USP6 and circCYFIP2 target oncoprotein GOLPH3 for deubiquitination and induce platinum resistance in colon cancer.

GOLPH3 has been identified as an oncoprotein, playing a crucial role on progression and chemoresistancein of colon adenocarcinoma (COAD). However, it is still unclear the regulation of GOLPH3 expression at protein level. We discovered ubiquitin-specific proteases 6 (USP6) directly regulated the deubiquitination of the GOLPH3 protein and enhanced its stability in COAD. Overexpression of USP6 promoted COAD cell viability, inhibited apoptosis, and accelerated the growth of transplanted tumors growth in vitro and in vivo by deubiquitinating GOLPH3. Additionally, circCYFIP2 showed high expression levels in DDP-resistant colon cancer cells, promoting the cell proliferation. Mechanically, circCYFIP2 binds to both GOLPH3 protein and USP6, strengthening the interaction between GOLPH3 and USP6, and consequently induced DDP resistance in vitro and in vivo. In conclusion, USP6 operates as a deubiquitinase, targeting the GOLPH3 protein in COAD and enhancing its stability. Meanwhile, circCYFIP2 is crucial for the deubiquitination of GOLPH3 protein mediated by USP6 and acts as a scaffold to confer platinum resistance. The discovery of circCYFIP2/USP6/GOLPH3 pathway offers a potential target for overcoming chemoresistance in COAD.

Risk factors for pharyngalgia and xerostomia undergoing supraglottic jet oxygenation and ventilation in gastrointestinal endoscopy: a retrospective study.

Supraglottic jet oxygenation/ventilation (SJOV) can reduce hypoxemia in sedated endoscopy but may increase minor side effects like pharyngalgia and xerostomia. This study aimed to identify risk factors for pharyngalgia/xerostomia with SJOV during gastrointestinal endoscopy. From January 1 to December 31, 2021, 5313 patients with propofol sedation and SJOV underwent gastrointestinal endoscopy or removal of gastrointestinal polyps was analyzed retrospectively. Data included patient characteristics, operation details, postoperative adverse events, and potential risk factors for postoperative adverse events. Parameters considered as potential risk factors were identified based on study results published previously and based on the researcher's idea and clinical experience. The patient factors and the incidence of pharyngalgia/xerostomia at 30 min post-procedure were assessed. Descriptive statistics were calculated using SPSS software. Evaluation potential risk factors using univariate and multivariate logistic regression. Pharyngalgia/xerostomia occurred in 18.7% of patients at 30 min after procedure. A multivariable analysis showed that procedure time and pharyngalgia/xerostomia within 2 weeks were independent risk factors. Procedure time had the strongest association with postoperative pharyngalgia/xerostomia (OR, 8.09 [95% CI, 4.197-6.312]). No factors were significantly associated with hypoxemia risk (1.7% incidence). There were no barotrauma or other serious morbidity or mortality. Procedure duration and recent pharyngalgia/xerostomia increased risk of pharyngalgia/xerostomia with SJOV during endoscopy. Limiting SJOV duration may reduce side effects in susceptible patients. No predictors of hypoxemia were identified.

NSUN5-FTH1 Axis Inhibits Ferroptosis to Promote the Growth of Gastric Cancer Cells.

Recent studies revealed that NOP2/Sun RNA methyltransferase 5 (NSUN5) - ferritin heavy chain (FTH1) pathway is associated with ferroptosis in stem cells, whereas its roles in gastric cancer are still unclear. Our study aims to investigate the roles of the NSUN5-FTH1 axis in gastric cancer (GC) and its molecular mechanisms. Stable cell lines were constructed on SGC7901 cells by using shRNAs and pcDNA3.1 expression vectors, respectively. CCK-8 kits were used to determine cell viability. Biochemicals assays were used to detect lipid reactive oxygen species (ROS) and intracellular Fe2+ levels. RNA immunoprecipitation assay, qPCR, and Western blotting were used to determine the changes in biomarkers. GC xenograft mouse model was established to confirm the observation in vivo. An elevation of NSUN5 was observed in GC tumor tissues. NSUN5 inhibited ferroptosis including decreasing cell viability and increasing levels of lipid ROS and Fe2+ in GC cells. Besides, a positive correlation was also observed between NSUN5 and FTH1. Interestingly, NSUN5 regulated the levels of FTH1, instead of FTH1 regulating NSUN5 in GC cells. NSUN5-FTH1 axis regulated erastin-induced ferroptosis in SGC7901 cells. Consistently, silencing NSUN5 or FTH1 inhibited the growth of the SGC7901 tumor in vivo. NSUN5-FTH1 axis promoted the growth of GC cells in part by the regulation of ferroptosis.

Auriculasin enhances ROS generation to regulate colorectal cancer cell apoptosis, ferroptosis, oxeiptosis, invasion and colony formation.

Colorectal cancer (CRC) is one of the most common malignant tumors in the digestive system, and Chinese herbal medicine plays an important role in tumor treatment. The in-depth study of auriculasin isolated from Flemingia philippinensis showed that auriculasin promoted reactive oxygen species (ROS) generation in a concentration-dependent manner; when ROS scavenger NAC was added, the effects of auriculasin in promoting ROS generation and inhibiting cell viability were blocked. Auriculasin induced CRC cell apoptosis, led to mitochondrial shrinkage, and increased the intracellular accumulation of Fe2+ and MDA. When auriculasin and NAC were added simultaneously, the levels of apoptosis, Fe2+ and MDA returned to the control group levels, indicating that auriculasin activated apoptosis and ferroptosis by inducing ROS generation. In addition, auriculasin promoted the expression of Keap1 and AIFM1, but significantly reduced the phosphorylation level of AIFM1, while NAC significantly blocked the regulation of Keap1 and AIFM1 by auriculasin, which indicates that auriculasin can also induce oxeiptosis through ROS. When Z-VAD-FMK, Ferrostatin-1, Keap1 siRNA, PGAM5 siRNA and AIFM1 siRNA were added respectively, the inhibitory effect of auriculasin on cell viability was significantly weakened, indicating that auriculasin inhibits cell viability by inducing apoptosis, ferroptosis and oxeiptosis. Auriculasin also inhibited the invasion and clone forming ability of CRC cells, while NAC blocked the above effects of auriculasin. Therefore, auriculasin can promote CRC cell apoptosis, ferroptosis and oxeiptosis by inducing ROS generation, thereby inhibiting cell viability, invasion and clone formation, indicating that auriculasin has a significant antitumor effect.

Golgi Phosphoprotein 3 Represents a Novel Tumor Marker for Gastric and Colorectal Cancers.

BACKGROUND: Early diagnosis is very important for the clinical treatment of gastric cancer (GC) and colorectal cancer (CRC). We aimed to detect Golgi phosphoprotein 3 (GOLPH3) and evaluate its diagnostic value. MATERIALS AND METHODS: Serum concentrations of GOLPH3 were detected by ELISA in 136 CRC patients, 102 GC patients, and 50 healthy controls at the Second Affiliated Hospital of Fujian Medical University from June 2016 to December 2019. Serum concentrations of CEA and CA19-9 were detected by ECLIA. RESULTS: Serum concentrations of GOLPH3, CEA, and CA19-9 were higher in GC and CRC patients than in healthy controls (P < 0.001). Serum GOLPH3 concentrations were increased in GC and CRC patients with tumors greater than 5 cm, poor differentiation, greater depth of tumor invasion, and increased lymphatic and distant metastases (P < 0.05). In the GC and CRC groups, the AUCs of GOLPH3 were higher than those of CEA and CA19-9 (P < 0.05), while the AUCs of the marker combination were higher than those of GOLPH3 (P < 0.05), and postoperative serum GOLPH3 levels were lower than preoperative levels (P < 0.001). Serum GOLPH3 concentrations in CRC patients correlated positively with CEA and CA19-9 concentrations (P < 0.05). CONCLUSION: Serum GOLPH3 concentrations in GC and CRC patients are related to TNM stage. GOLPH3 may represent a novel biomarker for the diagnosis of GC and CRC. The combination of serum GOLPH3, CEA, and CA19-9 concentrations can improve diagnostic efficiency for GC and CRC. GOLPH3 is expected to become an indicator for the early diagnosis and evaluation of surgical effects.

The Biological Characteristics of Novel H5N6 Highly Pathogenic Avian Influenza Virus and Its Pathogenesis in Ducks.

Clade 2.3.4.4 H5Nx highly pathogenic avian influenza viruses (HPAIVs) have caused outbreaks in poultry in the world. Some of these viruses acquired internal genes from other subtype avian influenza viruses (AIVs) such as H9 and H6 for the generation of novel reassortant viruses and continually circulated in poultry. Here, we applied a duck-origin virus DK87 and a chicken-origin virus CK66 to assess the biological characteristics of novel reassortant H5N6 HPAIVs and its pathogenesis in ducks. A genetic analysis indicated that the HA genes of the two H5N6 HPAIVs were closely related to the H5 viruses of clade 2.3.4.4 circulating in Eastern Asia and classified into H5 AIV/Eastern Asia (EA)-like lineage. Their NA genes fell into Eurasian lineage had close relationship with those of H5N6 viruses circulating in China, Laos, Vietnam, Japan, and Korea. All internal genes of DK87 were aggregated closely with H5 AIV/EA-like viruses. The internal genes (PB1, PA, NP, M, and NS) of CK66 were derived from H9N2 AIV/SH98-like viruses and the PB2 were derived from H5 AIV/EA-like viruses. These results indicate that clade 2.3.4.4 H5N6 AIVs have continually evolved and recombined with the H9N2 viruses circulating in Southern China. Pathogenicity test showed that the two viruses displayed a broader tissue distribution in ducks and caused no clinical signs. These results indicated that ducks were permissive for the replication of the chicken-origin reassortant virus CK66 without prior adaptation, but the duck-origin virus DK87-inoculated ducks showed significantly higher viral titers in some organs than the CK66-inoculated ducks at 5 day post-inoculated (DPI). The recovery of viruses from oropharyngea and cloacal swabs of contacted ducks indicated that they transmitted in native ducks by direct contact. Quantitative reverse transcription PCR (qRT-PCR) results revealed that the immune-relative genes (PRRs, IFNs, Mx-1, IL-6, and IL-8) in the lungs of inoculated ducks were expressed regardless of virus origin, but the expression of these genes was significantly higher in response to infection with the DK87 virus compared to the CK66 virus at 3 DPI. Overall, we should provide further insights into how clade 2.3.4.4 H5N6 AIVs undergo genetic and pathogenic variations to prevent outbreaks of this disease.

Protosappanin B Exerts Anti-tumor Effects on Colon Cancer Cells via Inhibiting GOLPH3 Expression.

Protosappanin B (PSB) is a key active component of Lignum Sappan extract. Although the antiproliferative effects of Lignum Sappan extract have been demonstrated in various cancer cells, relatively little is known about the effects of PSB on tumor progression. The aim of this study was to explore the anti-tumor effects of PSB on human colon cancer cells by regulation of intracellular signaling pathways and Golgi phosphoprotein 3 (GOLPH3) expression in vitro and in vivo. Our results showed that PSB effectively inhibited the viability and migration of SW620 cells and induced apoptosis, but had poor effect on HCT116 cells. Furthermore, PSB significantly reduced the expression of p-AKT, p-p70S6K, ß-catenin, and p-ERK1/2 proteins in SW620 cells, and this effect was reversed by the corresponding signaling pathway agonists. Interestingly, PSB could also suppress GOLPH3 expression of SW620 cells in a concentration-dependent manner, but SW620 cells transfected with lentiviral vectors overexpressing GOLPH3 can effectively resist the cytotoxic activity of PSB in vitro. The xenograft experiment of SW620 cells with LV-GOLPH3 confirmed that PSB distinctly inhibited the tumor growth via suppressing GOLPH3 expression. Collectively, these findings clarified a new anti-cancer mechanism of PSB through inhibition of GOLPH3 expression and intracellular signaling pathways in colon cancer cells. PSB may be a potential new drug for colon cancer.

MicroRNA-1276 Promotes Colon Cancer Cell Proliferation by Negatively Regulating LACTB.

PURPOSE: LACTB, regulated by a variety of microRNAs (miRNAs), is proven to be a tumor suppressor. However, there are few reports that LACTB in colon cancer cells is regulated by miRNA. Therefore, the aim of this study was to explore the miRNAs that regulate LACTB in colon cancer. PATIENTS AND METHODS: Data from TCGA were analyzed in starBase and GEPIA2, and Western blot and quantitative PCR (qPCR) were used to detect the expression of LACTB in colon cancer cell lines. MiRNAs targeting LACTB were predicted by MicroT-CDS, starBase, miRDB, mirDIP, and DIANA. The relationship between LACTB and miRNA was explored by dual-luciferase assay. MTT, propidium iodide (PI), Western blot, Annexin V-FITC/PI Kit, qPCR and transwell assay were used to detect the changes in cell proliferation, cell cycle, autophagy, apoptosis, epithelial-to-mesenchymal transition (EMT), cell migration, and invasiveness in colon cancer cells that overexpressed miR-1276 and/or LACTB. RESULTS: The results showed that the LACTB mRNA level was lower and the miR-1276 level was higher in colon cancer than in normal tissue. MiR-1276 inhibited the expression of LACTB. Furthermore, overexpression of miR-1276 in colon cancer cells increased proliferation, migration, invasiveness and EMT, and decreased autophagy and apoptosis. Supplementing LACTB suppressed these effects of miR-1276. CONCLUSION: In conclusion, miR-1276, which may be a potential therapy for colon cancer, inhibits cell growth and promotes apoptosis by targeting LACTB in colon cancer cells.

Tenacissoside H Induces Apoptosis and Inhibits Migration of Colon Cancer Cells by Downregulating Expression of GOLPH3 Gene.

OBJECTIVE: Tenacissoside H (TDH) is a Chinese medicine monomer extracted from Marsdenia tenacissima extract (MTE), which has been confirmed to have antitumor effects, but its mechanism is still unclear. The aim of this study was to investigate the effect and mechanism of TDH on human colon cancer LoVo cell proliferation and migration and explore the correlation of TDH treatment with the expression of GOLPH3 and cell signaling pathways in LoVo cells. METHODS: LoVo cells were treated with TDH at 0.1, 1, 10, and 100 µg/mL for 24, 48, and 72 h. The proliferation rate of LoVo cells was evaluated by MTT assay. Recombinant plasmid p-CMV-2-GOLPH3 was constructed, and p-CMV-2-GOLPH3 and p-CMV-2 empty plasmids were transfected into LoVo cells by lipofection. Western blotting was used to detect the transfection efficiency and the expression of p-p70S6K, p70S6K, ß-catenin, and GOLPH3. The apoptosis rate was analyzed with Annexin V-FITC/PI double-staining method, and cell migration assessed by transwell assay. RESULTS: TDH inhibited the proliferation of LoVo cells in a concentration-dependent manner. The IC50 of TDH treatment in LoVo cells at 24, 48, and 72 h was 40.24, 13.00, and 5.73 µg/mL, respectively. TDH treatment significantly induced apoptosis and suppressed the viability and migration of human colon cancer LoVo cells. The effect of TDH on induction of apoptosis and inhibition of migration in LoVo cells decreased significantly after activating the PI3K/AKT/mTOR and Wnt/ß-catenin signaling pathways with agonists. Additionally, the expression of GOLPH3 protein downregulated significantly in LoVo cells under TDH treatment. Overexpression of the GOLPH3 gene increased the expression of key proteins in PI3K/AKT/mTOR and Wnt/ß-catenin signaling pathways and blocked the antitumor activity of TDH. CONCLUSION: Collectively, the present results indicated that TDH can inhibit the proliferation vitality of colon cancer LoVo cells through downregulating GOLPH3 expression and activity of PI3K/AKT/mTOR and Wnt/ß-catenin signaling pathways.

Polymorphism of inhibin ßB gene and its relationship with litter size in sheep.

The inhibin ß(B) (INHBB) gene was studied as a candidate gene for the prolificacy of Small Tail Han and Hu sheep. According to the sequence of exon 1 and 2 of bovine INHBB gene, six pairs of primers were designed to detect single nucleotide polymorphisms of exon 1 and 2 of INHBB gene in both high (Small Tail Han and Hu sheep) and low prolificacy breeds (Dorset, Texel and German Mutton Merino sheep) by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). Three pairs of primers (primers 1-1, 1-2 and 1-3) were used to amplify the exon 1, and others (primers 2-1, 2-2 and 2-3) to the exon 2. Only the products amplified by primer 2-3 displayed polymorphism. For primer 2-3, three genotypes (AA, AB and BB) were detected in Hu sheep and only AA genotype in other breeds. In Hu sheep, frequency of AA, AB and BB genotypes was 0.636, 0.046 and 0.318, respectively. Sequencing revealed 276A > G mutation (based on the amplification region of primer 2-3) which did not cause any amino acid change because it lay in the 3' untranslated region. The ewes with genotype BB had 0.58 (P < 0.01) lambs more than those with AA in Hu sheep.