METTL3/IGF2BP2 axis affects the progression of colorectal cancer by regulating m6A modification of STAG3.

Colorectal cancer (CRC) is among the commonest malignant tumors of humans. Existing evidence has linked the poor prognosis of CRC with high expression of stromal antigen 3 (STAG3), but, the exact biological effect of STAG3 in CRC is still unclear. The aim of this research is to reveal the biological function and molecular mechanism of STAG3 in CRC. To investigate the differential expression of STAG3 in CRC tissues and cell lines compared to normal colon tissues and cell lines, Western blot (WB) and quantitative real-time PCR (qRT-PCR) techniques were utilized. STAG3 N6-methyladenosine (m6A) modification level were identified using m6A RNA immunoprecipitation (MeRIP). Additionally, the functional roles of methyltransferase-like protein 3 (METTL3) and insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) in CRC were explored by manipulating their levels via knockdown or overexpression. Cell proliferation was evaluated through Cell Counting Kit 8 (CCK-8) and clone formation experiments, while cell migration was assessed through wound healing experiments. Furthermore, cell apoptosis was detected using flow cytometry, and the protein expressions associated with proliferation and apoptosis were detected using WB. To identify the specific binding of target genes, RIP and pull-down assays were employed. Finally, the biological function of STAG3 in vivo was investigated through a xenotransplantation mouse tumor model. In CRC tissues and cell lines, STAG3 was up-regulated and accompanied by m6A methylation. Additionally, the expression of METTL3 was found to be upregulated in CRC tissues. Knocking down METTL3 resulted in a decrease in both the m6A level and protein expression of STAG3, inhibited cell proliferation and migration while promoting apoptosis, which were restored through STAG3 overexpression. Furthermore, online prediction indicated the interaction between STAG3 mRNA and IGF2BP2 protein, which was further verified by RIP experiments. IGF2BP2 downregulation led to decreased STAG3 protein expression, cell proliferation, and migration, but increased apoptosis. However, these impacts were reversed by STAG3 overexpression. Finally, subcutaneous tumor experiments conducted in nude mice also confirmed that METTL3 regulated CRC progression through STAG3 in vivo. The METTL3/IGF2BP2/STAG3 axis affects CRC progression in an m6A modification-dependent manner. This may guide targeted therapy in CRC patients.

Intestinal intussusception caused by intestinal duplication and ectopic pancreas: A case report and review of literature.

BACKGROUND: Intestinal intussusception caused by intestinal duplication and ectopic pancreas is extremely rare in the clinic and has not been reported previously. CASE SUMMARY: A 29-year-old man was admitted to the hospital for chronic abdominal pain and bloating. The preoperative diagnosis was intestinal obstruction and intussusception. Then, laparotomy, partial small intestinal resection and extraintestinal decompression were performed. Postoperative pathology confirmed intestinal duplication and ectopic pancreas. After surgery, the patient recovered well with no complications. No recurrence was observed after more than 5 mo of follow-up. CONCLUSION: We report a new case of a young male with intussusception caused by intestinal duplication and ectopic pancreas. Surgery is the main treatment for these conditions. This study aimed to raise awareness and provide information to improve the clinical management of this rare yet serious condition.

Prognostic Lnc-S100B-2 Affects Cell Apoptosis and Microenvironment of Colorectal Cancer through MLLT10 Signaling.

Long noncoding RNA (LncRNA) is closely associated with the development of colorectal cancer (CRC). The chip data and clinical information of GSE104364 and GSE151021 were downloaded by GEOquery. Limma and Kaplan-Meier analysis were performed. Lnc-S100B-2 was obtained, and high expression of Lnc-S100B-2 was predicted to be associated with a lower survival rate. Online software was adopted to predict downstream regulatory genes, and miR-331-3p and Mixed Lineage Leukemia Translocated to 10 (MLLT10) were screened and verified. After silencing Lnc-S100B-2 and MLLT10, the proliferative activity of CRC cells decreased, and the apoptosis rate increased. At the gene and protein levels, the expressions of PCNA, Ki67, and Bcl-2 were decreased in the sh-Lnc-S100B-2 group, sh-MLLT10 group, and sh-Lnc-S100B-2 + sh-MLLT10 group, while the expressions of cleaved caspase 3, caspase 9, and Bax were increased. In vivo, the volume and mass of the tumor decreased in the sh-Lnc-S100B-2 + sh-MLLT10 group. Proliferation and apoptosis-related index (PCNA, Ki67, cleaved caspase 3, caspase 9, Bax, and Bcl-2) expression level was also altered. Meanwhile, the infiltration of immune cells (CD3 (-), CD16 (+), and CD11b (+) cells) decreased. The expressions of epithelial-mesenchymal transformation (EMT) related indicators (E-cadherin, N-cadherin, Vimentin, ß-catenin, Snail, and Slug) were changed. E-cadherin and ß-catenin were increased in the sh-Lnc-S100B-2 + sh-MLLT10 group, while N-cadherin, vimentin, snail, and slug were decreased. In conclusion, our study found that the expression of Lnc-S100B-2 was dysregulated in CRC. Lnc-S100B-2 could affect cell apoptosis and the microenvironment of CRC through regulating MLLT10.

Exosomal transfer of circular RNA FBXW7 ameliorates the chemoresistance to oxaliplatin in colorectal cancer by sponging miR-18b-5p.

Circular RNA F-box and WD repeat domain containing 7 (circ-FBXW7) has been revealed to be involved in the tumorigenesis of colorectal cancer (CRC). Exosomes are critical mediators of intercellular communication. However, the role of exosomal circ-FBXW7 in the CRC oxaliplatin resistance remains unknown. Cell viability, apoptosis, motility, and drug efflux were measured by the cell counting kit-8 assay, flow cytometry, transwell assay, and atomic absorption spectrophotometry, respectively. The expression of circ-FBXW7 and microRNA (miR)-18b-5p was detected using the quantitative real-time polymerase chain reaction. Western blot was used to determine multidrug resistance protein 1 (MRP1), myeloid cell leukemia-1 (MCL-1), CD9, CD63, Caspase3, E-cadherin, and N-cadherin. Exosomes were isolated and captured using the ultracentrifugation method and transmission electron microscopy. The interaction between circ-FBXW7 and miR-18b-5p was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation assay. In vivo experiments were conducted using the murine xenograft model. Our results showed that circ-FBXW7 was decreased in oxaliplatin-resistant CRC patients and cells. circ-FBXW7 was secreted by circ-FBXW7-transfected FHC cells and could be transferred to resistant CRC cells through the exosome secretion. Subsequently, in vitro and in vivo studies demonstrated exosomal circ-FBXW7 led resistant cells sensitive to oxaliplatin, increased the oxaliplatin-induced apoptosis, inhibited oxaliplatin-induced epithelial-mesenchymal transition, and suppressed oxaliplatin efflux. miR-18b-5p was increased in oxaliplatin-resistant CRC patients and cells and was confirmed to be a target of circ-FBXW7. Immediately, the rescue assay showed exosome-mediated transfer of circ-FBXW7 enhanced oxaliplatin sensitivity by binding to miR-18b-5p in vitro and in vivo. To conclude, the circ-FBXW7 delivery by exosomes could ameliorate chemoresistance to oxaliplatin in CRC by directly binding to miR-128-3p, suggesting a promising therapeutic strategy for oxaliplatin-resistant CRC patients.

Bispecific and human disease-related anti-keratin rabbit monoclonal antibodies.

Rabbit antibodies may have favorable properties compared to mouse antibodies, including high affinities and better antigen recognition. We used a biochemical and reverse immunologic approach to generate and characterize rabbit anti-phospho-keratin and anti-keratin monoclonal antibodies (MAb). Human keratins 8 and 18 (K8/K18) were used as immunogens after isolation from cells pretreated with okadaic acid or pervanadate to promote Ser/Thr or Tyr hyperphosphorylation, respectively. Selected rabbit MAb were tested by immunofluorescence staining, immunoprecipitation, and 2-dimensional gels. Keratin phospho and non-phospho-mutants were used for detailed characterization of two unique antibodies. One antibody recognizes a K8 G61-containing epitope, an important epitope given that K8 G61C is a frequent mutation in human liver diseases. This antibody binds K8 that is not phosphorylated on S73, but its binding is ablated by G61 but not S73 mutation. The second antibody is bispecific in that it simultaneously recognizes two epitopes: one phospho (K8 pS431) conformation-independent and one non-phospho conformation-dependent, with both epitopes residing in the K8 tail domain. Therefore, a reverse immunologic and biochemical approach is a viable tool for generating versatile rabbit MAb for a variety of cell biologic applications including the potential identification of physiologic phosphorylation sites.

Elevation of seed alpha-tocopherol levels using plant-based transcription factors targeted to an endogenous locus.

Synthetic zinc finger transcription factors (ZFP-TFs) were designed to upregulate the expression of the endogenous Arabidopsis gamma-tocopherol methyltransferase (GMT) gene. This gene encodes the enzyme responsible for the conversion of gamma-tocopherol to alpha-tocopherol, the tocopherol species with the highest vitamin E activity. Five three-finger zinc finger protein (ZFP) DNA binding domains were constructed and proven to bind tightly to 9 bp DNA sequences located in either the promoter or coding region of the GMT gene. When these ZFPs were fused to a nuclear localization signal and the maize C1 activation domain, four of the five resulting ZFP-TFs were able to upregulate the expression of the GMT gene in leaf protoplast transient assays. Seed-specific expression of these ZFP-TFs in transgenic Arabidopsis produced several lines with a heritable elevation in seed alpha-tocopherol. These results demonstrate that engineered ZFP-TFs comprised of plant-derived elements are capable of modulating the expression of endogenous genes in plants.