Comprehensive prognostic and immune analysis of sterol O-acyltransferase 1 in patients with hepatocellular carcinoma.

BACKGROUND: Sterol O-acyltransferase 1 (SOAT1) is an important target in the diagnosis and treatment of liver cancer. However, the prognostic value of SOAT1 in patients with hepatocellular carcinoma (HCC) is still not clear. AIM: To investigate the correlation of SOAT1 expression with HCC, using RNA-seq and gene expression data of The Cancer Genome Atlas (TCGA)-liver hepatocellular carcinoma (LIHC) and pan-cancer. METHODS: The correlation between SOAT1 expression and HCC was analyzed. Cox hazard regression models were conducted to investigate the prognostic value of SOAT1 in HCC. Overall survival and disease-specific survival were explored based on TCGA-LIHC data. Biological processes and functional pathways mediated by SOAT1 were characterized by gene ontology (GO) analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes. In addition, the protein-protein interaction network and co-expression analyses of SOAT1 in HCC were performed to better understand the regulatory mechanisms of SOAT1 in this malignancy. RESULTS: SOAT1 and SOAT2 were highly expressed in unpaired samples, while only SOAT1 was highly expressed in paired samples. The area under the receiver operating characteristic curve of SOAT1 expression in tumor samples from LIHC patients compared with para-carcinoma tissues was 0.748, while the area under the curve of SOAT1 expression in tumor samples from LIHC patients compared with GTEx was 0.676. Patients with higher SOAT1 expression had lower survival rates. Results from GO/KEGG and gene set enrichment analyses suggested that the PI3K/AKT signaling pathway, the IL-18 signaling pathway, the calcium signaling pathway, secreted factors, the Wnt signaling pathway, the Jak/STAT signaling pathway, the MAPK family signaling pathway, and cell-cell communication were involved in such association. SOAT1 expression was positively associated with the abundance of macrophages, Th2 cells, T helper cells, CD56bright natural killer cells, and Th1 cells, and negatively linked to the abundance of Th17 cells, dendritic cells, and cytotoxic cells. CONCLUSION: Our findings demonstrate that SOAT1 may serve as a novel target for HCC treatment, which is helpful for the development of new strategies for immunotherapy and metabolic therapy.

Phenethyl isothiocyanate inhibits metastasis potential of non-small cell lung cancer cells through FTO mediated TLE1 m6A modification.

N6-methyladenosine (m6A) modification is a prevalent RNA epigenetic modification, which plays a crucial role in tumor progression including metastasis. Isothiocyanates (ITCs) are natural compounds and inhibit the tumorigenesis of various cancers. Our previous studies show that ITCs inhibit the proliferation and metastasis of non-small cell lung cancer (NSCLC) cells, and have synergistic effects with chemotherapy drugs. In this study, we investigated the molecular mechanisms underlying the inhibitory effects of ITCs on cancer cell metastasis. We showed that phenethyl isothiocyanate (PEITC) dose-dependently inhibited the cell viability of both NSCLC cell lines H1299 and H226 with IC50 values of 17.6 and 15.2 µM, respectively. Furthermore, PEITC dose-dependently inhibited the invasion and migration of H1299 and H226 cells. We demonstrated that PEITC treatment dose-dependently increased m6A methylation levels and inhibited the expression of the m6A demethylase fat mass and obesity-associated protein (FTO) in H1299 and H226 cells. Knockdown of FTO significantly increased m6A methylation in H1299 and H226 cells, impaired their abilities of invasion and migration in vitro, and enhanced the inhibition of PEITC on tumor growth in vivo. Overexpression of FTO promoted the migration of NSCLC cells, and also mitigated the inhibitory effect of PEITC on migration of NSCLC cells. Furthermore, we found that FTO regulated the mRNA m6A modification of a transcriptional co-repressor Transducin-Like Enhancer of split-1 (TLE1) and further affected its stability and expression. TCGA database analysis revealed TLE1 was upregulated in NSCLC tissues compared to normal tissues, which might be correlated with the metastasis status. Moreover, we showed that PEITC suppressed the migration of NSCLC cells by inhibiting TLE1 expression and downstream Akt/NF-κB pathway. This study reveals a novel mechanism underlying ITC's inhibitory effect on metastasis of lung cancer cells, and provided valuable information for developing new therapeutics for lung cancer by targeting m6A methylation.

Identifying the EMT-related signature to stratify prognosis and evaluate the tumor microenvironment in lung adenocarcinoma.

Background: Epithelial-mesenchymal transition (EMT) is a critical process in tumor invasion and metastasis. EMT has been shown to significantly influence the invasion, metastasis, and poor prognosis in lung adenocarcinoma (LUAD). This study aimed to develop a novel EMT-related prognostic model capable of predicting overall survival (OS) in patients with LUAD. Methods: A total of 283 LUAD patients from TCGA RNA-seq dataset were assigned to a training cohort for model building, and 310 LUAD patients from GEO RNA-seq dataset were assigned to a validation cohort. EMT genes were acquired from MsigDB database and then prognosis-related EMT genes were identified by univariate Cox regression. Lasso regression was then performed to determine the genes and the corresponding variables to construct a prognosis risk model from the training cohort. Furthermore, characteristics of the tumor microenvironment (TME), mutation status and chemotherapy responses were analyzed to assess the differences between the two risk groups based on the prognostic model. In addition, RT-qPCR was employed to validate the expression patterns of the 6 genes derived from the risk model. Results: A six-gene EMT signature (PMEPA1, LOXL2, PLOD2, MMP14, SPOCK1 and DCN) was successfully constructed and validated. The signature assigned the LUAD patients into high-risk and low-risk groups. In comparison with the low-risk group, patients in the high-risk group had a significantly lower survival rate. ROC curves and calibration curves for the risk model demonstrated reliable stratification and predictive ability. The risk model was robustly correlated with multiple TME characteristics. Besides, the data showed that patients in the low-risk group had more immune activities, higher stemness scores and cytolytic activity scores and higher TMB. In addition, RT-qPCR results revealed that PMEPA1, LOXL2, PLOD2, MMP14, and SPOCK1 were notably upregulated in LUAD tissues, while DCN was downregulated. Conclusion: Our study successfully developed a novel EMT-related signature to predict prognosis of LUAD patients and guide treatment strategies. The six genes derived from the prediction signature might play a potential role in antitumor immunity and serve as promising therapeutic targets in LUAD.

Theoretical Calculation and Experimental Verification Demonstrated the Impossibility of Finding Haptens Identifying Triphenylmethane Dyes and Their Leuco Metabolites Simultaneously.

Detection of triphenylmethane dyes (TDs), especially the widely used malachite green (MG) and crystal violet (CV), plays an important role in safety control of aquatic products. There are two chromatic forms of TDs: oxidized or reduced. Usually, only one form can be detected by reported ELISA antibodies. In this article, molecular shape superimposing and quantum mechanics calculation were employed to elucidate the differences between MG, CV, and their reduced chromatic forms (leucomalachite green, LMG and leucocrystal violet, LCV). A potential hapten was rationally designed and synthesized. Polyclonal antibodies were raised through immunizing New Zealand white rabbits and BALB/C mice. We tested the cross-reactivity ratios between the hapten and TDs. The cross-reactivity ratios were correlated with the difference in surface electrostatic potential. The determination coefficients (r²) of the correlations are 0.901 and 0.813 for the rabbit and mouse antibody, respectively. According to this linear model, the significant difference in the atomic charge seemed to make it impossible to find a hapten that can produce antibodies with good cross-reactivities with both reduced and oxidized TDs.

[Inhibitory effect of arsenic trioxide on invasion in human hepatocellular carcinoma SMMC-7721 cells and its mechanism].

AIM: To investigate the effects of arsenic trioxide (As(2);O(3);) on human hepatocellular carcinoma SMMC-7721 cells' migration and invasion and the underlying molecular mechanism. METHODS: The effects of As(2);O(3); on cell migration and invasion were examined by wound-healing and Transwell assays; The expressions of CD147 and MMP-2 at both mRNA and protein levels in SMMC-7721 cells treated with As(2);O(3); were determined by real-time PCR and Western blotting, respectively. RESULTS: The wound-healing assay showed that the scuffing distance of SMMC-7721 cells exposed to 2.0 µmol/L As(2);O(3); for 12 and 24 h was significantly wider as compared with the ones unexposed to As(2);O(3);, indicating As(2);O(3); could inhibit the migration in SMMC-7721 cells (P<0.05). The Transwel1 assay showed that the number of SMMC-7721 cells migrating through the Matrigel treated by 2.0 µmol/L As(2);O(3); for 12 and 24 h obviously decreased as compared with non-treated ones, indicating that As(2);O(3); could inhibit the invasion of SMMC-7721 cells (P<0.05). Real-time PCR showed that the expressions of CD147 and MMP-2 mRNA were down-regulated in SMMC-7721 cells treated by 2.0 µmol/L As(2);O(3); for 6, 12 and 24 h, respectively (P<0.05). Western blotting demonstrated that the expressions of CD147 and MMP-2 proteins decreased in SMMC-7721 cells exposed to 2.0 µmol/L As(2);O(3); for 12, 24 and 48 h, respectively (P<0.05). CONCLUSION: As(2);O(3); could inhibit the migration and invasion in human hepatocellular carcinoma SMMC-7721 cells, which may be related to the down-regulation of CD147 and MMP-2.

[Effect of Nivalenol and selenium on IL-1beta and TNF-alpha secretion in cultured chondrocytes].

AIM: To investigate the effect of Nivalenol(NIV) and Selenium(Se) on the levels of IL-1beta and TNF-alpha in the cultured chondrocytes. METHODS: Human chondrocytes cultured in vitro were treated with or without NIV and Se. The morphology of chondrocytes was observed by optic microscope. The DNA content was determined by UV Spectrophotometry. The levels of IL-1beta and TNF-alpha in cultured medium were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Hematoxylin & eosin staining indicated there was cell necrosis in the cartilage reconstructed in vitro from both NIV group and NIV+Se group. Compared with the group of NIV toxin, the damage of chondrocytes was less severe when Se was added. NIV could inhibit chondrocyte DNA synthesis. The content of DNA with NIV was lowest than that in other groups. The levels of IL-1beta and TNF-alpha with NIV were significantly higher than control group (P<0.05). After Se was added, the levels did not change significantly compared with the groups without Se. CONCLUSION: NIV toxin could superinduce IL-1beta and TNF-alpha secretion in chondrocytes, which may be the key mechanism of chondrocyte injury by NIV. Se can partially alleviate the effects of NIV on chondrocytes cultured in vitro.

[Development and identification of the multiple B cell epitope antigens of Schistosoma japonicum].

OBJECTIVE: To develop multiple B cell epitope antigens of Schistosoma japonicum and evaluate their antigenicity. METHODS: Bioinformatics software BioSun was used to predict B cell epitopes from Sj22.6, Sj14-3-3 and Sj26. The predicted epitopes P2, P6 and P7 were ligated to construct P2-P6-P7 and P6-P2-P7 multiepitope in random order, a 6 amino acid linker inserted between epitopes. Recombinant plasmids containing the two multiepitopes identified by enzyme digestion and sequencing were transformed into E. coli BL21. The expressed recombinant fusion proteins of E. coli BL21 induced with IPTG were purified with Ni2+ chelating HiTrap HP column. Their antigenicity was evaluated with Western-blotting. RESULT: The two multiple B cell epitopes P2-P6-P7 and P6-P2-P7 were successfully cloned into pET-32c(+) plasmid and fusion proteins were expressed. SDS-PAGE showed a single band and both of the recombinant fusion proteins were with Mr 20 400. The two proteins reacted with the sera of schistosomiasis patients but not with that of healthy people. CONCLUSION: Two multiple B cell epitope antigens were developed with potential diagnosis value.

Over-expressing GLT1 in a gpd2Delta mutant of Saccharomyces cerevisiae to improve ethanol production.

We constructed two recombinant strains of Saccharomyces cerevisiae in which the GPD2 gene was deleted using a one-step gene replacement method to minimize formation of glycerol and improve ethanol production. In addition, we also over-expressed the GLT1 gene by a two-step gene replacement method to overcome the redox-imbalancing problem in the genetically modified strains. The result of anaerobic batch fermentations showed that the rate of growth and glucose consumption of the KAM-5 (MATalpha ura3 gpd2Delta::RPT) strain were slower than the original strain, and the KAM-13 (MATalpha ura3 gpd2Delta::RPT P ( PGK ) -GLT1) strain, however, was indistinguishable compared to the original strain using the same criteria, as analyzed. On the other hand, when compared to the original strain, there were 32 and 38% reduction in glycerol formation for KAM-5 and KAM-13, respectively. Ethanol production increased by 8.6% for KAM-5 and 13.4% for KAM-13. Dramatic reduction in acetate and pyruvic acid was also observed in both mutants compared to the original strains. Although gene GPD2 is responsible for the glycerol synthesis, the mutant KAM-13, in which glycerol formation was substantially reduced, was able to cope and maintain osmoregulation and redox balance and have increased ethanol production under anaerobic fermentations. The result verified the proposed concept of increasing ethanol production in S. cerevisiae by genetic engineering of glycerol synthesis and over-expressing the GLT1 gene along with reconstituted nicotinamide adenine dinucleotide metabolism.

Overexpressing GLT1 in gpd1Delta mutant to improve the production of ethanol of Saccharomyces cerevisiae.

To improve ethanol production in Saccharomyces cerevisiae, two yeast strains were constructed. In the mutant, KAM-4, the GPD1 gene, which encodes a glycerol 3-phosphate dehydrogenase of S. cerevisiae to synthesize glycerol, was deleted. The mutant KAM-12 had the GLT1 gene (encodes glutamate synthase) placed under the PGK1 promoter while harboring the GPD1 deletion. Notably, overexpression of GLT1 by the PGK1 promoter along with GPD1 deletion resulted in a 10.8% higher ethanol production and a 25.0% lower glycerol formation compared to the wild type in anaerobic fermentations. The growth rate of KAM-4 was slightly lower than that of the wild type under the exponential phase whereas KAM-12 and the wild type were indistinguishable in the biomass concentration at the end of growth period. Meanwhile, dramatic reduction of formation of acetate and pyruvic acid was observed in all the mutants compared to the wild type.

[Expression and bioactivity of S1 scFv antibody against SAG1 of Toxoplasma gondii fused with green fluorescent protein].

OBJECTIVE: To construct, express and purify human scFv antibody (S1) against the recombinant SAG1 of Toxoplasma gondii and fused to green fluorescent protein (GFP), and observe its binding capacity to tachyzoite of Toxoplasma gondii. METHODS: The GFP gene amplified from vector pEGFP-N1 was subcloned into procaryotic expression vector pET-26b (+), then the S1 scFv antibody gene amplified from phagmid pIT-2-S1 was cloned into downstream of GFP gene. The recombinant plasmid pET-26b-GFPS1 proved by DNA sequencing was transformed into E. coli BL21, and induced for fusion expression of GFPS1 with IPTG, the green fluorescence of E. coli BL21 harboring plasmid pET-26b-GFPS1 was observed under the fluorescence microscope. The expressed GFPS1 was purified with Ni2+ chelating HiTrap HP column, and detected with SDS-PAGE. Toxoplasma tachyzoites were incubated with the recombinant GFPS1, and the binding bioactivity was observed under the fluorescence microscope. RESULTS: The fused gene of S1 and GFP was successfully cloned into procaryotic expression vector pET-26b proved by DNA sequencing. The green fluorescence of E. coli BL21 harboring plasmid pET-26b-GFPS1 was catched under the fluorescence microscope. The recombinant GFPS1 protein about Mr 53 000 was expressed in E. coli as inclusion body. The immunofluorescence detection verified that anti-rSAG1 scFv antibody S1 could specifically bind outer membrane of Toxoplasma tachyzoite. CONCLUSIONS: The purified rGFPS1 shows a strong binding capacity to outer membrane of Toxoplasma tachyzoite using GFP as a labeling protein, and the constructed pET-26b-GFP can also be used for research on other targeting molecules.

Improved production of ethanol by deleting FPS1 and over-expressing GLT1 in Saccharomyces cerevisiae.

To improve ethanol production in Saccharomyces cerevisiae, two yeast strains were constructed. In the mutant KAM-3, the FPS1 gene, which encodes a channel protein responsible for glycerol export, was deleted. The mutant KAM-11 had the GLT1 gene (encoding glutamate synthase) placed under the PGK1 promoter while having the FPS1 deletion. Growth rate and biomass concentration remained virtually unchanged with the mutant KAM-11, compared to that of the parent. Over-expression of GLT1 by the PGK1 promoter along with FPS1 deletion resulted in a 14% higher ethanol production and a 30% lower glycerol formation compared to the parental strain under anaerobic fermentation conditions. Furthermore, acetate and pyruvic acid formation was also reduced in order for cells to maintain redox balance.

[Optimized expression of a single-chain Fv antibody against human asialoglycoprotein receptor and determination of its affinity constant].

AIM: To express and purify a single chain Fv antibody (scFv) C1 against human hepatic asialoglycoprotein receptor (ASGPR) and to determine affinity constant of the purified scFv C1. METHODS: The specific anti-ASGPR phage clone C1 was transfected into E. coli HB2151. The single colony was chosen to be inoculated into 2 x TY medium and shaken (250 r/min) overnight at 37 degrees C. After 1 in 100 dilution in 2 x TY medium and induced for secreted expression, scFv C1 was induced at different concentrations (0.25, 0.5 and 1.0 mmol/L IPTG) overnight at 37 degrees C, 25 degrees C and 20 degrees C, respectively. The supernatant was precipitated with saturated ammonium sulfate and its sediment was analyzed by SDS-PAGE. In addition, the sediment was resuspended in 30 mL PBS and dialyzed against PBS overnight at 4 degrees C. The expressed scFv C1 was purified by Ni(2+) chelating HiTrap HP column and the purity of the purified scFv C1 was identified by SDS-PAGE. Then affinity constant of scFv C1 was determined by noncompetitive enzyme immunoassay. RESULTS: After induced with 0.5 mmol/L IPTG overnight at 25 degrees C, the amount of expressed scFv C1 increased greatly and its relative molecular mass was about 28,000, and it existed in culture supernant in soluble form. The purity of scFv C1 by nickel-agarose column was above 95% and its yield was about 0.8 mg/L. The affinity constant of the purified scFv C1 was confirmed to be (2.31+/-0.36)x10(-7) mol/L. CONCLUSION: The E. coli HB2151 infected with phage C1 clones may express soluble scFv C1 with low affinity, which has potential applications to gene therapy of hepatocellular carcinoma.

Effect of high intensity ultrasound on the allergenicity of shrimp.

The tropomyosin fraction of shrimp proteins is potentially responsible for allergic reaction in individuals with genetic predisposition to allergy. However, there are no efficient and safe methods to reduce its allergenicity. High intensity ultrasound is known to change the structure of proteins. This study is aimed at assessing high intensity ultrasound's effect on the allergenicity of shrimp allergen. Shrimp and purified shrimp allergen were treated with high intensity ultrasound for 30-180 min. Extracts of treated samples were analyzed by enzyme-linked immunosorbent assay (ELISA) with pool serum of shrimp allergy patients and polyclonal anti-allergen antibodies and by immunoblotting after polyacrylamide gel electrophoresis. Shrimp treated with high intensity ultrasound showed a decrease in allergenicity measured with ELISA. A linear relationship between the immune response induced by treated shrimp allergen and the applied treatment time was observed. The decrease in allergenicity was confirmed by immunoblot assays with shrimp allergic patients serum. Allergenicity of shrimp allergen extracted from treated shrimp was higher than that of purified shrimp allergen with the same treatment time. Gel-filtration HPLC was applied for analysis of shrimp allergen after treatment with high intensity ultrasound. Some fractions were appeared with increasing treatment time. The results suggested that high intensity ultrasound could be used to reduce the allergenicity of shrimp.

[Survivin antisense RNA enhances taxol-induced apoptosis in leukemia cell line HL-60].

OBJECTIVE: To explore the effects of survivin antisense RNA on taxol-induced apoptosis in leukemia cell line HL-60. METHODS: A survivin antisense eukaryotic vector pcDNA3-SVVas was transferred into HL-60 cells by electroporation. The live fraction was determined by trypan blue dye exclusion assay. Cell counting and MTT assay were performed to evaluate the sensibility of the transfected cells to taxol. Apoptosis was detected by DNA gel electrophoresis and nuclear staining. RESULTS: Two positive cell clones, HL-60 SVVas and HL-60 neo were obtained. Compared to HL-60 and HL-60 neo cells, HL-60 SVVas cells growth was significantly reduced (P < 0.05). By MTT assay, the IC(50) of taxol to HL-60 SVVas, HL-60 neo and HL-60 cells were (14.4 +/- 1.87) ng/ml, (31.9 +/- 6.38) ng/ml and (32.0 +/- 3.52) ng/ml, respectively, the difference was significant by statistic analysis (P < 0.01). Agarose gel electrophoresis of genomic DNA from HL-60 SVVas showed typical DNA ladder, but DNA from HL-60 neo and HL-60 did not. Nuclei become condense in HL-60 SVVas cells. CONCLUSION: Survivin antisense RNA could enhance taxol-induced apoptosis in leukemia cell line HL-60. This may lay an experimental foundation for further research of gene therapy in leukemia.