Quantitative proteomic analysis for high-throughput screening of differential glycoproteins in hepatocellular carcinoma serum

Objective: Hepatocellular carcinoma(HCC) is a leading cause of cancer-related deaths. Novel serum biomarkers are required to increase the sensitivity and specificity of serum screening for early HCC diagnosis. This study employed a quantitative proteomic strategy to analyze the differential expression of serum glycoproteins between HCC and normal control serum samples.Methods: Lectin affinity chromatography(LAC) was used to enrich glycoproteins from the serum samples. Quantitative mass spectrometric analysis combined with stable isotope dimethyl labeling and 2D liquid chromatography(LC) separations were performed to examine the differential levels of the detected proteins between HCC and control serum samples. Western blot was used to analyze the differential expression levels of the three serum proteins.Results: A total of 2,280 protein groups were identified in the serum samples from HCC patients by using the 2D LC-MS/MS method. Up to 36 proteins were up-regulated in the HCC serum, whereas 19 proteins were down-regulated. Three differential glycoproteins, namely, fibrinogen gamma chain(FGG), FOS-like antigen 2(FOSL2), and α-1, 6-mannosylglycoprotein 6-β-N-acetylglucosaminyltransferase B(MGAT5B) were validated by Western blot. All these three proteins were up-regulated in the HCC serum samples.Conclusion: A quantitative glycoproteomic method was established and proven useful to determine potential novel biomarkers for HCC.

mRNA and protein expression of autophagy marker molecule Beclin1 in osteosarcoma tissue and their correlation with pathological features

Objective: To discuss the mRNA and protein expression of autophagy marker molecule Beclin1 in osteosarcoma tissue and their correlation with pathological features. Methods: A total of 60 patients with osteosarcoma receiving surgical treatment in the hospital between April 2012 and January 2016 were selected, and the osteosarcoma tissue and adjacent normal tissue samples were collected during operation. Fluorescence quantitative PCR method was used to detect the expression of Beclin1 mRNA as well as proliferation and invasion gene expression in the tissue specimens, and the Beclin1 protein expression was detected by western-blot method. According to the median of Beclin1 mRNA and protein expression in osteosarcoma tissues, they were further grouped into high Beclin1 mRNA expression group (n=30) and low Beclin1 mRNA expression group (n=30) as well as high Beclin1 protein expression group (n=30) and low Beclin1 protein expression group (n=30). The differences in proliferation and invasion gene expression in osteosarcoma tissue with different Beclin1 mRNA and Beclin1 protein expression were compared. Results: Beclin1 mRNA and Beclin1 protein expression in osteosarcoma tissue were lower than those in adjacent normal tissue;proliferation gene KISS-1 mRNA expression in low Beclin1 mRNA expression group of osteosarcoma tissue was lower than that in high Beclin1 mRNA expression group while Six1, FoxM1, RIPK4 and STIM1 mRNA expression were higher than those in high Beclin1 mRNA expression group;invasion gene DLX1 mRNA expression was lower than that in high Beclin1 mRNA expression group while EFEMP1, MTA1, Notch1, HES1 and LEF-1 mRNA expression were higher than those in high Beclin1 mRNA expression group;proliferation gene KISS-1 mRNA expression in low Beclin1 protein expression group of osteosarcoma tissue was lower than that in high Beclin1 protein expression group while Six1, FoxM1, RIPK4 and STIM1 mRNA expression were higher than those in high Beclin1 protein expression group;invasion gene DLX1 mRNA expression was lower than that in high Beclin1 protein expression group while EFEMP1, MTA1, Notch1, HES1 and LEF-1 mRNA expression were higher than those in high Beclin1 protein expression group. Conclusion: Both mRNA and protein expression of autophagy marker molecule Beclin1 decrease in osteosarcoma tissue and are directly correlated with the proliferation and invasion activity of tumor cells.

Genome-wide analysis of AP2/ERF superfamily in Isatis indigotica

Objective AP2/ERF(APETALA2/ethylene-responsive factor)superfamily is one of the largest gene families in plants and has been reported to participate in various biological processes,such as the regulation of biosynthesis of active lignan.However,few studies have investigated the genome-wide role of the AP2/ERF superfamily in Isatis indigotica.This study establishes a complete picture of the AP2/ERF superfamily in I.indigotica and contributes valuable information for further functional characterization of IiAP2/ERF genes and supports further metabolic engineering.Methods To identify the IiAP2/ERF superfamily genes,the AP2/ERF sequences from Arabidopsis thaliana and Brassica rapa were used as query sequences in the basic local alignment search tool.Bioinformatic analyses were conducted to investigate the protein structure,motif composition,chromosome location,phylogenetic relationship,and interaction network of the IiAP2/ERF superfamily genes.The accuracy of omics data was verified by quantitative polymerase chain reaction and heatmap analyses.Results One hundred and twenty-six putative IiAP2/ERF genes in total were identified from the I.indigotica genome database in this study.By sequence alignment and phylogenetic analysis,the IiAP2/ERF genes were classified into 5 groups including AP2,ERF,DREB(dehydration-responsive element-binding factor),Soloist and RAV(related to abscisic acid insensitive 3/viviparous 1)subfamilies.Among which,122 members were unevenly distributed across seven chromosomes.Sequence alignment showed that I.indigotica and A.thaliana had 30 pairs of orthologous genes,and we constructed their interaction network.The comprehensive analysis of gene expression pattern in different tissues suggested that these genes may play a significant role in organ growth and development of I.indigotica.Members that may regulate lignan biosynthesis in roots were also preliminarily identified.Ribonucleic acid sequencing analysis revealed that the expression of 76 IiAP2/ERF genes were up-or down-regulated under salt or drought treatment,among which,33 IiAP2/ERF genes were regulated by both stresses.Conclusion This study undertook a genome-wide characterization of the AP2/ERF superfamily in I.indigotica,providing valuable information for further functional characterization of IiAP2/ERF genes and discovery of genetic targets for metabolic engineering.

Simultaneous Determination of Two Major Triterpenoid Saponins:Celosins Ⅰ and Ⅱ in Celosiae Semen by HPLC-ELSD

Objective To establish an analytical method for the simultaneous determination of celosin Ⅰ and celosin Ⅱ, two major triterpenoids in Celosia Semen and compare the contents of celosin Ⅰ and celosin Ⅱ from different habitats to screen the resources of elite germplasm for further applications. Methods A sensitive and simple high- performance liquid chromatography coupled with evaporative light-scattering detector（HPLC-ELSD） method was developed for the first time for the simultaneous determination of celosin Ⅰ and celosin Ⅱ. Using this method, 21 batches of Celosiae Semen were determined from different habitats in China. Results There was an obvious difference in the contents of celosin Ⅰ and celosin Ⅱ among Celosiae Semen species from various habitats across China. The crude drug from Yongzhou, Hunan province, Zhuhai, Guangdong province, and Nanning, Guangxi province showed the highest contents of all habitats, while Anguo, Hebei province, Haidian, Beijing, and Zhengzhou, Henan province showed the lowest content. The results also showed that geographical location had a great influence on the contents of the two compounds. The batches from lower latitudes were higher in contents of celosin Ⅰ and celosin Ⅱ. Conclusion The sun light may be a key factor influencing the contents of the two saponins, indicating that the environment has a great impact on the quality of Celosiae Semen.