CLIC1 Promotes the Progression of Gastric Cancer by Regulating the MAPK/AKT Pathways.

BACKGROUND/AIMS: Chloride intracellular channel 1 (CLIC1), which is a member of the chloride channel protein family, is associated with various human tumors. Recent studies have shown that CLIC1 is involved in the occurrence and development of gastric cancer (GC). However, the exact mechanism remains unclear in GC. METHODS: Effects of CLIC1 on the progression of GC in vivo and in vitro and the potential underlying mechanisms have been investigated by analysing 54 patients with GC, as well as human gastric cell lines SGC-7901 and MGC-803, utilizing proteomics, RT-PCR, Western blotting, flow cytometry, Cell invasion and migration assays and xenograft tumor models. RESULTS: Our study shows that CLIC1 knockdown by targeted-siRNA markedly inhibits GC cell invasion and migration and induces apoptosis in vitro. In total, 54 differentially expressed proteins were identified in GC cells SGC-7901 after CLIC1 silencing by isobaric tags for relative isotope labeled and absolute quantitation (iTRAQ) technology, including integrin α1 (ITGα1) and ITGα3. The expression levels of ITGα3, ITGαv, ITGß1 and Bcl-2 mRNA and protein were decreased significantly in GC cells after CLIC1 knockdown; ITGα1 and Fas were upregulated, but the level of survivin was not significantly different. GC growth and metabolism were decreased in vivo after CLIC1 silencing, but apoptosis was markedly increased. Further study showed that the expression levels of ITGα3, ITGαv and ITGß1, as well as AKT-phosphorylation, ERK-phosphorylation and p38-phosphorylation, were reduced in vivo after CLIC1 knockdown, while ITGα1 was upregulated. CONCLUSIONS: We speculate that CLIC1 may play an important role in the progression of GC, and its mechanism may be related to the regulation of integrin family proteins, which leads to the sequential regulation of the PI3K/AKT, MAPK/ERK and MAPK/p38 pathways.

Identification of CERS5 as a molecular biomarker in pan-cancer through multiple omics integrative analysis.

Cancer is a devastating disease that presents a major threat to human health. The protein CERS5 is responsible for synthesizing C16-ceramide, but its role in cancer is poorly understood. In this study, we examined the connection between CERS5 expression and pan-cancer prognosis, diagnosis, and the molecular mechanism involved. Kaplan-Meier survival analysis revealed variations among different cancer types. Functional enrichment analysis was conducted using gene set enrichment analysis (GSEA), and a network of protein-protein interaction (PPI) was constructed. The relationship between CERS5 and 22 immune infiltrating cell categories was detected using CIBERSORT. Single-cell analysis revealed elevated CERS5 levels in fibroblasts, which are vital in tumor immunity. The relationship between the expression of CERS5 and the immune-related genes, microsatellite instability, tumor mutational burden, and RNA modification genes in cancer were examined using the pan-cancer database. The role of CERS5 in immune regulation might be crucial to the tumor microenvironment. Pathway enrichment analysis indicated associations between CERS5 and extracellular matrix-receptor interaction, the WNT signaling pathway, and cell-cell junctions. Specifically, CERS5 was positively correlated with Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA4), Programmed Cell Death 1 (PDCD1), and Lymphocyte Activating 3 (LAG3) in stomach adenocarcinoma. In vitro, knockdown of CERS5 significantly hindered gastric cancer cells' ability to proliferate, migrate invade and increased apoptotic rate. We believe that CERS5 could be a promising target for future cancer research, contributing to the development of effective therapies.

Gastric residual volume, safety, and effectiveness of drinking 250 mL of glucose solution 2-3 hours before surgery in gastric cancer patients: a multicenter, single-blind, randomized-controlled trial.

Background: Carbohydrate drinking 2-3 hours before surgery has been widely adopted in colorectal operations. However, there is little direct evidence regarding its application in gastric cancer surgery. We aimed to evaluate the gastric residual volume, safety, and effectiveness of drinking 250 mL of 5% glucose solution 2-3 hours before elective gastric cancer surgery. Methods: We conducted an investigator-initiated, multicenter, randomized-controlled, parallel group, and equivalence trial. Eighty-eight patients with gastric adenocarcinoma were randomized into study or control group. Patients in the control group followed the traditional routine of 6-8 hours preoperative fasting, while those in the study group drank 250 mL of 5% glucose solution 2-3 hours before surgery. Immediately following tracheal intubation, gastric contents were aspirated through gastroscopy. The primary outcome was preoperative gastric residual volume. Results: Eighty-three patients were eventually analysed in the study (42 in the study group and 41 in the control group). Two groups were comparable at baseline characteristics. There were no statistical differences in residual gastric fluid volumes (35.86 ± 27.13 vs 27.70 ± 20.37 mL, P = 0.135) and pH values (2.81 ± 1.99 vs 2.66 ± 1.68, P = 0.708) between the two groups. Preoperative discomfort was significantly more decreased in the study group than in the control group (thirst score: 1.49 ± 1.23 vs 4.14 ± 2.07, P < 0.001; hunger score: 1.66 ± 1.18 vs 3.00 ± 2.32, P = 0.007). There was no statistical difference in the incidence of postoperative complications (19.05% vs 17.07%, P = 0.815). Conclusions: Drinking 250 mL of 5% glucose solution 2-3 hours before surgery in elective gastric cancer patients shows benefits in lowering thirst and hunger scores without increasing gastric residual volume and perioperative complications.

Elevation of preoperative serum hs-CRP is an independent risk factor for malnutrition in patients with gastric cancer.

Background: Recent studies have reported hypersensitive C-reactive protein (hs-CRP) linked to clinicopathological characteristics and nutritional status of the tumor, but its clinical significance in GC remains unclear. This study aimed to investigate the relationship between preoperative serum hs-CRP level and clinicopathological features and nutritional status in gastric cancer (GC) patients. Methods: The clinical data of 628 GC patients who met the study criteria were analyzed retrospectively. The preoperative serum hs-CRP level was divided into two groups (<1 mg/L and ≥1 mg/L) to evaluate clinical indicators. Nutritional Risk Screening and nutritional assessment of GC patients were performed by the Nutritional Risk Screening 2002 (NRS2002) and the Patient-Generated Subjective Global Assessment (PG-SGA), respectively. The data were subjected to chi-square test, univariate and multivariate logistic regression analyses, respectively. Results: The analysis of 628 GC cases revealed that 338 patients (53.8%) were on malnutrition risk(NRS2002≥3 points), and 526(83.8%) had suspected/moderate to severe malnutrition(PG-SGA≥ 2 points). Preoperative serum hs-CRP level was significantly correlated with age, tumor maximum diameter (TMD), peripheral nerve invasion (PNI), lymph-vascular invasion (LVI), depth of tumor invasion (DTI), lymph node metastasis (LNM), pTNM stage, body weight loss (BWL), body mass index (BMI), NRS2002 score, PG-SGA grade, hemoglobin (HB), total protein (TP), albumin (ALB), prealbumin (PAB) and total lymphocyte count (TLC). Multivariate logistic regression analysis revealed that hs-CRP (OR=1.814, 95%CI=1.174-2.803; P=0.007), age, ALB, BMI, BWL and TMD were independent risk factors for existing malnutritional risk in GC. Similarly, non-malnutrition and suspected/moderate to severe malnutrition groups presented that hs-CRP (OR=3.346, 95%CI=1.833-6.122; P< 0.001), age, HB, ALB, BMI and BWL were independent risk factors for malnutrition in GC. Conclusion: In addition to the generally used nutritional evaluation indicators such as age, ALB, BMI, and BWL, the hs-CRP level may be used as a nutritional screening and evaluation indicator for GC patients.

Circ_0008287 promotes immune escape of gastric cancer cells through impairing microRNA-548c-3p-dependent inhibition of CLIC1.

BACKGROUND: Analyses in silico suggested the upregulation of a circular RNA (circRNA), circ_0008287, in gastric cancer and possible interactions among microRNA (miR)-548c-3p, circ_0008287, and intracellular chloride channel protein 1 (CLIC1). This study aims to testify whether circ_0008287 can affect the immune escape of gastric cancer cells by regulating miR-548c-3p and CLIC1. METHODS: RT-qPCR was performed to determine the expression pattern of circ_0008287 in gastric cancer cells. Gain- and loss-of function assays were then performed to assess the effects of circ_0008287 on malignant phenotypes of cancer cells. Interactions among circ_0008287, miR-548c-3p and CLIC1 were verified by dual luciferase reporter gene, RIP and FISH assays. Effects of CLIC1 on IFN-γ secretion and apoptosis in CD8 + T cells were evaluated by flow cytometry following co-culture of CD8 + T cells with cancer cells overexpressing/silencing CLIC1. A gastric cancer mouse model was further developed for in vivo investigation on effects of circ_0008287 on tumorigenesis and tumor metastasis. RESULTS: circ_0008287, an upregulated circRNA in gastric cancer cells, augmented the viability as well as invasive and migratory potentials of gastric cancer cells. By competitively binding to miR-548c-3, circ_0008287 increased the expression of CLIC1, which impaired the function of CD8 + T cells and promoted their apoptosis. After downregulation of circ_0008287, in vivo tumorigenesis and metastasis were suppressed. CONCLUSION: Hence, this study suggests the promotive role of circ_0008287 in gastric cancer progression and immune escape and further elucidates the underlying circ_0008287/miR-548c-3p/CLIC1 regulatory axis.

Using a safe and effective fixative to improve the immunofluorescence staining of bacteria.

The emerging and development of green chemistry has once again drawn the researchers' attention to eliminating the use and generation of hazardous materials. Here we report the use of a safe and effective fixative, chlorine dioxide (ClO2), instead of traditional hazardous fixatives for the cross-linking of cellular proteins to improve immunofluorescence staining of bacteria. The concentration of ClO2needed for 100% fixation is 50µg ml-1, which is much lower than that of traditional fixatives (1000-10000µg ml-1). The ClO2mediated cross-linking can preserve the integrity of bacterial cells and prevent cell loss through lysis. Meanwhile, lysozyme can permeabilize the bacterial cells, allowing the labelled antibodies to diffuse to their intracellular target molecules. By usingE. coliO157:H7/RP4 as a gram-negative bacteria model, immunofluorescence staining assays for both intracellular protein and surface polysaccharide were carried out to investigate the effect of ClO2fixation on the staining. The results demonstrated that ClO2fixation could prevent the target antigens from cracking off the bacteria without damage on the interaction between the antibodies and antigens (either for polysaccharide or protein). As a safe and effective fixative, ClO2has potential practical applications in immunofluorescence staining and fluorescencein situhybridization for single bacteria/cell analysis.

Identification and verification of the prognostic value of the glutathione S-transferase Mu genes in gastric cancer.

Gastric cancer (GC) is one of the most frequently diagnosed gastrointestinal cancer types in the world. Novel prognostic biomarkers are required to predict the progression of GC. Glutathione S-transferase Mu (GSTM) belongs to a family of phase II enzymes that have been implicated in a number of cancer types. However, the prognostic value of the GSTM genes has not been previously investigated in GC. The Cancer Genome Atlas (TCGA) was used to evaluate mRNA expression levels of GSTMs in GC tissue samples. Overall survival (OS) rates, hazard ratios (HRs) and 95% CIs were calculated using the Cox logistic regression model and Kaplan-Meier (KM) analysis was performed. In addition, the KM plotter online database was used to validate mRNA expression and the prognostic value of GSMT family members in patients with GC. To predict the function of GSTM genes in these patients, several bioinformatics tools, including the Database for Annotation, Visualization and Integrated Discovery, gene multiple association network integration algorithm, Search Tool for the Retrieval of Interacting Genes/Proteins, Gene Set Enrichment Analysis (GSEA), nomogram and genome-wide co-expression analysis were used. In the present study, high expression of GSTM5 was indicated to be strongly associated with lower OS in patients with GC, according to the TCGA and KM plotter online databases (HR=1.47, 95% CI: 1.06-2.04, P=0.021; and HR=1.69, 95% CI: 1.42-2.01, P=1.6×10-9, respectively). The results from the GSEA and genome-wide co-expression analysis indicated that GSTM5 expression associated with several biological process terms, including 'adhesion', 'angiogenesis', 'apoptotic process', 'cell growth', 'proliferation', 'migration', 'Hedgehog signaling', 'MAPK signaling' and the 'TGF-ß signaling pathway'. In conclusion, the present results indicated that GSTM5 may serve as a biomarker for GC prognosis and may be a potential therapeutic target for GC.

Development and Validation of the Chinese Version of the Quality of Recovery-40 Questionnaire.

PURPOSE: The present study aimed to develop the official Chinese version of the QoR-40 (QoR-40C) and to test its reliability, validity, and responsiveness. PATIENTS AND METHODS: A systematic translation procedure was established and performed to develop the QoR-40C from the original English QoR-40 version. After the pilot study, 223 surgical patients were administered  the QoR-40C at four time points. The validity, reliability, and responsiveness were assessed to validate the QoR-40C. RESULTS: The test-retest reliability of the QoR-40C in the morning and afternoon of the third day after surgery was 0.917 (P < 0.001). The split-half reliability for all domains was 0.938 in the morning of the third day after surgery. The median item-to-own dimension and total score of Cronbach's α for internal consistency of the QoR-40C at different assessment time points were more than 0.70. All the correlation coefficients between each subscale and the QoR-40 total score showed good correlation and were greater than those for other subscales in the morning of the third day after surgery. Furthermore, in the morning of the third day after surgery, the QoR-40C total score was moderately positively correlated with the SF-36 score (ρ = 0.575, P < 0.001), while the QoR-40C score was negatively correlated with the visual analogue scale (VAS) score (ρ = -0.299, P < 0.001). The factor loadings of each item were within the required range. A statistically significant difference was observed in the QoR-40C total scores before and after the surgery (P < 0.001) with the standardized responsive mean (SRM) of 0.51. CONCLUSION: The QoR-40C showed good reliability, validity, and responsiveness and was appropriate to be used as a quality of life measurement questionnaire for patients after surgery in China.

Effects of siRNA-mediated silencing of myeloid cell leukelia-1 on the biological behaviors and drug resistance of gastric cancer cells.

OBJECTIVE: This study was to investigate the effects of siRNA mediated silencing of myeloid cell leukelia-1 (Mcl-1) on the biological behaviors and drug resistance of human drug-resistant gastric cancer (GC) cell lines, and to explore the potential mechanisms. METHODS: siRNA targeting Mcl-1 mRNA were designed and independently transfected into SGC-7901/VCR and SGC-7901/DDP. Cell proliferation and drug sensitivity were examined by MTT assay. Cell apoptosis and cell cycle were detected by flow cytometry. Cell Invasion and migration abilities were detected by transwell chamber assays. The expressions of drug-resistance-related genes and apoptosis-related proteins were detected by quantitative real-time PCR and Western blot assay, respectively. RESULTS: siRNA effectively inhibited the Mcl-1 expression, lowered the proliferation rate (P<0.05), raised the apoptosis rate (P<0.05), and arrested cells in S-phase (P<0.05). After inhibiting Mcl-1, the cell migration and invasion decreased (P<0.05), the resistance to VCR, DDP and 5-Fu was reversed to different extents (P<0.05), TS mRNA expression increased significantly (P<0.05), MDR1 remained unchanged (P>0.05), but DPD and TOP2A decreased significantly (P<0.05). Following Mcl-1 silencing, Bcl-2 was over-expressed in VCR-siRNA group, but the expressions of Fas and survivin reduced markedly (P<0.05); Bcl-2 and Fas expressions decreased significantly in DDP-siRNA group (P<0.05), but survivin expression remained unchanged. CONCLUSION: Mcl-1 is implicated in the proliferation, invasion, apoptosis and drug resistance of GC cells, and may be a promising target for the therapy of GC.