Mechanism and Application of Surface-Charged Ferrite Nanozyme-Based Biosensor toward Colorimetric Detection of l-Cysteine.

Peroxidase-like nanozymes with robust catalytic capacity and detection specificity have been proposed as substitutes to natural peroxidases in biochemical sensing. However, the catalytic activity enhancement, detection mechanism, and application of nanozyme-based biosensors toward l-cysteine (l-Cys) detection still remain significant challenges. In this work, a doped ferrite nanozyme with well-defined structure and surface charges is fabricated by a two-step method of continuous flow coprecipitation and high-temperature annealing. The resulted ferrite nanozyme possesses an average size of 54.5 nm and a zeta-potential of 6.45 mV. A high-performance biosensor is manufactured based on the peroxidase-like catalytic feature of the doped ferrite. The ferrite nanozyme can oxidize the 3,3',5,5'-tetramethylbenzidine (TMB) with the assistance of H2O2 because of the instinctive capacity to decompose H2O2 into ·OH. The Michaelis-Menten constants (0.0911 mM for TMB, 0.140 mM for H2O2) of the ferrite nanozyme are significantly smaller than those of horseradish peroxidase. A reliable colorimetric method is established to selectively analyze l-Cys via a facile mixing-and-detecting methodology. The detection limit and linear range are 0.119 µM and 0.2-20 µM, respectively. Taking the merits of the ferrite nanozyme-based biosensors, the l-Cys level in the human serum can be qualitatively detected. It can be anticipated that the surface-charged ferrite nanozyme shows great application prospects in the fields of bioanalytical chemistry and point-of-care testing.

Exosomal lncRNA SNHG10 derived from colorectal cancer cells suppresses natural killer cell cytotoxicity by upregulating INHBC.

BACKGROUND: Exosome-mediated crosstalk between cancer cells and immune cells contributes to tumor growth. In this study, we investigated the mechanism underlying the exosome-mediated immune escape of colorectal cancer (CRC) cells from natural killer (NK) cells via the transfer of long noncoding RNAs (lncRNAs). METHODS: An epithelial-mesenchymal transition (EMT) model of SW480 cells was established by transforming growth factor beta (TGF-ß), followed by the assessment of the effect of EMT-derived exosomes (EMT-exo) on the functions of NK cells. RNA sequencing was performed to identify exosomal lncRNAs and target genes. The function of exosomal lncRNAs in tumor growth was further verified in vivo. RESULTS: EMT-exo suppressed the proliferation, cytotoxicity, IFN-γ production, and perforin-1 and granzyme B secretion of NK cells. RNA sequencing revealed that SNHG10 expression was upregulated in EMT-exo compared with that in non-EMT-exo. Moreover, SNHG10 expression was upregulated in tumor tissues in CRC, which was associated with poor prognosis. Overexpression of SNHG10 in exosomes (oe-lnc-SNHG10 exo) significantly suppressed the viability and cytotoxicity of NK cells. Transcriptome sequencing of NK cells revealed that the expression levels of 114 genes were upregulated in the oe-lnc-SNHG10 exo group, including inhibin subunit beta C (INHBC), which was involved in the TGF-ß signaling pathway. Si-INHBC treatment abrogated the effect of oe-lnc-SNHG10 exo on NK cells. oe-lnc-SNHG10 exo induced tumor growth and upregulated INHBC expression in mice and downregulated the expression of perforin, granzyme B, and NK1.1 in tumor tissues. CONCLUSIONS: The CRC cell-derived exosomal lncRNA SNHG10 suppresses the function of NK cells by upregulating INHBC expression. This study provides evidence that exosomal lncRNAs contribute to immune escape by inducing NK cell inhibition and proposes a potential treatment strategy for CRC.

Long Noncoding RNA GAPLINC Promotes Cells Migration and Invasion in Colorectal Cancer Cell by Regulating miR-34a/c-MET Signal Pathway.

BACKGROUND: Gastric adenocarcinoma predictive long intergenic noncoding RNA (GAPLINC) has been detected in colorectal cancer (CRC) cells and reportedly performs many functions related to tumor proliferation and metastasis. Aim The present study aimed to comprehensively explore the biological functions of GAPLINC and their underlying mechanism in CRC cell. METHODS: The human cancer LncRNA PCR array was used to detect the differentially expressed long noncoding RNAs in human CRC samples. Real-time PCR, dual-luciferase assay, RNA pull-down assay, Transwell assay, and western blot analysis were performed to explore the molecular mechanism underlying GAPLINC functions related to migration and invasion of a human CRC cell line (HCT116). RESULTS: Compared to the non-cancerous tissues, GAPLINC expression was obviously increased in CRC tissues. In HCT116, silencing of GAPLINC weakened cell migration and invasion, while overexpression of GAPLINC significantly promoted cell migration and invasion. Through dual-luciferase, RNA pull-down, and Transwell assays, we verified that miR-34a was the downstream molecule of GAPLINC and that miR-34a negatively regulated the migration and invasion of HCT116 cell. Furthermore, we found that GAPLINC positively regulated the miR-34a target gene c-MET in CRC tissues. CONCLUSIONS: Our findings revealed that GAPLINC was up-regulated in CRC tissues and was involved in the migration and invasion of CRC cells by regulating miR-34a/c-MET signaling pathway.

Integrated Bioinformatics Analysis to Identify Abnormal Methylated Differentially Expressed Genes for Predicting Prognosis of Human Colon Cancer.

OBJECTIVE: To identify the value of key differentially expressed genes (DEGs) regulated by differentially methylated regions (DMRs) in predicting the prognosis of human colon cancer. MATERIALS AND METHODS: RNA sequencing data and DNA methylation data of 455 colon adenocarcinoma (COAD) cases and 41 normal controls were downloaded from The Cancer Genome Atlas (TCGA). Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed by the DAVID database. To identify the hub genes regulated by methylation, univariate Cox and multivariate Cox regression analyses were carried out. A nomogram based on the risk score was built to identify the power of the hub genes to predict prognosis in patients with colon cancer. RESULTS: A total of 133 DEGs regulated by DMRs were identified through analyzing RNA sequencing data and DNA methylation data from TCGA. GO functional enrichment and KEGG pathway enrichment analysis showed the genes involved in the initiation and progression of colon cancer. Univariate Cox regression analysis and multivariate Cox regression analysis focused on the seven hub genes (CDH4, CR2, KRT85, LGI4, NPAS4, RUVBL1 and SP140) associated with overall survival, the expression of which negatively correlated with their methylation level. The risk score and nomogram model showed that the hub genes served as potential biomarkers for the prognosis prediction of patients with colon cancer. CONCLUSION: Our findings suggest that the DEGs regulated by DMRs are involved in the carcinogenesis and development of colon cancer, and the aberrantly methylated DEGs associated with overall survival of patients may be potential diagnostic and therapeutic targets for colon cancer.

Characterization of rhodamine 123 low staining cells and their dynamic changes during the injured-repaired progress induced by 5-FU.

OBJECTIVE: To investigate the characterization of intestinal epithelial stem cells stained by Rhodamine 123 (Rho) and analyze the dynamic changes of intestinal epithelial stem cells during the injured-repaired progress induced by 5-FU. METHODS: Mucosal cells were obtained from adult C57BL/6J mice. The Rho stained cells were sorted using FACS. The mouse model of intestinal mucosal injured-repaired was established by injecting 5-FU and sacrificed at different time post-injection, and the middle intestines were used for detecting the percentage of Rho low staining cell fraction by FACS and detecting the expression of the intestinal epithelial stem cells marker-musashi-1 (msi-1) by RT-PCR and immunohistochemistry. RESULTS: The Rho stained intestinal mucosal cells were divided into three fractions: Rho low staining fraction (12.35%), Rho middle staining fraction (35.5%) and Rho strong staining fraction (50.5%). The cells in Rho low staining fraction expressed rich msi-1 and most of which were in the G0/G1 phase of cell cycle. After treatment of 5-FU, the intestinal mucous were damaged, although the number of msi-1 positive cells has a little decrease, there was no statistical difference among the mice at different time after injection (P>0.05). However, the percentage of msi-1 positive cells increased significantly after injection (P<0.01), and the percentage of msi-1 positive cells decreased gradually during the repaired procedure of the intestinal mucous. There was significant positive correction between the percentage of msi-1 positive cells and the percentage of Rho low staining cell fraction (r=0.867, p<0.01) after 5-FU injection. CONCLUSIONS: The Rho low staining cell fraction from intestinal mucous contained rich intestinal epithelial stem cells, and the intestinal epithelial stem cell which expressed msi-1 played a key role in repairing the damage of intestinal mucous induced by 5-FU.

[Anti-microRNA-221 enhances radiosensitivity of colorectal carcinoma cells by up-regulating PTEN].

OBJECTIVE: To investigate the effect of knocking-down microRNA-221 (miR-221) expression on the radiosensitivity of human colorectal carcinoma cells. METHODS: Human colorectal carcinoma-derived cell line Caco2 was transfected with miR-221 antisense oligonucleotides (anti-miR-221) via Lipofectamine 2000. Real-time quantitative PCR was performed to detect the expression of miR-221 and PTEN mRNA in Caco2 cells. The changes in the protein expression of PTEN in the transfected cells were detected by Western blotting. The cell death after transfection and irradiation was detected by flow cytometry. RESULTS: Transfection with anti-miR-221 caused a significant reduction in miR-221 expression (P<0.05) and up-regulated PTEN protein expression (P<0.05) in Caco2 cells. The percentage of cell death was significantly increased in anti-miR-221 group and anti-miR-221 with irradiation group (P<0.01). Anti-miR-221 significantly enhanced the radiosensitivity of Caco2 cells, which was partially reversed by PTEN-siRNA. CONCLUSION: Anti-miR-221 can enhance the radiosensitivity of colorectal carcinoma cells by up-regulating the expression of PTEN.

[Clinical study of different bowel preparations on changes of gut flora in patients undergoing colorectal resection].

OBJECTIVE: To compare the impact of traditional and fast bowel preparation on the changes of gut flora in the patients following colorectal resection. METHODS: Sixty patients undergoing colorectal resection from March 2010 to March 2011 in the Nanfang Hospital were randomly divided into the control group(n=27, 3 days of bowel preparation) and the experimental group(n=33, 1 day of bowel preparation). Fresh feces were collected before bowel preparation and on the first defecation after surgery. The postoperative changes in gut flora and septic complications were observed. RESULTS: Gut flora disturbance was found in both groups. The postoperative population of Bifidobacterium and Lactobacillus decreased significantly(P<0.05), and the decrease was more significant in the experimental group compared to the control group(P<0.05), while E.coli and Staphylococcus were much higher than the preoperative level(P<0.05), which was more significant in the control group. The incidence of postoperative infection was 9.1%(3/33) in the experimental group, which was significantly lower than 29.6%(8/27) in the control group(P<0.05). CONCLUSION: Fast bowel preparation is effective in reducing gut flora disturbance and the incidence of postoperative infection.