Neutrophil extracellular traps induced by the hypoxic microenvironment in gastric cancer augment tumour growth.

BACKGROUND: Inflammation-related predisposition to cancer plays an essential role in cancer progression and is associated with poor prognosis. A hypoxic microenvironment and neutrophil infiltration are commonly present in solid tumours, including gastric cancer (GC). Neutrophil extracellular traps (NETs) have also been demonstrated in the tumour immune microenvironment (TIME), but how NETs affect GC progression remains unknown. Here, we investigated the role of NET formation in the TIME and further explored the underlying mechanism of NETs in GC tumour growth. METHODS: Hypoxia-induced factor-1α (HIF-1α), citrulline histone 3 (citH3) and CD66b expression in tumour and adjacent nontumor tissue samples was evaluated by western blotting, immunofluorescence and immunohistochemical staining. The expression of neutrophil-attracting chemokines in GC cells and their hypoxic-CM was measured by qRT‒PCR and ELISA. Neutrophil migration under hypoxic conditions was evaluated by a Transwell assay. Pathway activation in neutrophils in a hypoxic microenvironment were analysed by western blotting. NET formation was measured in vitro by immunofluorescence staining. The protumour effect of NETs on GC cells was identified by Transwell, wound healing and cell proliferation assays. In vivo, an lipopolysaccharide (LPS)-induced NET model and subcutaneous tumour model were established in BALB/c nude mice to explore the mechanism of NETs in tumour growth. RESULTS: GC generates a hypoxic microenvironment that recruits neutrophils and induces NET formation. High mobility group box 1 (HMGB1) was translocated to the cytoplasm from the nucleus of GC cells in the hypoxic microenvironment and mediated the formation of NETs via the toll-like receptor 4 (TLR4)/p38 MAPK signalling pathway in neutrophils. HMGB1/TLR4/p38 MAPK pathway inhibition abrogated hypoxia-induced neutrophil activation and NET formation. NETs directly induced GC cell invasion and migration but not proliferation and accelerated the augmentation of GC growth by increasing angiogenesis. This rapid tumour growth was abolished by treatment with the NET inhibitor deoxyribonuclease I (DNase I) or a p38 MAPK signalling pathway inhibitor. CONCLUSIONS: Hypoxia triggers an inflammatory response and NET formation in the GC TIME to augment tumour growth. Targeting NETs with DNase I or HMGB1/TLR4/p38 MAPK pathway inhibitors is a potential therapeutic strategy to inhibit GC progression. Video Abstract.

Circular RNA circ_0000592 elevates ANXA4 expression via sponging miR-1179 to facilitate tumor progression in gastric cancer.

Increasing evidence indicated that dysregulated circular RNAs were implicated in the progression of multiple malignancies. However, the function of circ_0000592 in gastric cancer (GC) progression and its associated mechanism remain poorly understood. Quantitative real-time PCR and Western blot assay were performed to detect RNA and protein expression. Cell proliferation, migration and invasion were analyzed by 5-Ethynyl-2'-deoxyuridine staining assay, Transwell migration assay and Transwell invasion assay, respectively. The glucose/lactate assay kit was used to assess the rates of glucose consumption and lactate production. The interaction between microRNA-1179 (miR-1179) and circ_0000592 or Annexin A4 (ANXA4) was confirmed by dual-luciferase reporter assay and RNA pull-down assay. Xenograft tumor model was established to investigate the effect of circ_0000592 on tumor growth in vivo. Circ_0000592 expression was elevated in GC tissues and cells. Circ_0000592 knockdown hampered cell proliferation, migration, invasion and glycolysis of GC cells. MiR-1179 was a direct target of circ_0000592, and circ_0000592 silencing-mediated effects in GC cells were partly reversed by the knockdown of miR-1179. MiR-1179 interacted with the 3' untranslated region (3'UTR) of ANXA4. Circ_0000592 silencing reduced ANXA4 expression partly by upregulating miR-1179 in GC cells. ANXA4 overexpression partly overturned circ_0000592 knockdown-induced effects in GC cells. Circ_0000592 depletion markedly suppressed xenograft tumor growth in vivo. Circ_0000592 contributed to GC progression through regulating miR-1179/ANXA4 axis, which provided novel potential biomarkers and therapeutic targets for GC treatment.

Multiple Pathway-Dephosphorylated ASK-1 Confers Temozolomide-Resistance to Human Glioma Cells.

AIM: Temozolomide (TMZ) resistance contributes to the unfavorable prognosis of glioma, however, the mechanism of resistance is unknown. ASK-1 has various functions in many tumors, but its function in glioma is poorly understood. This study aimed to elucidate the function of ASK-1 and the role of its modulators in the induction of TMZ resistance in glioma and the underlying mechanism. MATERIAL AND METHODS: ASK-1 phosphorylation, the IC50 of TMZ, cell viability, and apoptosis were assessed in the U87 and U251 glioma cell lines and the derived TMZ-resistant cell lines U87-TR and U251-TR. We then blocked ASK-1 function, either with an inhibitor or by overexpression of multiple ASK-1 upstream modulators, to further explore the role of ASK-1 in TMZ-resistant glioma. RESULTS: TMZ-resistant glioma cells showed high IC50 values of TMZ, high survival, and low levels of apoptosis following the TMZ challenge. ASK-1 phosphorylation, but not protein expression, was higher in U87 and U251 cells than in TMZ-resistant glioma cells exposed to TMZ. The addition of the ASK-1 inhibitor selonsertib (SEL) resulted in the dephosphorylation of ASK-1 in U87 and U251 cells after the TMZ challenge. SEL treatment increased the TMZ resistance of U87 and U251 cells, as evidenced by the increased IC50 and cell survival rate and low apoptosis rate. Overexpression of some ASK-1 upstream suppressors [Thioredoxin (Trx), protein phosphatase 5 (PP5), 14-3-3, and cell division cycle 25C (Cdc25C)] led to various degrees of ASK-1 dephosphorylation and a TMZ-resistant phenotype in U87 and U251 cells. CONCLUSION: Dephosphorylation of ASK-1 induced TMZ resistance in human glioma cells, and several ASK-1 upstream suppressors, including Trx, PP5, 14-3-3, and Cdc25C, are involved in this phenotypic change induced by dephosphorylation of ASK-1.

MicroRNA-574-3p Regulates HIF-α Isoforms Promoting Gastric Cancer Epithelial-Mesenchymal Transition via Targeting CUL2.

BACKGROUND: Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide. MicroRNAs (miRNAs) have been widely validated as potential biomarkers for cancer treatment and diagnosis. AIMS: This paper intends to study the effect and specific mechanism of miR-574-3p/CUL2 axis in GC. METHODS: The miR-574-3p expression in GC tissues and cell lines was analyzed by reverse transcription polymerase chain reaction (RT-PCR). GC cell (N87) proliferation, migration and invasion were determined by the Brdu assay and Transwell assay, respectively. The tumor xenotransplantation model was established in vivo to test the effect of miR-574-3p or Cullin 2 (CUL2) on tumor growth. The relationship between miR-574-3p and CUL2 was predicated by bioinformatic analysis and verified by dual-luciferase reporter assay and RIP experiment. The expression of CUL2, hypoxia-induced transcription factor-1α (HIF-1α) as well as E-cadherin, Snail and Vimentin was monitored by western blot and immunohistochemistry. RESULTS: miR-574-3p was overexpressed in GC tissues and cells. Forced upregulation of miR-574-3p enhanced proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of GC cells (N87), while downregulation of miR-574-3p resulted in reverse effects. Additionally, miR-574-3p promoted N87 cells growth and EMT in vivo. CUL2 was negatively regulated by miR-574-3p in N87 cells, and upregulation of CUL2 repressed the malignant behaviors of N87 cells. Moreover, CUL2 directly interacted with HIF-1α and suppressed HIF-1α expression both in vitro and in vivo. CONCLUSIONS: miR-574-3p targeted CUL2 to upregulate HIF-1α, thus facilitating the progression of GC.

Association of CYP24A1 gene polymorphism with colorectal cancer in the Jiamusi population.

BACKGROUND: The population in Jiamusi has been reported to have the highest prevalence of colorectal cancer (CRC) in China. The genetic causal-effect for this occurrence among the residents remains unclear. Given the long cold seasons with people wearing more clothes and reduced UV exposure, we aimed to study the association between the vitamin D metabolism-related gene CYP24A1 polymorphism and CRC susceptibility. METHOD: A case-control study was conducted that included 168 patients with CRC and 710 age-matched healthy individuals as the control group. Plausible susceptible variations were sought and clinical phenotypic-genotype association analysis was performed. RESULTS: Overall, two CYP24A1 polymorphisms, rs6013905 AX (P = 0.02, OR = 1.89, 95%CI: 1.09-3.29) and rs2762939 GX (P = 0.02, OR = 1.52, 95%CI: 1.08-2.13) were significantly associated with CRC in the Jiamusi population. In the female group, three CYP24A1 polymorphisms, rs6013905 AX (P = 0.04, OR = 2.59, 95%CI: 1.03-6.49), rs2762939 GX (P = 0.01, OR = 2.35, 95%CI: 1.25-4.42), and rs6068816 GG (P = 0.05, OR = 1.89, 95%CI: 0.99-3.59) carriers were significantly associated with CRC. In clinical phenotypic-genotype analysis, rs6013905 GG (P = 0.05, OR = 4.00, 95%CI: 0.92-17.48) and rs2762939 GX (P = 0.03, OR = 4.87, 95%CI: 1.00-23.69) carriers were significantly associated with poorly differentiated CRC, while CYP24A1 rs6068816 AX was significantly associated with the tumor type (P = 0.02, OR = 2.08, 95%CI: 1.10-3.96) and location (P = 0.04, OR = 2.24, 95%CI: 1.05-4.77). CONCLUSION: CYP24A1 gene polymorphism may be a genetic risk factor attributable to the highest prevalence of CRC in Jiamusi people. Individuals with CYP24A1 gene polymorphism may have an increased barrier for vitamin D absorption, thus contributing to the risk of CRC development.

Construction and Characterization of a Synergistic lncRNA-miRNA Network Reveals a Crucial and Prognostic Role of lncRNAs in Colon Cancer.

Non-coding RNAs such as long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have been found to be indispensable factors in carcinogenesis and cancer development. Numerous studies have explored the regulatory functions of these molecules and identified the synergistic interactions among lncRNAs or miRNAs, while those between lncRNAs and miRNAs remain to be investigated. In this study, we constructed and characterized an lncRNA-miRNA synergistic network following a four-step approach by integrating the regulatory pairs and expression profiles. The synergistic interactions with more shared regulatory mRNAs were found to have higher interactional intensity. Through the analysis of nodes in the network, we found that lncRNAs played roles that are more central and had similar synergistic interactions with their neighbors when compared with miRNAs. In addition, known colon adenocarcinoma (COAD)-related RNAs were found to be enriched in this synergistic network, with higher degrees, betweenness, and closeness. Finally, we proposed a risk score model to predict the clinical outcome for COAD patients based on two prognostic hub lncRNAs, MEG3 and ZEB1-AS1. Moreover, the hierarchical networks of these two lncRNAs could contribute to the understanding of the biological mechanism of tumorigenesis. For each lncRNA-miRNA interaction in the hub-related subnetwork and two hierarchical networks, we performed RNAup method to evaluate their binding energy. Our results identified two important lncRNAs with prognostic roles in colon cancer and dissected their regulatory mechanism involving synergistic interaction with miRNAs.

Impact of Colon-Specific DNA Methylation-Regulated Gene Modules on Colorectal Cancer Patient Survival.

BACKGROUND Colorectal cancer (CRC) is the second most commonly diagnosed cancer in females and the third in males worldwide. Although existing evidence explained some critical functions of the single genetic abnormality in the pathogenesis of CRC, the function of interactors involved in the colon-specific regulatory network, especially DNA methylation regulated network is still poorly understood. MATERIAL AND METHODS In this work, matched gene expression and DNA methylation samples of CRC patients were retrieved. Differential gene expression and methylation analyses were performed. In addition, gene expression and DNA methylation were integrated into a colon-specific regulatory gene network, detecting the epigenetically regulated gene modules which drive CRC through an underlying epigenetic mechanism. Finally, the colon-specific DNA methylation-regulated gene modules were validated using an independent set of CRC patients. RESULTS Differential gene expression analysis demonstrated the upregulation of the cell cycle and DNA replication and downregulation of cGMP-PKG signaling pathway and calcium signaling pathway in CRC. Differentially methylated regions (DMRs) showed the different levels of methylation in promoters, CpG islands, and genes in CRC. In addition, gene expression and DNA methylation were integrated into a colon-specific regulatory gene network, detecting 8 epigenetically regulated gene modules which drive CRC through an underlying epigenetic mechanism. Interestingly, 2 of the colon-specific DNA methylation-regulated gene modules showed a significant predictive ability for the survival of an independent set of CRC patients. CONCLUSIONS The results of this study could open a new era and aid the development of novel therapeutic targets for the treatment of CRC patient.

Effect of different doses of ulinastatin on cellular immunity and hepatorenal functions in patients undergoing laparoscopic colorectalcarcinoma surgery.

The effect of different doses of ulinastatin on cellular immunity and hepatorenal functions in patients undergoing laparoscopic colorectal-carcinoma surgery was observed and analyzed. The 200 patients with laparoscopic colorectal-carcinoma surgery in our hospital were selected as research subjects and divided into 4 groups containing equal patients, namely, saline group, 0.5 x 104U/kg ulinastatin group, 1 x 104U/kg ulinastatin group, and 1.5 x 104U/kg ulinastatin group, which were denoted as group A, group B, group C and group D, respectively. The treatment effect of patients in 4groups was observed and compared. By observing the Narcotrend cerebral state index (NT index), the results showed that NT index at tracheal intubation, pneumoperitoneum beginning, pneumoperitoneum 30min, resection of tumor, end of operation in 4 groups was significantly lower than that at preoperative anesthesia (T0) (p<0.05); differences in hepatorenal values (AST, ALT, BUN and Cr) among 4 groups at T0 were of no statistical significance (P>0.05); each index in T cell subsets in the postoperative third days (T1) was significantly lower than that at T0; indexes of T cell subgroup of group B, C and D at T1 were higher than that of group A at T1 (p<0.05). For 4 groups, the difference in liver and kidney function indicators at T1 and T0 was of no statistical significance, p>0.05. Different doses of ulinastatin have a certain effect on cellular immunity in patients undergoing laparoscopic colorectal-carcinoma surgery and do not significantly affect hepatorenal function.

Downregulation of TBC1 Domain Family Member 24 (BC1D24) Inhibits Breast Carcinoma Growth via IGF1R/PI3K/AKT Pathway.

BACKGROUND TBC1 domain family member 24 (TBC1D24) pathogenic mutations affect its binding to ARF6 and then result in severe impairment of neuronal development. However, there are no reports about the expression and function of TBC1D24 in cancer. The aim of the present study was to evaluate the effect of proliferation, migration, and invasion after silencing TBC1D24 expression in breast cancer MCF-7 cells, and to elucidate the potential mechanism of TBC1D24 in breast cancer. MATERIAL AND METHODS The expression of TBC1D24 in breast cancer tissues and the adjacent non-tumor tissues was determined by S-P immunohistochemistry. The malignant behavior, including proliferation, migration, and invasion ability, was determined after silencing TBC1D24 in breast cancer MCF-7 cells. The expression of IGF1R was determined after silencing TBC1D24. The expression of TBC1D24 and IGF1R was detected after transfecting miR-30a mimics or inhibitors. The effect of TBC1D24 on MCF-7 cells growth in vivo was evaluated by a tumor xenograft study. RESULTS TBC1D24 expression was elevated and was associated with poor outcome in breast carcinoma. TBC1D24 high expression was significantly correlated with unfavorable OS and RFS for breast cancer patients (p<0.05). Silencing TBC1D24 inhibited the proliferation, migration, and invasion ability of MCF-7 cells. TBC1D24 and IGF1R expression were decreased when transfected with miR-30a mimics. However, TBC1D24 and IGF1R expression were increased when transfected with miR-30a inhibitors (p<0.05). Knockdown of TBC1D24 inhibited the expression of IGF1R, PI3K, and p-AKT (p<0.05). Knockdown of TBC1D24 abolished tumorigenicity of MCF-7 cells. The average volume and weight of tumors was lower after silencing TBC1D24 expression (P<0.05). CONCLUSIONS Silencing TBC1D24 inhibited MCF-7 cells growth in vitro and in vivo. TBC1D24 promoted breast carcinoma growth through the IGF1R/PI3K/AKT pathway. TBC1D24 is a potential therapeutic target for breast cancer.

Long non-coding RNA ZFAS1 is an unfavourable prognostic factor and promotes glioma cell progression by activation of the Notch signaling pathway.

Survival of patients with glioma remains poor, which is largely attributed to active carcinogenesis. Accumulating evidence indicates that long non-coding RNAs (lncRNAs) play key roles in tumor initiation and progression. However, the function of lncRNA ZFAS1 in glioma is still unclear. In the current study, we found that ZFAS1 was upregulated in glioma tissues and cell lines. High ZFAS1 expression in glioma tissues was significantly correlated with advanced tumor stage and poor overall survival. Furthermore, in vitro assays demonstrated that ZFAS1 inhibition significantly suppressed glioma cell proliferation, migration and invasion. Importantly, we further confirmed that epithelial-mesenchymal transition (EMT) and the Notch signaling pathway was inactivated in the glioma cells after ZFAS1 knockdown. Thus, our findings indicated that ZFAS1 could exhibit a tumor oncogenic role in glioma progression by regulating EMT and Notch signaling pathway. LncRNA ZFAS1 might serve as a therapeutic target for the treatment of glioma patients.