Micro-RNA-27a/b negatively regulates hepatic gluconeogenesis by targeting FOXO1.

In the context of hepatic insulin resistance, hepatic gluconeogenesis is abnormally increased, which results in increased hepatic glucose production and hyperglycemia, but the underlying mechanisms remain to be fully elucidated. Micro-RNAs (miRNAs) have been identified as critical regulators of diabetes and other metabolic disorders. In this study, we found that the expressions of miRNA-27 family members miRNA-27a and miRNA-27b (miR-27a/b) decreased significantly in the livers of diabetic mice. Moreover, the levels of miR-27a/b increased in the serum of patients with type 2 diabetes. Our present results showed that inhibition of miR-27a/b expression led to increased hepatic protein levels of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase and enhanced hepatic gluconeogenesis in vitro and in vivo. Overexpression of miR-27a/b suppressed hepatic glucose output and alleviated hyperglycemia in diabetic mice. Further study revealed that forkhead box O1 (FOXO1) is a downstream target of miR-27a/b. Taken together, we found novel evidence suggesting that miR-27a/b contributes to hepatic gluconeogenesis through targeting FOXO1 and provided novel mechanistic insight into the pathophysiology of insulin resistance.

Hepatic miR-181b-5p Contributes to Glycogen Synthesis Through Targeting EGR1.

BACKGROUND/AIM: The miR-181 family plays an important role in the regulation of various cellular functions. However, whether miR-181b-5p mediates hepatic insulin resistance remains unknown. In this study, we investigated the effect of miR-181b-5p on the regulation of hepatic glycogen synthesis. METHODS: The miR-181b-5p levels in the livers of diabetic mice were detected by real-time PCR. The glycogen levels and AKT/GSK pathway activation were examined in human hepatic L02 cells and HepG2 cells transfected with miR-181b-5p mimic or inhibitor. The potential target genes of miR-181b-5p were evaluated using a luciferase reporter assay and Western blot analysis. EGR1-specific siRNA and pCMV-EGR1 were used to further determine the role of miR-181b-5p in hepatic glycogen synthesis in vitro. Hepatic inhibition of miR-181b-5p in mice was performed using adeno-associated virus 8 (AAV8) vectors by tail intravenous injection. RESULTS: The miR-181b-5p levels were significantly decreased in the serum and livers of diabetic mice as well as the serum of type 2 diabetes patients. Importantly, inhibition of miR-181b-5p expression impaired the AKT/GSK pathway and reduced glycogenesis in hepatocytes. Moreover, upregulation of miR-181b-5p reversed high-glucose-induced suppression of glycogenesis. Further analysis revealed that early growth response 1 (EGR1) was a downstream target of miR-181b-5p. Silencing of EGR1 expression rescued miR-181b-5p inhibition-reduced AKT/GSK pathway activation and glycogenesis in hepatocytes. Hepatic inhibition of miR-181b-5p led to insulin resistance in C57BL/6 J mice. CONCLUSION: We demonstrated that miR-181b-5p contributes to glycogen synthesis by targeting EGR1, thereby regulating PTEN expression to mediate hepatic insulin resistance.

Expression and effects of TSP50 in mouse embryo and cardiac myocyte development.

TSP50, a testis-specific gene encoding a serine protease-like protein, was specifically expressed in the spermatocytes of testes but abnormally activated and expressed in many different kinds of cancers. Here, we aimed to analyze the expression of TSP50 in mouse embryo and its function in early embryonic development. Firstly, the distribution of TSP50 in oocytes and embryonic development was characterized by immunofluorescence, RT-PCR and western blotting, and the results showed that TSP50 was detected at all studied stages with a dynamic expression pattern. When overexpressed TSP50 in zygotes by microinjection, the zygotes development was highly accelerated. On the contrary, knocking down TSP50 expression by RNA interference greatly retarded the zygote development. Furthermore, TSP50 expression at embryonic day 6.5 (E6.5), day 8.5 (E8.5) and day 10.5 (E10.5) were increasingly enhanced, However, the expression of TSP50 decreased gradually in the development and differentiation of cardiac myocyte from E12.5 to postnatal (P0). Additionally, we found that TSP50 expression was decreased during cardiac myocyte differentiation of P19 cells. Overexpression of TSP50 could decrease the expression of GATA-4, and knockdown of TSP50 markedly increase the expression of GATA-4. Taken together, our data indicate that TSP50 may play an important role during the process of mouse embryonic development as well as myocardial cell differentiation.

20(S)-25-methoxyl-dammarane-3ß, 12ß, 20-triol negatively regulates activation of STAT3 and ERK pathways and exhibits anti-cancer effects in HepG2 cells.

The pro-inflammatory cytokine interleukin 6 (IL-6), via activating its downstream JAK/STAT3 and Ras/ERK signaling pathways, is involved in cell growth, proliferation and anti-apoptotic activities in various malignancies. To screen inhibitors of IL-6 signaling, we constructed a STAT3 and ERK dual-pathway responsive luciferase reporter vector (Co.RE). Among several candidates, the natural compound 20(S)-25-methoxyl-dammarane-3ß, 12ß, 20-triol (25-OCH3-PPD, GS25) was identified to clearly inhibit the luciferase activity of Co.RE. GS25 was confirmed to indeed inhibit activation of both STAT3 and ERK pathways and expression of downstream target genes of IL-6, and to predominantly decrease the viability of HepG2 cells via induction of cell cycle arrest and apoptosis. Interestingly, GS25 showed preferential inhibition of HepG2 cell viability relative to normal liver L02 cells. Further investigation showed that GS25 could not induce apoptosis and block activation of STAT3 and ERK pathways in L02 cells as efficiently as in HepG2 cells, which may result in differential effects of GS25 on malignant and normal liver cells. In addition, GS25 was found to potently suppress the expression of endogenous STAT3 at a higher concentration and dramatically induce p38 phosphorylation in HepG2 cells, which could mediate its anti-cancer effects. Finally, we demonstrated that GS25 also inhibited tumor growth in HepG2 xenograft mice. Taken together, these findings indicate that GS25 elicits its anti-cancer effects on HepG2 cells through multiple mechanisms and has the potential to be used as an inhibitor of IL-6 signaling. Thus, GS25 may be developed as a treatment for hepatocarcinoma with low toxicity on normal liver tissues as well as other inflammation-associated diseases.

TSP50 Attenuates DSS-Induced Colitis by Regulating TGF-ß Signaling Mediated Maintenance of Intestinal Mucosal Barrier Integrity.

The integrity of the intestinal mucosal barrier is crucial for protecting the intestinal epithelium against invasion by commensal bacteria and pathogens, thereby combating colitis. The investigation revealed that the absence of TSP50 compromised the integrity of the intestinal mucosal barrier in murine subjects. This disruption facilitated direct contact between intestinal bacteria and the intestinal epithelium, thereby increasing susceptibility to colitis. Mechanistic analysis indicated that TSP50 deficiency in intestinal stem cells (ISCs) triggered aberrant activation of the TGF-ß signaling pathway and impeded the differentiation of goblet cells in mice, leading to impairment of mucosal permeability. By inhibiting the TGF-ß pathway, the functionality of the intestinal mucosal barrier is successfully restored and mitigated colitis in TSP50-deficient mice. In conclusion, TSP50 played a crucial role in maintaining the intestinal mucosal barrier function and exhibited the preventive effect against the development of colitis by regulating the TGF-ß signaling pathway.

Cancer/testis antigens: promising immunotherapy targets for digestive tract cancers.

Digestive tract cancers, including esophageal, gastric, and colorectal cancers, are the major cause of death among cancer patients worldwide due to the heterogeneity of cancer cells, which limits the effectiveness of traditional treatment methods. Immunotherapy represents a promising treatment strategy for improving the prognosis of patients with digestive tract cancers. However, the clinical application of this approach is limited by the absence of optimal targets. Cancer/testis antigens are characterized by low or absent expression in normal tissues, but high expression in tumor tissues, making them an attractive target for antitumor immunotherapy. Recent preclinical trials have shown promising results for cancer/testis antigen-targeted immunotherapy in digestive cancer. However, practical problems and difficulties in clinical application remain. This review presents a comprehensive analysis of cancer/testis antigens in digestive tract cancers, covering their expression, function, and potential as an immunotherapy target. Additionally, the current state of cancer/testis antigens in digestive tract cancer immunotherapy is discussed, and we predict that these antigens hold great promise as an avenue for breakthroughs in the treatment of digestive tract cancers.

UPS: Opportunities and challenges for gastric cancer treatment.

Gastric cancer remains the fourth most frequently diagnosed malignancy and the fifth leading cause of cancer-related mortality worldwide owning to the lack of efficient drugs and targets for therapy. Accumulating evidence indicates that UPS, which consists of E1, E2, and E3 enzymes and proteasome, plays an important role in the GC tumorigenesis. The imbalance of UPS impairs the protein homeostasis network during development of GC. Therefore, modulating these enzymes and proteasome may be a promising strategy for GC target therapy. Besides, PROTAC, a strategy using UPS to degrade the target protein, is an emerging tool for drug development. Thus far, more and more PROTAC drugs enter clinical trials for cancer therapy. Here, we will analyze the abnormal expression enzymes in UPS and summarize the E3 enzymes which can be developed in PROTAC so that it can contribute to the development of UPS modulator and PROTAC technology for GC therapy.

Colorectal cancer organoid models uncover oxaliplatin-resistant mechanisms at single cell resolution.

PURPOSE: Oxaliplatin-based chemotherapy is a standard treatment for advanced colorectal cancer (CRC) patients. However, chemoresistance-induced resistance is an essential cause for mortality. Therefore, it is necessary to study the mechanism of drug resistance in CRC. METHODS: Here, we established two strains of patient-derived organoids (PDOs) selected from oxaliplatin-resistant and treatment-naïve CRC patients. To dissect the drug-resistant mechanisms, these CRC-PDOs were subjected to single-cell RNA sequencing (scRNA-Seq). RESULTS: We found that the drug sensitivity test outcome from these organoids subjected to oxaliplatin and 5-FU exposure was consistent with the clinic readout. CRC-PDOs well recapitulated the morphology and histology of their parental biopsies based on HE and IHC staining of pathological biomarkers. The scRNA-Seq data filtered drug-resistant cell populations and related signaling pathways (e.g. oxidative phosphorylation and ATP metabolic process). The data also revealed several putative drug resistant-driven genes (STMN1, VEGFA and NDRG1) and transcription factors (E2F1, BRCA1, MYBL2, CDX2 and CDX1). CONCLUSION: We generated an oxaliplatin-resistant CRC organoid model that was employed to provide potential therapeutic targets for treating CRC patients exhibiting oxaliplatin-resistance.

Modaline sulfate promotes Oct4 expression and maintains self-renewal and pluripotency of stem cells through JAK/STAT3 and Wnt signaling pathways.

BACKGROUND: Stem cells have been extensively explored for a variety of regenerative medical applications and they play an important role in clinical treatment of many diseases. However, the limited amount of stem cells and their tendency to undergo spontaneous differentiation upon extended propagation in vitro restrict their practical application. Octamer-binding transcription factor-4 (Oct4), a transcription factor belongs to the POU transcription factor family Class V, is fundamental for maintaining self-renewal ability and pluripotency of stem cells. METHODS: In the present study, we used the previously constructed luciferase reporters driven by the promoter and 3'-UTR of Oct4 respectively to screen potential activators of Oct4. Colony formation assay, sphere-forming ability assay, alkaline phosphatase (AP) activity assay and teratoma-formation assay were used to assess the role of modaline sulfate (MDLS) in promoting self-renewal and reinforcing pluripotency of P19 cells. Immunofluorescence, RT-PCR, and western blotting were used to measure expression changes of stem-related genes and activation of related signaling pathways. RESULTS: We screened 480 commercially available small-molecule compounds and discovered that MDLS greatly promoted the expression of Oct4 at both mRNA and protein levels. Moreover, MDLS significantly promoted the self-renewal capacity of P19 cells. Also, we observed that the expression of pluripotency markers and alkaline phosphatase (AP) increased significantly in MDLS-treated colonies. Furthermore, MDLS could promote teratoma formation and enhanced differentiation potential of P19 cells in vivo. In addition, we found that in the presence of LIF, MDLS could replace feeder cells to maintain the undifferentiated state of OG2-mES cells (Oct4-GFP reporter gene mouse embryonic stem cell line), and the MDLS-expanded OG2-mES cells showed an elevated expression levels of pluripotency markers in vitro. Finally, we found that MDLS promoted Oct4 expression by activating JAK/STAT3 and classic Wnt signaling pathways, and these effects were reversed by treatment with inhibitors of corresponding signaling pathways. CONCLUSIONS: These findings demonstrated, for the first time, that MDLS could maintain self-renewal and pluripotency of stem cells.

TSP50 promotes hepatocyte proliferation and tumour formation by activating glucose-6-phosphate dehydrogenase (G6PD).

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a common malignant tumour with high morbidity and mortality. Metabolic regulation by oncogenes is necessary for tumour growth. Testes-specific protease 50 (TSP50) has been found to promote cell proliferation in multiple tumour types. However, the mechanism that TSP50 promotes HCC progression are not known. METHODS: Hepatocyte proliferation was analysed by MTT and BrdU incorporation after TSP50 transfection. Furthermore, LC-MS/MS, co-immunoprecipitation and GST pull-down assays were performed to analyse protein(s) binding to TSP50. Moreover, the site-specific mutation of G6PD was used to reveal the key site critical for G6PD acetylation mediated by TSP50. Finally, the role of G6PD K171 acetylation regulated by TSP50 in cell proliferation and tumour formation was investigated. RESULTS: Our data suggest that the overexpression of TSP50 accelerates hepatocyte proliferation. In addition, G6PD is an important protein that binds to TSP50 in the cytoplasm. TSP50 activates G6PD activity by inhibiting the acetylation of G6PD at the K171 site. In addition, TSP50 promotes the binding of G6PD to SIRT2. Furthermore, the K171ac of G6PD regulated by TSP50 is required for TSP50-induced cell proliferation in vitro and tumour formation in vivo. Additionally, according to The Cancer Genome Atlas (TCGA) programme, TSP50 and G6PD are negatively correlated with the survival of HCC patients. CONCLUSIONS: Collectively, our findings demonstrate that TSP50-induced cell proliferation and tumour formation are mediated by G6PD K171 acetylation.

Ethyl-p-methoxycinnamate enhances oct4 expression and reinforces pluripotency through the NF-κB signaling pathway.

Pluripotent stem cells are have therapeutic applications in regenerative medicine and drug discovery. However, the differentiation of stem cells in vitro hinders their large-scale production and clinical applications. The maintenance of cell pluripotency relies on a complex network of transcription factors; of these, octamer-binding transcription factor-4 (Oct4) plays a key role. This study aimed to construct an Oct4 gene promoter-driven firefly luciferase reporter and screen small-molecule compounds could maintain cell self-renewal and pluripotency. The results showed that ethyl-p-methoxycinnamate (EPMC) enhance the promoter activity of the Oct4 gene, increased the expression of Oct4 at both mRNA and protein levels, and significantly promoted the colony formation of P19 cells. These findings suggesting that EPMC could reinforce the self-renewal capacity of P19 cells. The pluripotency markers Oct4, SRY-related high-mobility-group-box protein-2, and Nanog were expressed at higher levels in EPMC-induced colonies. EPMC could promote teratoma formation and differentiation potential of P19 cells in vivo. It also enhanced self-renewal and pluripotency of human umbilical cord mesenchymal stem cells and mouse embryonic stem cells. Moreover, it significantly activated the nuclear factor kappa B (NF-κB) signaling pathway via the myeloid differentiation factor 88-dependent pathway. The expression level of Oct4 decreased after blocking the NF-κB signaling pathway, suggesting that EPMC promoted the expression of Oct4 partially through the NF-κB signaling pathway. This study indicated that EPMC could maintain self-renewal and pluripotency of stem cells.

Alantolactone suppresses APOC3 expression and alters lipid homeostasis in L02 liver cells.

A high level of APOC3 expression is an independent risk factor for some lipid metabolism-related diseases, such as cardiovascular disease (CVD), nonalcoholic fatty liver disease (NAFLD) and atherosclerosis (AS). This suggests that down-regulating APOC3 expression is a potential way of regulating lipid levels. In this study, we used luciferase reporter screening to identify a natural compound, alantolactone (ALA), that can inhibit the promoter activity of APOC3. ALA decreased APOC3 expression at both mRNA and protein levels. Then we pretreated L02 liver cells with oxLDL to investigate the function of ALA in lipid homeostasis. Intriguingly, ALA attenuated oxLDL-induced foam cell formation by reducing total cholesterol (TC) and triglyceride (TG) contents. Furthermore, these results could be reversed by overexpressing APOC3 protein. ALA inhibited tyrosine phosphorylation (Tyr705pho) of STAT3 to down-regulate APOC3 expression. Intriguingly, overexpression of a wild-type STAT3 or a constitutively active form of STAT3 (STAT3-C) up-regulated APOC3 expression and partly reversed the effect of ALA in oxLDL-induced L02 cells. Overexpression of wild-type STAT3 also increased TC but not TG contents in L02 cells. However, overexpression of STAT3-C significantly increased TC and TG contents, and the effect of ALA was partly attenuated by STAT3-C, although this was not statistically significant. These results suggest that ALA attenuates lipid accumulation through down-regulation of APOC3 expression, at least in part by inhibiting STAT3 signaling.

Prohibitin 2 localizes in nucleolus to regulate ribosomal RNA transcription and facilitate cell proliferation in RD cells.

Prohibitin 2 (PHB2), as a conserved multifunctional protein, is traditionally localized in the mitochondrial inner membrane and essential for maintenance of mitochondrial function. Here, we investigated the role of PHB2 in human rhabdomyosarcoma (RMS) RD cells and found substantial localization of PHB2 in the nucleolus. We demonstrated that PHB2 knockdown inhibited RD cell proliferation through inducing cell cycle arrest and suppressing DNA synthesis. Meanwhile, down-regulation of PHB2 also induced apoptosis and promoted differentiation in fractions of RD cells. In addition, PHB2 silencing led to altered nucleolar morphology, as observed by transmission electron microscopy, and impaired nucleolar function, as evidenced by down-regulation of 45S and 18S ribosomal RNA synthesis. Consistently, upon PHB2 knockdown, occupancy of c-Myc at the ribosomal DNA (rDNA) promoter was attenuated, while more myoblast determination protein 1 (MyoD) molecules bound to the rDNA promoter. In conclusion, our findings suggest that nucleolar PHB2 is involved in maintaining nucleolar morphology and function in RD cells by regulating a variety of transcription factors, which is likely to be one of the underlying mechanisms by which PHB2 promotes tumor proliferation and represses differentiation. Our study provides new insight into the pathogenesis of RMS and novel characterizations of the highly conserved PHB2 protein.