ZEB2 alleviates Hirschsprung's-associated enterocolitis by promoting the proliferation and differentiation of enteric neural precursor cells via the Notch-1/Jagged-2 pathway.

BACKGROUND: Hirschsprung's-associated enterocolitis (HAEC) is a prevalent complication of Hirschsprung's disease (HSCR). Zinc finger E-box binding homeobox 2 (ZEB2) and Notch-1/Jagged-2 are dysregulated in HSCR, but their role in HAEC progression remains poorly understood. We aimed to explore the role and underlying mechanism of enteric neural precursor cells (ENPCs) and the ZEB2/Notch-1/Jagged-2 pathway in HAEC development. METHODS: Colon tissues were collected from HSCR and HAEC patients. ENPCs were isolated from the HAEC group and stimulated by lipopolysaccharide (LPS). The expressions of ZEB2/Notch-1/Jagged-2 were measured using RT-qPCR and Western blot. Immunofluorescence and cell counting kit-8 assays were performed to assess the differentiation and proliferation of ENPCs. Inflammatory factors were measured by ELISA kits. Co-immunoprecipitation and bioinformatic analysis were used to explore the interaction between ZEB2 and Notch-1. Small interfering RNA and overexpression vectors were used to investigate the role and mechanism of ZEB2 and Notch-1 in regulating ENPCs' proliferation and differentiation during HAEC progression. RESULTS: We observed increased LPS in the colon tissues of HAEC, with downregulated ZEB2 expression and upregulated Notch-1/Jagged-2 expression. ZEB2 interacts with Notch-1. LPS treatment downregulated ZEB2 expression, upregulated Notch-1/Jagged-2 expression, and induced proliferation and differentiation disorders in ENPCs, which were reversed by the knockdown of Notch-1. Furthermore, overexpression of ZEB2 inhibited Notch-1/Jagged-2 signaling and ameliorated inflammation and dysfunction in LPS-induced ENPCs. Notch-1 overexpression enhanced LPS-induced dysfunction, but this effect was antagonized by the overexpression of ZEB2. CONCLUSION: Overexpression of ZEB2 ameliorates LPS-induced ENPCs' dysfunction via the Notch-1/Jagged-2 pathway, thus playing a role in HAEC.

ALKBH5 facilitates the progression of infantile hemangioma by increasing FOXF1 expression in a m6A-YTHDF2 dependent manner to activate HK-2 signaling.

Alkylation repair homolog protein 5 (ALKBH5) is reported to participate in infantile hemangioma (IH) progression. However, the underlying mechanism of ALKBH5 in IH remains unclear. Using qRT-PCR and Western blotting, ALKBH5, forkhead box F1 (FOXF1) and hexokinase 2 (HK-2) expressions in IH tissues and IH-derived endothelial cells XPTS-1 were assessed. The Me-RIP assay was used to analyze FOXF1 m6A level. CCK8, colony formation, flow cytometry and transwell assays were employed to determine IH cell viability, proliferation, apoptosis, migration and invasion. The interactions between YTH (YT521-B homology) domain 2 (YTHDF2), FOXF1 and HK-2 were analyzed by RIP, dual luciferase reporter gene assay and/or ChIP assay. The in vivo IH growth was evaluated in immunocompromised mice. FOXF1 was overexpressed in IH tissues, and its silencing inhibited IH cell proliferation, migration and invasion whereas promoting cell apoptosis in vitro. ALKBH5 upregulation facilitated FOXF1 mRNA stability and expression in IH cells in a m6A-YTHDF2-dependent manner. FOXF1 downregulation reversed the impact of ALKBH5 upregulation on IH cellular phenotypes. It also turned out that FOXF1 positively regulated HK-2 expression in IH cells through interacting with the HK-2 promoter. HK-2 upregulation abolished FOXF1 knockdown's inhibition on IH cell aggressive behaviors. ALKBH5 or FOXF1 silencing suppressed IH tumor development via HK-2 signaling in immunocompromised mice. ALKBH5 promoted FOXF1 expression m6A-YTHDF2 dependently, which in turn elevated HK-2 expression, thereby accelerating IH development.

Electrochemical detection of glutathione based on accelerated CRISPR/Cas12a trans-cleavage with MnO2 nanosheets.

The CRISPR/Cas12a system is accelerated by glutathione-mediated reduction of MnO2 nanosheets. By monitoring the trans-cleavage of the DNA probe, an electrochemical method for glutathione assay is fabricated, with the detection limit of 3.5 pM. It provides a promising tool for plasma analysis with satisfactory performance, indicating the broad application prospects of this glutathione assay.

Extracellular vesicle-packaged circBIRC6 from cancer-associated fibroblasts induce platinum resistance via SUMOylation modulation in pancreatic cancer.

BACKGROUND: Cancer-associated fibroblasts (CAFs) play pivotal roles in chemoresistance of pancreatic ductal adenocarcinoma (PDAC). However, the underlying mechanisms are poorly understood. Revealing the cross-talk network between tumor stroma and pancreatic cancer and developing effective strategies against oxaliplatin resistance are highly desired in the clinic. METHODS: High-throughput sequence was used to screened the key circRNAs transmitted by extracellular vesicles (EVs) from CAFs to pancreatic cancer cells. The associations between EV-packaged circBIRC6 and chemotherapy responsiveness were validated in a cohort of 82 cases of advanced PDAC patients. Then, the effects of EV-packaged circBIRC6 on CAF-induced oxaliplatin resistance were investigated by flow cytometry, colony formation, viability of pancreatic cancer organoids in vitro and by xenograft models in vivo. RNA pulldown, RNA immunoprecipitation, and sites mutation assays were used to reveal the underlying mechanism. RESULTS: We identified a circRNA, circBIRC6, is significantly upregulated in CAF-derived EVs and is positively associated with oxaliplatin-based chemoresistance. In vitro and in vivo functional assays showed that CAF-derived EV-packaged circBIRC6 enhance oxaliplatin resistance of pancreatic cancer cells and organoids via regulating the non-homologous end joining (NHEJ) dependent DNA repair. Mechanistically, circBIRC6 directly binds with XRCC4 and enhanced the interaction of XRCC4 with SUMO1 at the lysine 115 residue, which facilitated XRCC4 chromatin localization. XRCC4K115R mutation dramatically abrogated the EV-packaged circBIRC6 induced effect. Moreover, combination of antisense oligonucleotide inhibitors against circBIRC6 with Olaparib dramatically suppressed chemoresistance in patient-derived xenograft models. CONCLUSIONS: Our study revealed that EV-packaged circBIRC6 confer oxaliplatin resistance in PDAC by mediating SUMOylation of XRCC4, introducing a promising predictive and therapeutic target for PDAC on oxaliplatin resistance.

Thoracoscopic Versus Open Repair for Oesophageal Atresia: A Retrospective Cohort Study of 359 Patients at a Single Center.

BACKGROUND: This study aimed to define the effectiveness of thoracoscopic versus open repair of gross type C oesophageal atresia (EA) based on the experience of a single centre over a decade. METHODS: This retrospective cohort study included patients who were admitted to Hunan Children's Hospital between January, 2010 and December, 2021 and underwent repair surgery for type C EA. RESULTS: A total of 359 patients underwent type C EA repair during the study period, of which 142 were completed via an open approach and 217 were attempted via a thoracoscopic approach (seven converted to open surgery). There were no differences in the demographics or comorbidities between the patients of thoracoscopy and thoracotomy (open repair) groups. The median operating time was 109 [90, 133] min in the thoracoscopic surgery group, which was slightly shorter than that in the open repair group (115 [102, 128] min, p = 0.059). Anastomotic leakage occurred in 41 (18.9%) and 35 (24.6%) infants in the thoracoscopic and open surgery groups, respectively (p = 0.241). Thirteen patients (3.6%) died in the hospital without significant differences in the repair approach. With a median follow-up of 23.7 months, 38 (13.6%) participants had one or more anastomotic strictures requiring dilatation, without significant differences in the repair approach (p = 0.994). CONCLUSIONS: Thoracoscopic repair of congenital EA is safe, and has perioperative and medium-term outcomes similar to those of open surgery. This technique is recommended only in hospitals with experienced teams of endoscopic paediatric surgeons and anaesthesiologists.

Assembly of DNA triangular pyramid frustum for ultrasensitive quantification of exosomal miRNA.

Early screening of biomarkers benefits therapy and prognosis of cancers. MiRNAs encapsulated in tumor-derived exosomes are emerging biomarkers for early diagnosis of cancers. Nevertheless, traditional methods suffer certain drawbacks, which hamper their wide applications. In this contribution, we have developed a convenient electrochemical approach for quantification of exosomal miRNA based on the assembly of DNA triangular pyramid frustum (TPF) and strand displacement amplification. Four single-stranded DNA helps the formation of primary DNA triangle with three thiols for gold electrode immobilization at the bottom and three amino groups on overhangs for the capture of silver nanoparticles. On the other hand, target miRNA induced strand displacement reaction produces abundant specific DNA strands, which help the DNA structural transition from triangle to TPF. Amino groups are thus hidden and the declined silver stripping current can be used for the evaluation of target miRNA concentration. This biosensor exhibits excellent analytical performances and successfully achieves analysis of exosomal miRNAs from cells and clinical serum samples.

HNF1A regulates oxaliplatin resistance in pancreatic cancer by targeting 53BP1.

DNA double­strand break repair is critically involved in oxaliplatin resistance in pancreatic ductal adenocarcinoma (PDAC). Hepatocyte nuclear factor 1 homeobox A (HNF1A) has received increased attention regarding its role in cancer progression. The present study explored the role of HNF1A in oxaliplatin resistance in PDAC. The results revealed that HNF1A expression was negatively associated with oxaliplatin chemoresistance in PDAC tissues and cell lines. HNF1A inhibition promoted the proliferation, colony formation and stemness of PDAC cells, and suppressed their apoptosis. Furthermore, HNF1A inhibition switched nonhomologous end joining to homologous recombination, thereby enhancing genomic stability and oxaliplatin resistance. Mechanistically, HNF1A transcriptionally activates p53­binding protein 1 (53BP1) expression by directly interacting with the 53BP1 promoter region. Upregulation of HNF1A and 53BP1 induced significant inhibition of PDAC growth and oxaliplatin resistance in patient­derived PDAC xenograft models and orthotopic models. In conclusion, the findings of the present study suggested that HNF1A/53BP1 may be a promising PDAC therapeutic target for overcoming oxaliplatin resistance.

Cancer-associated fibroblast-induced lncRNA UPK1A-AS1 confers platinum resistance in pancreatic cancer via efficient double-strand break repair.

The tumor stroma of pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundant and heterogeneous population of cancer-associated fibroblasts (CAFs), which are critically involved in chemoresistance. However, the underlying mechanism of CAFs in chemoresistance is unclear. Here, we show that CAFR, a CAF subset derived from platinum-resistant PDAC patients, assumes an iCAF phenotype and produces more IL8 than CAFS isolated from platinum-sensitive PDAC patients. CAFR-derived IL8 promotes oxaliplatin chemoresistance in PDAC. Based on long noncoding RNA (lncRNA) profiling in tumor cells incubated with CAF-CM, we found that UPK1A-AS1, whose expression is directly induced by IL8/NF-kappa B signaling, functions as a chemoresistance-promoting lncRNA and is critical for active IL8-induced oxaliplatin resistance. Impressively, blocking the activation of UPK1A-AS1 expression increases the oxaliplatin sensitivity of tumor cells in vivo. Mechanistically, UPK1A-AS1 strengthens the interaction between Ku70 and Ku80 to facilitate nonhomologous end joining (NHEJ), thereby enhancing DNA double-strand break (DSB) repair. Clinically, UPK1A-AS1 expression is positively correlated with IL8 expression, a poor chemotherapeutic response and a shorter progression-free survival (PFS) time in advanced PDAC patients. Collectively, our study reveals a lncRNA-mediated mechanism of CAF-derived paracrine IL8-dependent oxaliplatin resistance and highlights UPK1A-AS1 as a potential therapeutic target.

ALKBH5 promotes the progression of infantile hemangioma through regulating the NEAT1/miR-378b/FOSL1 axis.

Our work aims to investigate long non-coding RNA (lncRNA) N6-methyladenosine (m6A) modification and its role in infantile hemangioma (IH). The mRNA and protein expression levels were assessed using quantitative real-time polymerase chain reaction, western blot and immunohistochemistry. Me-RIP assay was performed to evaluate lncRNA NEAT1 m6A levels. Cell proliferation, migration and invasion were evaluated using cell counting kit-8 assay, transwell migration and invasion assay, respectively. Photo-activatable ribonucleoside-enhanced crosslinking and immunoprecipitation assay was conducted to verify the binding relationship between lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) and ALKBH5 (an RNA demethylase). The binding relationship between lncRNA NEAT1, microRNA (miR)-378b and FOS-like antigen 1 (FOSL1) was verified using dual-luciferase reporter gene assay and/or RNA immunoprecipitation assay. ALKBH5, lncRNA NEAT1 and FOLS1 expression was elevated in IH tissues, while miR-378b was downregulated. ALKBH5 knockdown suppressed cell proliferation, migration and invasion of IH cells, while promoting cell apoptosis. ALKBH5 promoted lncRNA NEAT1 expression by reducing the m6A modification of lncRNA NEAT1. In addition, miR-378b was the target of lncRNA NEAT1, and its overexpression reversed the promotion effect of lncRNA NEAT1 overexpression on IH cell tumor-like behaviors. Moreover, FOLS1 was the target of miR-378b, and its overexpression reversed the inhibitory effect of miR-378b overexpression on IH cell tumor-like behaviors in vitro. ALKBH5 might have great potential as therapeutic target for IH, since ALKBH5 silencing suppressed IH progression by regulation of the NEAT1/miR-378b/FOSL1 axis.

circCUL2 induces an inflammatory CAF phenotype in pancreatic ductal adenocarcinoma via the activation of the MyD88-dependent NF-κB signaling pathway.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is characterized by clusters of cancer cells surrounded by a dense desmoplastic stroma. However, little is known about stromal cell heterogeneity in the pancreatic tumor microenvironment. METHODS: We conducted circRNA profiling in primary fibroblasts by high-throughput sequencing and detected circCUL2 levels in PDAC tissues by qRT-PCR. We subsequently investigated the effect of circCUL2 on inflammatory cancer-associated fibroblast (iCAF) activation, heterogeneity and protumor activity by ELISA, flow cytometry, colony formation and transwell assays in vitro and by xenograft models in vivo. The regulatory effect of circCUL2 on miR-203a-3p/MyD88/IL6 was examined by RNA pulldown, FISH, and luciferase reporter assays. RESULTS: We identified that circCUL2 was specifically expressed in cancer-associated fibroblasts (CAFs) but not in cancer cells. Moreover, the enrichment of circCUL2 in tumor tissues was significantly correlated with the poor prognosis of PDAC patients. Upregulation of circCUL2 expression in normal fibroblasts (NFs) induced the iCAF phenotype, and then iCAFs promoted PDAC progression through IL6 secretion in vitro. Furthermore, circCUL2-transduced NFs promoted tumorigenesis and metastasis of PDAC cells in vivo, which was blocked by an anti-IL6 antibody. Mechanistically, circCUL2 functioned as a ceRNA and modulated the miR-203a-3p/MyD88/NF-κB/IL6 axis, thereby further activating the STAT3 signaling pathway in pancreatic cancer cells to induce PDAC progression. CONCLUSIONS: We showed that the circCUL2/miR-203a-5p/MyD88/NF-κB/IL6 axis contributes to the induction of iCAFs and established a distinct fibroblast niche for PDAC progression, which could help the development of strategies that selectively target tumor-promoting CAFs in PDAC.

circFARP1 enables cancer-associated fibroblasts to promote gemcitabine resistance in pancreatic cancer via the LIF/STAT3 axis.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are critically involved in gemcitabine (GEM) resistance in pancreatic ductal adenocarcinoma (PDAC). However, the underlying mechanism by which CAFs promote chemotherapy resistance remains unexplored. Here, we explored the role of circRNAs in CAF-induced GEM resistance in PDAC. METHODS: circRNA sequencing and quantitative real-time PCR (qRT-PCR) were utilized to screen CAF-specific circRNAs. The effects of CAF circFARP1 expression on GEM resistance in tumor cells were assessed in vitro and in vivo. RNA-seq, RNA pulldown, RNA immunoprecipitation, and luciferase reporter assays were used to screen the downstream target and underlying mechanism of circFARP1. RESULTS: circFARP1 (hsa_circ_0002557), a CAF-specific circRNA, was positively correlated with GEM chemoresistance and poor survival in an advanced PDAC cohort. Silencing or overexpressing circFARP1 in CAFs altered the ability of CAFs to induce tumor cell stemness and GEM resistance via leukemia inhibitory factor (LIF). Mechanistically, we found that circFARP1 directly binds with caveolin 1 (CAV1) and blocks the interaction of CAV1 and the E3 ubiquitin-protein ligase zinc and ring finger 1 (ZNRF1) to inhibit CAV1 degradation, which enhances LIF secretion. In addition, circFARP1 upregulated LIF expression by sponging miR-660-3p. Moreover, high circFARP1 levels were positively correlated with elevated serum LIF levels in PDAC and poor patient survival. Decreasing circFARP1 levels and neutralizing LIF significantly suppressed PDAC growth and GEM resistance in patient-derived xenograft models. CONCLUSIONS: The circFARP1/CAV1/miR-660-3p/LIF axis is critical for CAF-induced GEM resistance in PDAC. Hence, circFARP1 may be a potential therapeutic target for PDAC.

Circ-ITCH overexpression promoted cell proliferation and migration in Hirschsprung disease through miR-146b-5p/RET axis.

BACKGROUND: Hirschsprung disease (HSCR) is a congenital intestinal disease caused by the abnormal proliferation and migration of enteric nerve cells (ENCC). Research suggested critical roles for circular RNA (circRNA) itchy E3 ubiquitin protein ligase (ITCH) in gastrointestinal malignancies progression. However, the function of circ-ITCH in HSCR remains poorly defined. METHODS: The related genes expression in 30 HSCR patients and 30 controls without HSCR were detected using qRT-PCR. Cell proliferation was assessed by CCK-8 assay and EdU assay. Cell migration was detected with wound-healing assay and transwell assay. The interactions among circ-ITCH, miR-146b-5p, and RET were confirmed by Dual luciferase reporter assay. RESULTS: Circ-ITCH and RET expressions were downregulated in HSCR patients and cells, while the miR-146b-5p expression was upregulated. Circ-ITCH overexpression facilitated cell proliferation, migration, and activated MAPK pathway, which were reversed by circRNA-ITCH knockdown. Circ-ITCH negatively regulated miR-146b-5p expression. MiR-146b-5p overexpression abolished the promoting effects of circ-ITCH overexpression on cell proliferation and migration. MiR-146b-5p inhibited RET expression. RET overexpression eliminated the inhibitory effects of miR-146b-5p overexpression on cell proliferation and migration. CONCLUSION: Circ-ITCH overexpression facilitated cell proliferation and migration in HSCR by regulating miR-146b-5p/RET/MAPK axis. IMPACT: The expressions of Circ-ITCH and RET were markedly reduced in HSCR, while miR-146b-5p expression was increased in HSCR. Circ-ITCH overexpression enhanced the proliferative and migratory abilities of SH-SY5Y and 293T cells. Circ-ITCH negatively regulated miR-146b-5p expression.

[Effect of enhancer of zeste homolog 2 on the expression of glial cell line-derived neurotrophic factor family receptor α-1 in the colon tissue of children with Hirschsprung's disease].

OBJECTIVE: To study the expression levels of glial cell line-derived neurotrophic factor family receptor α-1 (GFRα1) and enhancer of zeste homolog 2 (EZH2) in the intestinal tissue of children with Hirschsprung's disease (HSCR), as well as the role of EZH2 in the regulation of GFRα1 gene expression and the pathogenesis of HSCR. METHODS: The samples of colon tissue with spasm from 24 children with HSCR after radical treatment of HSCR were selected as the experimental group, and the samples of necrotized colon tissue from 18 children with neonatal necrotizing enterocolitis after surgical resection were selected as the control group. Real-time PCR and Western blot were used to measure the expression levels of GFRα1 and EZH2 in colon tissue in both groups. Human neuroblastoma SH-SY5Y cells were divided into an EZH2 over-expression group and a negative control group. The cells in the EZH2 over-expression group were transfected with pCMV6-EZH2 plasmid, and those in the negative control group were transfected with pCMV6 plasmid. The expression levels of EZH2 and GFRα1 were measured after transfection. RESULTS: Compared with the control group, the experimental group had significant reductions in the mRNA and protein expression levels of GFRα1 and EZH2 in colon tissue (P<0.05), and the protein expression of EZH2 was positively correlated with that of GFRα1 (r=0.606, P=0.002). Compared with the negative control group, the EZH2 over-expression group had significant increases in the expression levels of EZH2 and GFRα1 after SH-SY5Y cells were transfected with EZH2 over-expression plasmid (P<0.05). CONCLUSIONS: Low expression of EZH2 in the colon tissue of children with HSCR may be one of the causes of inadequate expression of GFRα1 and onset of HSCR.

Suppression of experimental abdominal aortic aneurysm in a rat model by the phosphodiesterase 3 inhibitor cilostazol.

BACKGROUND: The present experiments sought to determine whether cilostazol, a selective inhibitor of cyclic adenosine monophosphate (cAMP) phosphodiesterase 3 (PDE3), suppressed elastase-induced abdominal aortic aneurysm (AAA) development in a rat model. METHODS: Male Sprague-Dawley rats (n = 16/each group) were randomly distributed into three groups: sham-, saline-, and cilostazol-. Rats of saline and cilostazol groups underwent intra-aortic elastase perfusion to induce AAAs, while rats of sham-group were perfused with saline. Rats of cilostazol-group received cilostazol treatment (100 mgkg(-1)d(-1)) for the entire experimental period. The areas of the lumen of the aortas at the segment with maximum diameter were measured preperfusion and on d 7, 14 after perfusion. Systolic blood pressure was measured by tail-cuff technique. Aortic tissue samples were harvested on d 14 after intra-aortic perfusion and evaluated by reverse transcription-polymerase chain reaction and Western blot for matrix metalloproteinase-2, 9 (MMP-2, 9), by immunohistochemistry for nuclear factor kappa B (NF-κB), and by Gomori aldehyde fuchsin for elastin. Activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and level of reactive oxygen species (ROS) in these samples were also measured. RESULTS: On d 14, rats of saline-group had significantly increased aortic sizes compared with sham-group (P < 0.01), while, cilostazol treatment significantly reduced this increase (cilostazol- versus saline-, P < 0.01) without affecting blood pressure (P > 0.05). The expression of both MMP-2 and MMP-9 and the destruction of elastic fibers in aortic tissues were significantly decreased by cilostazol treatment (P < 0.05), probably through the suppression of NF-κB activation (P < 0.01). Consistently, cilostazol significantly inhibited NADPH oxidase activity (P < 0.01), accompanied by a reduced level of ROS (P < 0.01). CONCLUSION: Cilostazol retards experimental AAAs development independently of blood pressure reduction possibly by inhibiting proteolysis, inflammation, and oxidative stress. Selective PDE3 inhibition may offer an additional method to pharmacologically inhibit AAAs.