ALKBH5 promotes hepatocellular carcinoma cell proliferation, migration and invasion by regulating TTI1 expression.

The objective of this research was to investigate the potential mechanisms of AlkB homolog 5, RNA demethylase (ALKBH5) in hepatocellular carcinoma (HCC). We used The Cancer Genome Atlas (TCGA), Kruskal-Wallis method and Kaplan-Meier (KM) survival analysis to study the expression of ALKBH5 and its correlation with clinical factors in HCC. In vitro experiments verified the expression of ALKBH5 and its effect on HCC cell phenotype. We screened differentially expressed genes (DEGs) from HCC patients associated with ALKBH5. Through this screening we identified the downstream gene TTI1 which is associated with ALKBH5 and investigated its function using Gene Expression Profiling Interaction Analysis (GEPIA) along with univariate Cox proportional hazards regression analysis. Finally, we analyzed the functions of ALKBH5 and TTI1 in HCC cells. Across numerous pan-cancer types, we observed significant overexpression of ALKBH5. In vitro experiments confirmed ALKBH5 as an oncogene in HCC, with its knockdown leading to suppressed cell proliferation, migration, and invasion. Bioinformatics analyses also demonstrated a significant positive correlation between ALKBH5 and TTI1. TTI1, highly expressed in cells, showed promising prognostic ability for patients. Further experiments confirmed that suppressing TTI1 impeded cell growth and movement, with this effect partially offset by increased ALKBH5 expression. Conversely, promoting these cellular processes was observed with TTI1 overexpression, but was dampened by decreased ALKBH5 expression. In conclusion, our findings suggest that ALKBH5 may influence proliferation, migration and invasion of HCC by modulating TTI1 expression, providing a new direction for treating HCC.

Gut microbiota alteration after cholecystectomy contributes to post-cholecystectomy diarrhea via bile acids stimulating colonic serotonin.

Post-cholecystectomy diarrhea (PCD) is highly prevalent among outpatients with cholecystectomy, and gut microbiota alteration is correlated with it. However, how and to what extent changed fecal bacteria contributes to diarrhea are still unrevealed. Humanized gut microbiome mice model by fecal microbiota transplantation was established to explore the diarrhea-inducible effects of gut microbiota. The role of microbial bile acids (BAs) metabolites was identified by UPLC/MS and the underlying mechanisms were investigated with selective inhibitors and antagonists as probes. These mice transplanted with fecal microbiome of PCD patients (PCD mice) exhibited significantly enhanced gastrointestinal motility and elevated fecal water content, compared with these mice with fecal microbiome of NonPCD patients and HC. In analyzing gut microbiota, tryptophan metabolism was enriched in PCD microbiome. In addition, overabundant serotonin in serum and colon, along with elevated biosynthesis gene and reduced reuptake gene, and highly expressed 5-HT receptors (5-HTRs) in colon of PCD mice were found, but not in small intestine. Notably, diarrheal phenotypes in PCD mice were depleted by tryptophan hydroxylase 1 inhibitor (LX1606) and 5-HTRs selective antagonists (alosetron and GR113808). Furthermore, increased microbial secondary BAs metabolites of DCA, HDCA and LCA were revealed in feces of PCD mice and they were found responsible for stimulating 5-HT level in vitro and in vivo. Intriguingly, blocking BAs-conjugated TGR5/TRPA1 signaling pathway could significantly alleviate PCD. In conclusion, altered gut microbiota after cholecystectomy contributes to PCD by promoting secondary BAs in colon, which stimulates colonic 5-HT and increases colon motility.

Effect of aging on acute pancreatitis through gut microbiota.

Background: Compared to younger people, older people have a higher risk and poorer prognosis of acute pancreatitis, but the effect of gut microbiota on acute pancreatitis is still unknown. We aim to investigate the effect of aging gut microbiota on acute pancreatitis and explore the potential mechanism of this phenomenon. Methods: Eighteen fecal samples from healthy adult participants, including nine older and nine younger adults were collected. C57BL/6 mice were treated with antibiotics for fecal microbiota transplantation from older and younger participants. Acute pancreatitis was induced by cerulein and lipopolysaccharide in these mice. The effect of the aged gut microbiota was further tested via antibiotic treatment before or after acute pancreatitis induction. Results: The gut microbiota of older and younger adults differed greatly. Aged gut microbiota exacerbated acute pancreatitis during both the early and recovery stages. At the same time, the mRNA expression of multiple antimicrobial peptides in the pancreas and ileum declined in the older group. Antibiotic treatment before acute pancreatitis could remove the effect of aging gut microbiota, but antibiotic treatment after acute pancreatitis could not. Conclusion: Aging can affect acute pancreatitis through gut microbiota which characterizes the deletion of multiple types of non-dominant species. This change in gut microbiota may potentially regulate antimicrobial peptides in the early and recovery stages. The level of antimicrobial peptides has negative correlations with a more severe phenotype.

Disordered Gut Microbiota Correlates With Altered Fecal Bile Acid Metabolism and Post-cholecystectomy Diarrhea.

Post-cholecystectomy diarrhea (PCD) is a common complication of gallbladder removal, and gut microbiota changes have been determined in PCD patients. Bile acid diarrhea (BAD) is supposed to be the main pathogenic factor for PCD due to the disrupted fecal bile acid metabolism in diarrheal patients. However, the profiling of bile acid metabolite alteration in PCD is unclear and whether changed gut microbiota and fecal bile acid metabolism are correlated is also underdetermined. The fecal bile acid metabolites from fecal samples were profiled by targeted UPLC/MS (ultra-high-performance liquid chromatography coupled with a triple-quadrupole mass spectrometer) and the composition of fecal bile acid metabolites in PCD patients was demonstrated to be distinct from those in Non-PCD and HC groups. In addition, the quantification of bile acid excretion in feces of diarrheal patients was significantly elevated. Furthermore, 16S rRNA sequencing results revealed that PCD patients had the lowest operational taxonomic units (OTU) and significant reduction in microbial richness and evenness. Bacterial composition was remarkably shifted in PCD patients, which mainly lay in dominated phyla Firmicutes and Bacteroidota. Besides, the co-abundance network among genus bacteria declined in PCD. Among the genera, Prevotella, Enterococcus, and Erysipelotrichaceae_UCG-003 were enriched, but Alistipes, Bacteroides, Ruminococcus, and Phascolarctobacterium were reduced. Moreover, these disease-linked genera were closely associated with several diarrheal phenotypes. Notably, changed bile acid metabolites exhibited strong correlations with gut microbiota as well. Conclusively, this study reveals associations between PCD-linked microbes and bile acid metabolites, which may synergistically correlate to postoperative diarrhea.

SLC41A3 Exhibits as a Carcinoma Biomarker and Promoter in Liver Hepatocellular Carcinoma.

Liver Hepatocellular Carcinoma (LIHC) is the fifth widely occurred carcinoma, which is thought to be the second primary contributor of carcinoma-associated death. There are almost 788,000 death tolls worldwide. Solute carrier family 41 member 3 (SLC41A3) is a member of solute carrier family 41, and it is the key point of numerous researches. Our research attempted to explore the links between SLC41A3 and LIHC through public databases. Higher expression of SLC41A3 displayed an intimate association with higher pathological stages and poorer prognosis. GO and KEGG analysis revealed the possible regulatory pathways of SLC41A3. Additionally, we carried out cell functional experiments to determine the expression of SLC41A3 in the cell lines of LIHC, as well as the effects of its silence on cell proliferation, migration, and invasion. Our data showed that SLC41A3 was greatly increased in the cell lines of LIHC. Moreover, silencing SLC41A3 impeded LIHC cell proliferation, migration, and invasion in vitro. Collectively, our study demonstrated that highly expressed SLC41A3 was a probable indication of LIHC occurrence, and SLC41A3 could be regarded as a prospective target in the treatment of LIHC.

RORγt inhibitor SR1001 alleviates acute pancreatitis by suppressing pancreatic IL-17-producing Th17 and γδ-T cells in mice with ceruletide-induced pancreatitis.

The management of acute pancreatitis (AP) remains a challenge to clinicians worldwide for limited effective interventions. Retinoid orphan receptor gamma t (RORγt) is a therapeutic target for several diseases; however, it is unclear whether inhibiting RORγt can ameliorate AP. The relative expression of RORγt, IL-17 and IL-23 in the peripheral blood mononuclear cells of AP patients was measured by RT-PCR. An AP mouse model was induced by ceruletide, and SR1001 was injected before ceruletide administration. RORγt+ cells, T helper 17 cells (Th17), regulatory T cells (Tregs) and γδ T cells were assessed in the pancreas and spleen by flow cytometry. Higher RORγt expression in patients indicated the potential role of RORγt in AP progression. Analyses of the IL-17/IL-23 axis confirmed its role. SR1001 significantly alleviated AP histologically in the mouse model. Serum levels of amylase, IL-6, TNFalpha, IL-17 and IL-23 decreased upon SR1001 treatment. SR1001 selectively decreased the number of RORγt+, Th17, Tregs and γδ T cells in the pancreas but not the spleen. Collectively, these results showed that SR1001 exerted therapeutic effects on AP by suppressing IL-17-secreting Th17 and γδ T cells in the pancreas. Thus, SR1001 may be a promising drug for the treatment of AP in the clinic.

Role of Asxl2 in non­alcoholic steatohepatitis­related hepatocellular carcinoma developed from diabetes.

The present study investigated the mechanism(s) of non­alcoholic steatohepatitis­related hepatocellular carcinoma (NASH­HCC) developed from diabetes. Streptozotocin and a high­fat diet (STZ­HFD) were used to induce NASH­HCC in ApoE­/­ mice. Mouse liver functions were evaluated by H&E staining, liver/body weight and serum biochemical analysis. The expression levels of inflammation­associated factors were determined by RT­qPCR. Gene expression profiles related to molecular functions and pathways of NASH­HCC were examined by principal component analysis, heatmap, gene ontology and KEGG pathway enrichment analysis. Differentially expressed genes (DEGs) in tumor tissues were confirmed by RT­qPCR. The expression of Asxl2 in human NASH­HCC, other HCC tissues and HCC cells was measured by western blot (WB analysis) and RT­qPCR. For SNU­182 cells transfected with siAsxl2 or Hep3B cells with Asxl2 overexpression, cell proliferation, cell cycle, migration and invasion were respectively determined by CCK­8 assays, flow cytometry, wounding healing and Transwell assays. The expression levels of cell metastasis­ and cycle­related proteins were determined by WB analysis and RT­qPCR. NASH­HCC model mice exhibited tumor protrusion with severe steatosis. The blood glucose concentration, serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and low­density lipoprotein (LDL), total bile acid (TBA) and the levels of interleukin (IL)­6, tumor necrosis factor (TNF)­α, glypican 3 (GPC3) and transforming growth factor (TGF)­ß were all increased in NASH­HCC model mice. DEGs were mainly related to chromosome organization, the cell cycle and the mitogen­activated kinase (MAPK) pathway. Asxl2 was significantly downregulated in HCC tissues and cells, and this regulated cell growth, migration and invasion. The gene expression pattern, related molecular functions and signaling pathways of NASH­HCC differed from those of normal liver tissues. Additionally, the downregulation of Asxl2 may play a potential role in development of NASH­HCC in patients with diabetes.

LncRNA MYLK-AS1 facilitates tumor progression and angiogenesis by targeting miR-424-5p/E2F7 axis and activating VEGFR-2 signaling pathway in hepatocellular carcinoma.

BACKGROUND: Long non-coding RNAs (lncRNAs) are crucial in the invasion, angiogenesis, progression, and metastasis of hepatocellular carcinoma (HCC). The lncRNA MYLK-AS1 promotes the growth and invasion of HCC through the EGFR/HER2-ERK1/2 signaling pathway. However, the clinical significance of MYLK-AS1 in HCC still needs to be further determined. METHODS: Bioinformatic analysis was performed to determine the potential relationship among MYLK-AS1, miRNAs and mRNAs. A total of 156 samples of normal liver and paired HCC tissues from HCC patients were used to evaluate MYLK-AS1 expression by qRT-PCR. Human HCC cell lines were used to evaluate the colony formation, cell proliferation, migration, invasion, cell cycle and apoptosis after transfection of lentiviral short-hairpin RNAs (shRNAs) targeting MYLK-AS1 or MYLK-AS1 vectors. The competitive endogenous RNA (ceRNA) mechanism was clarified using fluorescence in situ hybridization (FISH), Western blotting, qPCR, RNA binding protein immunoprecipitation (RIP), and dual luciferase reporter analysis. RESULTS: MYLK-AS1 up-regulation was detected in the HCC tumor tissues and cell lines associated with the enhancement of the angiogenesis and tumor progression. The down-regulation of MYLK-AS1 reversed the effects on angiogenesis, proliferation, invasion and metastasis in the HCC cells and in vivo. MYLK-AS1 acted as ceRNA, capable of regulating the angiogenesis in HCC, while the microRNA miR-424-5p was the direct target of MYLK-AS1. Promoting the angiogenesis and the tumor proliferation, the complex MYLK-AS1/miR-424-5p activated the VEGFR-2 signaling through E2F7, whereas the specific targeting of E2F transcription factor 7 (E2F7) by miR-424-5p, was indicated by the mechanism studies. CONCLUSIONS: MYLK-AS1 and E2F7 are closely related to some malignant clinicopathological features and prognosis of HCC, thus the MYLK-AS1/ miR-424-5p/E2F7 signaling pathway might represent a promising treatment strategy to combat HCC.