glycerophosphate+vitamin C+dexamethasone), (4) high glucose classical induction group (sodium glycerophosphate+vitamin C+dexamethasone), (5) low glucose+drynaria total flavonoid group, and (6) high glucose with drynaria total flavonoid group. Alkaline phosphate (ALP) test kit was used to examine the level of ALP. The ALP staining positive rate was examined with modified calcium cobalt method. Alizarin red staining was adopted to observe the number of calcium nodes. Immunohistochemistry was used to detect type I collagen level. Advanced glycosylation end products (AGEs) were tested by ELISA. RESULTS: The A value indicating the ALP activity, ALP staining positive rate, calcium node number, and type I collagen expression score of the low glucose+drynaria total flavonoid group were (0.439±0.024), 48.7%, (9.75±1.71) nodes/HP, and (2.21±0.07) respectively, all significantly higher than those of the sodium glycerophosphate+vitamin C+dexamethasone [(0.385±0.029), 35.0%, (6.25±0.96) nodes/HP, and (1.93±0.13) respectively, all P<0.05]. The A value, ALP staining positive rate, calcium node number, and type I collagen expression score of the high glucose with drynaria total flavonoid group were (0.352±0.022), 25.3%, (4.50±1.29)/HP, and (1.70±0.03) respectively, all significantly higher than those of the sodium glycerophosphate+vitamin C+dexamethasone [(0.139±0.013), 22.7%, (3.25±1.50)/HP, and (1.28±0.27) respectively, all P<0.05]. The AGE expression levels of the high glucose classical induction group and high glucose+drynaria total flavonoid group were both significantly higher than those of the low glucose classical induction group and low glucose+drynaria total flavonoid group (both P<0.05). There were no significant differences in the AGE level among the low glucose control, low glucose classical induction, and low glucose+drynaria total flavonoid groups (all P<0.05); and among the high glucose control, high glucose classical induction, and high glucose+drynaria total flavonoid groups (all P<0.05). However, the AGE levels of the high glucose groups were all significantly higher than those of the corresponding low glucose groups (all P<0.05). Glucose increased the AGE levels dose- and time-dependently. The concentrations of AGEs were significantly negatively correlated with the expression of type I collagen (r=-0.410, P<0.05). CONCLUSIONS: Drynaria total flavonoid promotes the osteogenic differentiation of BMSCs and relieves the inhibitory effect of osteogenic differentiation by glucose at high concentration. Thus drynaria total flavonoid may provide a potential therapy for diabetic osteoporosis.

Effects of two low phytic acid mutations on seed quality and nutritional traits in soybean (Glycine max L. Merr).

Reduction of phytic acid in soybean seeds has the potential to improve the nutritional value of soybean meal and lessen phosphorus pollution in large scale animal farming. The objective of this study was to assess the effect of two new low phytic acid (LPA) mutations on seed quality and nutritional traits. Multilocation/season comparative analyses showed that the two mutations did not affect the concentration of crude protein, any of the individual amino acids, crude oil, and individual saturated fatty acids. Among other traits, Gm-lpa-TW75-1 had consistently higher sucrose contents (+47.4-86.1%) and lower raffinose contents (-74.2 to -84.3%) than those of wild type (WT) parent Taiwan 75; Gm-lpa-ZC-2 had higher total isoflavone contents (3038.8-4305.4 microg/g) than its parent Zhechun # 3 (1583.6-2644.9 microg/g) in all environments. Further tests of homozygous F(3) progenies of the cross Gm-lpa-ZC-2 x Wuxing # 4 (WT variety) showed that LPA lines had a mean content of total isoflavone significantly higher than WT lines. This study demonstrated that two LPA mutant genes have no negative effects on seed quality and nutritional traits; they instead have the potential to improve a few other properties. Therefore, these two mutant genes are valuable genetic resources for breeding high quality soybean varieties.