IGF2BP2 promotes colorectal cancer progression by upregulating the expression of TFRC and enhancing iron metabolism.

BACKGROUND: Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive system, ranking third for morbidity and mortality worldwide. At present, no effective control method is available for this cancer type. In tumor cells, especially iron metabolization, is necessary for its growth and proliferation. High levels of iron are an important feature to maintain tumor growth; however, the overall mechanism remains unclear. METHODS: We used western blotting, immunohistochemistry (IHC) and real-time quantitative PCR to analyze the expression of IGF2BP2 in cell lines and tissues. Further, RNA-sequencing, RNA immunoprecipitation and methylated RNA immunoprecipitation experiments explored the specific binding of target genes. Moreover, the RNA stability assay was performed to determine the half-life of genes downstream of IGF2BP2. In addition, the Cell Counting Kit-8, colony formation assay, 5-ethynyl-2'-deoxyuridine assay and flow cytometry were used to evaluate the effects of IGF2BP2 on proliferation and iron metabolism. Lastly, the role of IGF2BP2 in promoting CRC growth was demonstrated in animal models. RESULTS: We observed that IGF2BP2 is associated with iron homeostasis and that TFRC is a downstream target of IGF2BP2. Further, overexpression of TFRC can rescue the growth of IGF2BP2-knockdown CRC cells. Mechanistically, we determined that IGF2BP2 regulates TFRC methylation via METTL4, thereby regulating iron metabolism and promoting CRC growth. Furthermore, using animal models, we observed that IGF2BP2 promotes CRC growth. CONCLUSION: IGF2BP2 regulates TFRC mRNA methylation via METTL4, thereby regulating iron metabolism and promoting CRC growth. Our study highlights the key roles of IGF2BP2 in CRC carcinogenesis and the iron transport pathways.

Transcriptome Based System Biology Exploration Reveals Homogeneous Tumorigenicity of Alimentary Tract Malignancy.

Malignancies of alimentary tract include esophageal carcinoma (ESCA), stomach adenocarcinoma (STAD), colon adenocarcinoma (COAD), and rectum adenocarcinoma (READ). Despite of their similarities in cancer development and progression, there are numerous researches concentrating on single tumor but relatively little on their common mechanisms. Our study explored the transcriptomic data of digestive tract cancers from The Cancer Genome Atlas database, yielding their common differentially expressed genes including 1,700 mRNAs, 29 miRNAs, and 362 long non-coding RNAs (lncRNAs). There were 12 mRNAs, 5 miRNAs, and 16 lncRNAs in the core competitive endogenous RNAs network by RNA-RNA interactions, highlighting the prognostic nodes of SERPINE1, hsa-mir-145, and SNHG1. In addition, the weighted gene co-expression network analysis (WGCNA) illustrated 20 gene modules associated with clinical traits. By taking intersections of modules related to the same trait, we got 67 common genes shared by ESCA and READ and screened 5 hub genes, including ADCY6, CXCL3, NPBWR1, TAS2R38, and PTGDR2. In conclusion, the present study found that SERPINE1/has-mir-145/SNHG1 axis acted as promising targets and the hub genes reasoned the similarity between ESCA and READ, which revealed the homogeneous tumorigenicity of digestive tract cancers at the transcriptome level and led to further comprehension and therapeutics for digestive tract cancers.

POMOF/SWNT Nanocomposites with Prominent Peroxidase-Mimicking Activity for l-Cysteine "On-Off Switch" Colorimetric Biosensing.

In order to explore novel colorimetric biosensors with high sensibility and selectivity, two new Keggin polyoxometalates (POMs)-based Cu-trz (1,2,4-triazole) metal-organic frameworks (MOFs) with suitable specific surface areas and multiple active sites were favorably fabricated; then single-walled carbon nanotubes (SWNTs) were merged with new POMOFs to construct POMOF/SWNT nanocomposites. Herein, POMOF/SWNT nanocomposites as peroxidase mimics were explored for the first time, and the peroxidase-mimicking activity of the prepared POMOF/SWNT nanocomposites is heavily dependent on the mass ratio of POMOFs and SWNTs, in which the maximum activity is achieved at the mass ratio of 2.5:1 (named PMNT-2). More importantly, PMNT-2 exhibits the lowest limit of detection (0.103 µM) among all reported materials to date and the assumable selectivity toward l-cysteine (l-Cys) detection. With these findings, a convenient, sensitive, and effective "on-off switch" colorimetric platform for l-Cys detection has been successfully developed, providing a promising prospect in the biosensors and clinical diagnosis fields.

[ShRNA-mediated silencing of MDM2 inhibits growth of HepG2 hepatocellular carcinoma cells xenografted in nude mice].

OBJECTIVE: To construct a short hairpin (sh)RNA targeting the gene encoding the MDM2 oncoprotein in order to investigate its role in human hepatocellular carcinoma (HCC) and its potential for use as a gene therapy strategy to inhibit HCC growth in vivo. METHODS: Small interfering (si)RNAs were designed targeting the MDM2 gene (siMDM2-1 and siMDM2-2) and unrelated sequences (negative control) and cloned into the expression plasmid pGCSilencer-U6-neo-GFP. A HCC mouse model was established by subcutaneous inoculation of HepG2 cells (2 x 10(6) in 0.2 ml) into 20 nude mice. The inoculated mice were divided into four equal groups for tumor-localized injections of saline, negative control siRNA plasmid, siMDM2-1 plasmid, and siMDM2-2 plasmid. Tumor growth was observed daily (by caliper measurement) for one month, when mice were sacrificed by cervical dislocation. The tumor mass was resected for analysis of tumor inhibition rate (% = [(average tumor weight of control group - average tumor weight of treatment group) / average tumor weight of control group x 100]) and effects on MDM2 and p53 mRNA and protein expression (by reverse transcription- PCR and western blotting, both normalized to beta-actin). Significance of between-group differences was assessed by one-way ANOVA or LSD test; pairwise comparisons were made by the Chi-squared test. RESULTS: siMDM2-1 and siMDM2-2 suppressed the xenografted tumor growth remarkably (60.6% and 54.6% inhibition rates, respectively), significantly reduced the expression ofMDM2 gene (62.8% and 61.6%) and protein (60.7% and 59.5%), and significantly increased p53 gene (47.1% and 45.6%) and protein (45.9% and 44.3%) (all, P < 0.05). CONCLUSION: shRNA-mediated silencing of the MDM2 gene effectively inhibits HCC tumorigenesis of subcutaneously xenografted HepG2 cells in nude mice, and the mechanism may involve p53.

[Prokaryotic expression, purification and identification of NY-ESO-1/GST fusion protein in E.coli].

AIM: To construct an expression plasmid for NY-ESO-1 gene and identify the expression of recombinant protein NY-ESO-1/GST in E.coli. METHODS: NY-ESO-1 segment was amplified from the testis cDNA library by RT-PCR and cloned into the prokaryotic expression vector pGEX4T-1 downstream tagged by GST to construct the expression plasmid pGEX-4T1-NY-ESO-1. The recombinant vector was transformed to BL21 (DE3) and NY-ESO-1/GST fusion protein was induced expression by IPTG. The protein was purified by urea elution and identified by SDS-PAGE and Western blotting. RESULTS: The NY-ESO-1 segment was successfully amplified and its sequence was identical with that published in GenBank. The BL21 (DE3) pLysS containing the pGEX-4T1-NY-ESO-1 expressed a M(r); 44 000 fusion protein under the induction of IPTG. The purity of the protein was 90%. Western blotting proved that NY-ESO-1/GST had a specific reaction with anti-GST mAb. CONCLUSION: The prokaryotic expression vector of NY-ESO-1 has been constructed and the fusion protein NY-ESO-1/GST of high purity is successfully expressed.