miR-365 regulates liver cancer stem cells via RAC1 pathway.

Liver cancer stem cells (CSCs) were involved in tumorigenesis, progression, recurrence, and drug resistance of hepatocellular carcinoma (HCC). miR-365 was downregulated in hepatocellular carcinoma and inhibited HCC cell proliferation and invasion. However, the role of miR-365 in liver cancer stem cells was unknown. Herein, we observed a remarkable decrease of miR-365 expression in CD133 or EpCAM-positive liver CSCs as well as in CSC-enriched hepatoma spheres. Up-regulated miR-365 suppressed liver CSC expansion by inhibiting the dedifferentiation of hepatoma cells and decreasing the self-renewal ability of liver CSCs. Mechanistically, bioinformatic and luciferase reporter analysis identified Ras-related C3 botulinum toxin substrate 1 (RAC1) as a direct target of miR-365. Overexpression of miR-365 in hepatoma cells downregulated the RAC1 mRNA and protein expression. RAC1 also could promote the expansion of liver CSCs. The special RAC1 inhibitor EHop-106 or RAC1 overexpression abolished the discrepancy in liver CSC proportion and the self-renewal capacity between miR-365 overexpression hepatoma cells and control cells, which further confirmed that RAC1 was required in miR-365-suppressed liver CSCs expansion. miR-365 was downregulated in liver CSCs and could inhibit HCC cells dedifferentiation and liver CSCs expansion by targeting RAC1 signaling.

The influence of low-dose s-ketamine on postoperative delirium and cognitive function in older adults undergoing thoracic surgery.

BACKGROUND: Postoperative delirium (POD) and cognitive dysfunction (POCD) are common complications following thoracic surgery, particularly in patients aged 65 years and above. These complications can significantly affect recovery and increase healthcare costs. This study investigates the effects of low-dose S-ketamine on reducing POD and POCD in this patient demographic. METHODS: In this retrospective cohort study, medical records of patients aged ≥ 65 years who underwent elective thoracic surgery from January 2019 to August 2023 were reviewed. Patients were categorized into S-ketamine and Control groups based on intraoperative S-ketamine exposure. POD was assessed using the Confusion Assessment Method (CAM), while cognitive function was evaluated using the Montreal Cognitive Assessment (MoCA) at baseline, 1 week, 1 month, and 6 months post-surgery. Intraoperative and postoperative parameters, including hemodynamic stability, blood loss, pain scores, and ICU stay length, were also recorded. RESULTS: The study comprised 140 participants, with 70 in each group. The S-ketamine group demonstrated a significantly lower incidence of POD at 7 days post-surgery (12.0% vs. 26.7%, P < 0.001), and reduced POCD at 1 month (18.7% vs. 36.0%, P < 0.05) and 6 months (10.7% vs. 21.3%, P < 0.05). The Ketamine group had a significantly higher median MoCA score compared to the Control group both at 1 month (P = 0.021) and 6 months (P = 0.007). Adverse events, such as infection, bleeding, and respiratory failure, showed no significant differences between the groups, suggesting a safe profile for S-ketamine. CONCLUSION: Administering low-dose S-ketamine during thoracic surgery in patients aged 65 years and above significantly reduces the incidence of POD and POCD, highlighting its neuroprotective potential. These findings advocate for the inclusion of S-ketamine in anesthetic protocols to improve postoperative outcomes and reduce healthcare costs in this patient population.

miR-365 inhibits the progression of gallbladder carcinoma and predicts the prognosis of Gallbladder carcinoma patients.

Gallbladder carcinoma (GBC) is one of the most common fatal biliary tract tumors in the world. Its 3-year survival rate is 30% and the recurrence rate remains very high. miR-365 was downregulated in numerous tumors and worked as tumor suppressor gene. However, the role of miR-365 in GBC was unclear. In this study, our results found that the expression of miR-365 in GBC tissues was reduced rather than that in non-cancerous tissues. miR-365 overexpression inhibited the proliferation, metastasis and expansion of GBC CSCs. Mechanically, bioinformatic and luciferase reporter analysis identified Ras-related C3 botulinum toxin substrate 1 (RAC1) as a direct target of miR-365. Overexpression of miR-365 in GBC cells reduced the RAC1 mRNA and protein expression. The special RAC1 inhibitor EHop-106 abolished the discrepancy of growth, metastasis and self-renewal ability between miR-365-overexpression GBC cells and their control cells, which further demonstrated that RAC1 was involved in miR-365-disrupted GBC cells growth, metastasis and self-renewal. More importantly, reduced expression of miR-365 was a predictor of poor prognosis of GBC patients. In conclusion, miR-365 inhibited GBC cell growth, metastasis and self-renewal capacity by directly targeting RAC1, and may therefore prove to be a novel prognosis biomarker for GBC patients.

Effects of miR-330-3p on Invasion, Migration and EMT of Gastric Cancer Cells by Targeting PRRX1-Mediated Wnt/ß-Catenin Signaling Pathway.

BACKGROUND: miRNA, as a biological marker, had more and more attention in recent years due to the important role it plays in cancer. Currently, there are extensive studies on miRNAs, among which miR-330-3p is reported to be implicated in the pathophysiological processes of various cancers. However, little progress has been made in the mechanism of miR-330-3p in gastric cancer. OBJECTIVE: To explore the expression and relevant mechanism of miR-330-3p and PRRX1 in gastric cancer (GC). METHODS: Forty-five GC patients (study group), from whom paired GC and paracancerous tissues were collected, and another 45 healthy subjects (control group) who underwent physical examination during the same period were enrolled. In addition, GC cells and human gastric mucosa cells were purchased, and miR-330-3p-mimics, miR-330-3p-inhibitor, miR-NC, si-PRRX1, and sh-PRRX1 were transfected into MKN45, SGC7901 cell. QRT-PCR was employed to assess the miR-330-3p and PRRX1 expressions in the samples, and the cell expressions of PRRX1, GSK-3ß, p-GSK-3ß, ß-catenin, p-ß-catenin, cyclin D1, N-cadherin, E-cadherin and vimentin were evaluated by Western blot (WB). MTT, Transwell and wound-healing experiments were adopted to detect cell proliferation, invasion and migration. RESULTS: MiR-330-3p was under-expressed, while PRRX1 was highly expressed in the serum of patients, both of which had an area under the curve (AUC) of more than 0.9. MiR-330-3p and PRRX1 were associated with tumor diameter, TNM staging, lymph node metastasis and differentiation of GC patients. Overexpression of miR-330-3p and inhibition of PRRX1 expression could suppress epithelial-mesenchymal transition (EMT), proliferation, invasion and apoptosis of cells. What is more, WB assay showed that overexpressed miR-330-3p and inhibited PRRX1 could inhibit the expression levels of p-GSK-3ß, ß-catenin, cyclin D1, N-cadherin and vimentin proteins, while elevating GSK-3ß, p-ß-catenin and E-cadherin protein expressions. Dual-luciferase reporter assay confirmed that there was a targeting relation between miR-330-3p and PRRX1. Furthermore, rescue experiments revealed that the cell proliferation, invasion, migration did not differ significantly between co-transfected miR-330-3p-mimics+sh-PRRX1, miR-330-3p-inhibitor+si-PRRX1 groups of MKN45 and SGC7901 and the miR-NC group (without transfected sequences). CONCLUSION: Overexpressed miR-330-3p can promote cell EMT, proliferation, invasion and apoptosis through inhibiting PRRX1-mediated Wnt/ß-catenin signaling pathway, which is expected to be a potential therapeutic target for GC.

Application of multiple Roux-en-Y hepaticojejunostomy reconstruction by formation of bile hilar duct lake in the operation of hilar cholangiocarcinoma.

BACKGROUND: Hilar cholangiocarcinoma is the most common malignant tumor of the extrahepatic bile duct. Until now, radical resection has been the most effective method for the long-term survival of patients with the disease. However, many problems have emerged in the field of hepatobiliary surgery for a long time, including complex surgical procedures, low resection rate, and postoperative complications. We have adopted the "multiple Roux-en-Y hepaticojejunostomy reconstruction by formation of a bile duct lake" technique in the treatment of hilar cholangiocarcinoma since 2008, and obtained satisfactory short- and long-term results. AIM: To examine the feasibility of the application of multiple Roux-en-Y hepaticojejunostomy reconstruction by formation of a bile duct lake in the operation of hilar cholangiocarcinoma. METHODS: A retrospective analysis was performed for the clinical data, surgical methods, and results of 76 patients with hilar cholangiocarcinoma who were treated with hilar bile duct lake-forming multiple Roux-en-Y hepaticojejunostomy reconstruction at Gansu Provincial Hospital. RESULTS: In all 76 cases, the operation was successful and no operative death occurred. The mean (range) operation time was 215.4 ± 53.5 min (124-678 min), and the amount of bleeding during the operation was 428.2 ± 63.8 mL (240-2200 mL). The overall 1-year survival rate was 78.9%, and the 3-year survival rate was 32.8%. CONCLUSION: The multiple Roux-en-Y hepaticojejunostomy reconstruction technique with formation of a bile duct lake is safe and effective for the surgical treatment of hilar cholangiocarcinoma.

Effect of postoperative early enteral nutrition on clinical outcomes and immune function of cholangiocarcinoma patients with malignant obstructive jaundice.

BACKGROUND: Most cholangiocarcinoma patients with malignant obstructive jaundice (MOJ) have varying degrees of malnutrition and immunodeficiency preoperatively. Therefore, perioperative nutritional support has important clinical significance in the treatment of cholangiocarcinoma. AIM: To investigate the effects of postoperative early enteral nutrition (EEN) on immunity function and clinical outcomes of cholangiocarcinoma patients with MOJ. METHODS: This prospective clinical study included 60 cholangiocarcinoma patients with MOJ who underwent surgery. The patients were randomly divided into an experimental group and a control group according to the nutrition support modes. The control group received postoperative total parenteral nutrition (TPN), whereas the experimental group received postoperative EEN and parenteral nutrition (PN; EEN + PN). The clinical outcomes, postoperative immune function, incidences of surgical site infection and bile leakage, intestinal function recovery time, average hospitalization days, and hospitalization expenses of the two groups were assessed on postoperative days (PODs) 1, 3, and 7. RESULTS: The CD3+T, CD4+T, CD8+T, and CD4+T/CD8+T cell count and the immunoglobulin (Ig) G, IgM, and IgA levels in the EEN + PN group were significantly higher than those in the TPN group on PODs 3 and 7 (P < 0.05), whereas no significant differences in the CD3+T, CD4+T, CD8+T, and CD4+T/CD8+T cell counts and IgG, IgM, and IgA levels before operation and on POD 1 were found between the two groups (P > 0.05). The intestinal function recovery time and postoperative hospital stay were shorter (P < 0.001 for both) in the EEN + PN group than in the TPN group. The hospitalization expenses of the EEN + PN group were lower than those of the TPN group (P < 0.001). However, the incidence of abdominal distension was higher than in the EEN + PN group than in the TPN group (P < 0.05). The incidence rates of biliary leakage and surgical site infection were not significantly different between the two groups (P > 0.05). CONCLUSION: A postoperative EEN program could reduce the incidence of postoperative complications and improve the clinical outcomes and immune functions of cholangiocarcinoma patients with MOJ and is thus beneficial to patient recovery.

Knockdown of alpha-fetoprotein expression inhibits HepG2 cell growth and induces apoptosis.

AIMS: To explore the biological roles of alpha-fetoprotein (AFP), a tumor-associated antigen in human hepatocellular carcinoma (HCC). MATERIALS AND METHODS: After knockdown of AFP in HepG2 cells by transfection of specific Stealth™ RNAi, the expression of AFP were detected by reverse transcription polymerase chain reaction at mRNA level and by enzyme-linked immunosorbent assay at the protein level. Then, the effect of silenced AFP on cell proliferation was assessed by dimethylthiazolyl-2,5-diphenyl-tetrazolium bromide assay, and apoptosis assessment with Hoechst33258 and flow cytometry (double stain with fluorescein isothiocyanate/propidium iodide), the roles of AFP in the cell cycle regulation were assessed by flow cytometry. We also detected the expression of some key proteins related to apoptosis pathway by Western immunoblot analysis. RESULTS: After the transfection for 48 h, the expression of AFP gene was almost abolished, the cell proliferation was inhibited by 47.61%, the number of cells undergoing early apoptosis was significantly increased to 59.47%; cell cycle was arrested with the increase of G0/G1 phase cells from 45.3% to 58.4%. Inhibition of AFP expression also results in decreasing of transforming growth factor-ß (TGF-ß), mutant P53 expression, and increasing of Bax/Bcl-2 ratio, activation of caspase-3. CONCLUSIONS: The results suggest that AFP may positively regulate cell proliferation by enhancing the apoptosis resistance via effect on TGF-ß and p53/Bax/caspase-3 signaling pathway in HepG2 cells. As such, the knockdown of AFP gene should be further investigated in vivo as a novel approach to HCC treatment.

Mir-30d increases intracellular survival of Helicobacter pylori through inhibition of autophagy pathway.

AIM: To determine if mir-30d inhibits the autophagy response to Helicobacter pylori (H. pylori) invasion and increases H. pylori intracellular survival. METHODS: The expression of mir-30d was detected by quantitative polymerase chain reaction (PCR), and autophagy level was examined by transmission electron microscopy, western blot, and GFP-LC3 puncta assay in human AGS cells and GES-1 cells. Luciferase reporter assay was applied to confirm the specificity of mir-30d regulation on the expression of several core molecules involved in autophagy pathway. The expression of multiple core proteins were analyzed at both the mRNA and protein level, and the intracellular survival of H. pylori after different treatments was detected by gentamicin protection assay. RESULTS: Autophagy level was increased in AGS and GES-1 cells in response to H. pylori infection, which was accompanied by upregulation of mir-30d expression (P < 0.05, vs no H. pylori infection). In the two gastric epithelial cell lines, mimic mir-30d was found to repress the autophagy process, whereas mir-30d inhibitor increased autophagy response to H. pylori invasion. mir-30d mimic decreased the luciferase activity of wild type reporter plasmids carrying the 3' untranslated region (UTR) of all five tested genes (ATG2B, ATG5, ATG12, BECN1, and BNIP3L), whereas it had no effect on the mutant reporter plasmids. These five genes are core genes of autophagy pathway, and their expression was reduced significantly after mir-30d mimic transfection (P < 0.05, vs control cells without mir-30d mimic treatment). Mir-30d mimic transfection and direct inhibition of autophagy increased the intracellular survival of H. pylori in AGS cells. CONCLUSION: Mir-30d increases intracellular survival of H. pylori in gastric epithelial cells through inhibition of multiple core proteins in the autophagy pathway.

Inhibition of c-Myc by let-7b mimic reverses mutidrug resistance in gastric cancer cells.

Chemotherapy is one of the few effective choices for patients with advanced or recurrent gastric cancer (GC). However, the development of mutidrug resistance (MDR) to cancer chemotherapy is a major obstacle to the effective treatment of advanced GC. Additionally, the mechanism of MDR remains to be determined. In the present study, we tested IC50 of cisplatin (DDP), vincristine (VCR) and 5-fluorouracil (5-FU) in SGC7901, SGC7901/DDP and SGC7901/VCR gastric cancer cells using an MTT assay. The expression of let-7b and c-Myc in these cells was detected by qPCR and western blot analysis. The relationship between let-7b and c-Myc was explored using a luciferase reporter assay. Transfection of let-7b mimic or inhibitor was used to confirm the effect of let-7b on drug sensitivity in chemotherapy via the regulation of c-Myc expression. We found that the expression of let-7b was lower in chemotherapy-resistant SGC7901/DDP and SGC7901/VCR gastric cancer cells than that in chemotherapy-sensitive SGC7901 cells. By contrast, the expression of c-Myc was higher in SGC7901/DDP and SGC7901/VCR cells than that in SGC7901 cells. Furthermore, we confirmed that let-7b suppresses c-Myc gene expression at the mRNA and protein levels in a sequence-specific manner, while transfection of let-7b mimic increases drug sensitivity in chemotherapy-resistant SGC7901/DDP and SGC7901/VCR cells by targeting downregulation of c-Myc. In SGC7901 drug-sensitive cells, however, the sensitivity of chemotherapy was significantly decreased following let-7b inhibitor transfection. The present study results demonstrated that let-7b increases drug sensitivity in chemotherapy­resistant SGC7901/DDP and SGC7901/VCR gastric cancer cells by targeting the downregulation of c-Myc and that, let-7b mimic reverses MDR by promoting cancer stem cell differentiation controlled by double-negative autoregulatory loops (Lin28/let-7 and Myc/let-7) and a double-positive autoregulatory loop (Lin28/Lin28B/Myc) existing in GC cells, which remains to be confirmed.

Lipopolysaccharide-induced bacterial translocation is intestine site-specific and associates with intestinal mucosal inflammation.

The present study aimed to determine whether any specific intestinal site or intestinal mucosal inflammation is highly correlated with bacterial translocation (BT). Enterostomy tubes were surgically placed in adult male Sprague-Dawley rats 5 days before induction of experimental model. After surgery, sterile water containing kanamycin (25 mg/L) was injected into each intestinal segment through the tubes for 3 days. Green fluorescent protein (GFP)-transfected Escherichia coli (n = 30 for lipopolysaccharide (LPS) group, and n = 30 for control group) or 0.9 % saline (n = 30 for blank group) were injected into each intestinal segment through the tubes for two consecutive days. Rats were then subjected to LPS-induced endotoxemia; lactulose and mannitol were injected into each intestinal segment through the tubes simultaneously. At 6 h after LPS injection, BT to distant organs and integrity of tight junctions (TJ) were examined by fluorescence and electron microscopy, respectively. The urinary excretion ratio of lactulose/mannitol (L/M) and intestinal mucosal cytokine levels were assessed. We found that the intestinal permeability, reflected by translocation rates of GFP-labeled E. coli, the levels of open TJ, the excretion ratio of L/M, and the inflammatory cytokine levels were higher in the LPS group than in the control and blank groups. The endotoxemia ileum showed the highest levels of both intestinal permeability and inflammatory cytokine, while the colon showed the lowest. The present study of endotoxemia rats suggests that LPS increases gut paracellular permeability and induces BT. The ileum is the site of greatest BT risk, while the colon is the lowest, and the difference in risk between these sites is correlated with intestinal mucosal inflammation.