Tian-Yun Wang

A mini review of MAR-binding proteins

Genomic DNA encompasses several levels of organization, the nuclear matrix mediates the formation of DNA loop domains that are anchored to matrix attachment regions (MARs). By means of specific interaction with MAR binding proteins (MARBPs), MAR plays an important regulation role in enhancing transgene expression, decreasing expression variation among individuals of different transformants and serving as the replication origin. Through these years, some MARBPs have been identified and characterized from humans, plants, animals and algae so far and the list is growing. Most of MARBPs exist in a co-repressor/co-activator complex and involve in chromosome folding, regulation of gene expression, influencing cell development and inducing cell apoptosis. This review covers recent advances that have contributed to our understanding of MARBPs.

Enhanced expression of transgene in CHO cells using matrix attachment region

The expression of transgenes in mammalian cells is often at a low level mainly due to position effects from the neighboring chromatin context. To improve this, we have constructed a vector pCAM, which contains chloramphenicol acetyltransferase (CAT) reporter gene cassettes, driven by SV40 early promoter and flanked by two human β‐globin MARs in cis. We transfected this vector into the Chinese hamster ovary (CHO) cell line, and found that the level of CAT gene expression with MAR was effectively increased, about 5.493‐fold higher than those without MARs. Moreover, the variations of CAT expression among individuals of transformants were decreased 2.670‐fold. Our result also showed that MAR could increase the proportion of positive colonies in recombinants.

Simple, Time-Saving Dye Staining of Proteins for Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis Using Coomassie Blue

A fixation-free and fast protein-staining method for sodium dodecyl sulfate–polyacrylamide gel electrophoresis using Coomassie blue is described. The protocol comprises staining and quick washing steps, which can be completed in 0.5 h. It has a sensitivity of 10 ng, comparable with that of conventional Coomassie Brilliant Blue G staining with phosphoric acid in the staining solution. In addition, the dye stain does not contain any amount of acid and methanol, such as phosphoric acid. Considering the speed, simplicity, and low cost, the dye stain may be of more practical value than other dye-based protein stains in routine proteomic research.

Expression profile analysis of long non-coding RNA associated with vincristine resistance in colon cancer cells by next-generation sequencing.

Vincristine (VCR) is widely used in tumor treatment. However, long-term use of this drug can make tumor cells resistant to it. Furthermore, the mechanisms underlying resistance development are unclear. The aim of this study was to investigate the long non-coding RNAs (lncRNAs) associated with colon cancer drug resistance using next-generation sequencing. A cDNA library of HCT-8 VCR-resistant colon cancer cell was established through PCR amplification. Using HiSeq 2500 sequencing and bioinformatic methods, we identified lncRNAs showing different expression levels in drug-resistant and non-resistant cells, and constructed expression profiles of the lncRNA differences. The pretreatment of data was quality controlled using FastQC software. Transcription of lncRNA was calculated using Fragments Per Kilobase of transcript per Million fragments mapped (FPKM). To reveal the potential functions of these lncRNAs, we applied GO analysis to study the differentially expressed lncRNAs. Total transcript number was higher in resistant cells than in non-resistant colon cancer cells, and high-quality transcripts constituted the major portion of the total. In addition, 121 transcripts showed significantly different expression in VCR-resistant and non-resistant cells. Of these, we observed 23 up-regulated and 20 down-regulated lncRNAs (fold change >10.0). This is the first report of the expression profile of lncRNA of VCR-resistant colon cancer cells. Abnormal lncRNA expression was associated with VCR resistance in colon cancer cells and these expression differences may play a key role in VCR resistance of these cells.

Matrix attachment region combinations increase transgene expression in transfected Chinese hamster ovary cells

Matrix attachment regions (MARs) are cis-acting DNA elements that can increase transgene expression levels in a CHO cell expression system. To investigate the effects of MAR combinations on transgene expression and the underlying regulatory mechanisms, we generated constructs in which the enhanced green fluorescent protein (eGFP) gene flanked by different combinations of human β-interferon and β-globin MAR (iMAR and gMAR, respectively), which was driven by the cytomegalovirus (CMV) or simian virus (SV) 40 promoter. These were transfected into CHO-K1 cells, which were screened with geneticin; eGFP expression was detected by flow cytometry. The presence of MAR elements increased transfection efficiency and transient and stably expression of eGFP expression under both promoters; the level was higher when the two MARs differed (i.e., iMAR and gMAR) under the CMV but not the SV40 promoter. For the latter, two gMARs showed the highest activity. We also found that MARs increased the ratio of stably transfected positive colonies. These results indicate that combining the CMV promoter with two different MAR elements or the SV40 promoter with two gMARs is effective for inducing high expression level and stability of transgenes.

Impact of Different Promoters on Episomal Vectors Harbouring Characteristic Motifs of Matrix Attachment Regions.

We previously demonstrated that the characteristic sequence of matrix attachment regions (MARs) allows transgenes to be maintained episomally in CHO cells. In the present study, six commonly used promoters from human cytomegalovirus major immediate-early (CMV), simian vacuolating virus 40 (SV40), Rous sarcoma virus, Homo sapiens ubiquitin C, phosphoglycerate kinase, and β-globin, respectively, were evaluated to determine their effects on transgene expression and stability in CHO cells stably transfected via the episomal vector harbouring characteristic MAR motifs. The CHO cells were transfected with vectors and then screened using G418, after which the stably transfected cells were split into two and further cultured either in the presence or absence of G418. Of the six promoters, the CMV promoter yielded the highest transgene expression levels and the highest transfection efficiency, whereas the SV40 promoter maintained transgene expression more stably during long-term culture than the other promoters did. The CMV and SV40 promoter-containing vectors were furthermore episomally maintained and conferred sustained eGFP expression in the cells even under nonselective conditions. On the basis of these findings, we conclude that the CMV promoter performs best in terms of yielding both high expression levels and high levels of stability using this episomal vector system.

Molecular characterization of a human matrix attachment region that improves transgene expression in CHO cells.

Chinese hamster ovary (CHO) cells offer many advantages for recombinant gene expression, including proper folding and post-translational modification of the recombinant protein. However, due to positional effects resulting from the neighboring chromatin, transgenes are often expressed at low levels in these cells. While previous studies demonstrated that matrix attachment regions (MARs) can be utilized to increase transgene expression by buffering transgene silencing, the mechanism by which this occurs is poorly understood. We therefore performed a deletion analysis of the human β-globin MAR sequence to characterize the regions that are necessary to enhance transgene expression in CHO cells. Our results indicate that of the six β-globin MAR fragments tested (MAR-1-6; nucleotides 1-540, 420-1020, 900-1500, 1380-1980, 1860-2460, and 2340-2999, respectively), MAR-2, followed by MAR-3, was the most effective region for promoting stable and elevated transgene expression. Meanwhile, bioinformatic analyses demonstrated that these fragments encode a MAR-like motif and several transcription factor binding sites, including special AT-rich binding protein 1 (SATB1), CCAAT-enhancer-binding proteins (C/EBP), CCCTC-binding factor (CTCF), and Glutathione (GSH) binding motifs, indicating that these elements may contribute to the MAR-mediated enhancement of transgene expression. In addition, we found that truncated MAR derivatives yield more stable transgene expression levels than transgenes lacking the MAR. We concluded that the MAR-mediated transcriptional activation of transgenes requires a specific AT-rich sequence, as well as specific transcription factor-binding motifs.

RETRACTED ARTICLE: Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells

In the present study, six commonly used promoters, including cytomegalovirus major immediate-early (CMV), the CMV enhancer fused to the chicken beta-actin promoter (CAG), human elongation factor-1α (HEF-1α), mouse cytomegalovirus (mouse CMV), Chinese hamster elongation factor-1α (CHEF-1α), and phosphoglycerate kinase (PGK), a CMV promoter mutant and a CAG enhancer, were evaluated to determine their effects on transgene expression and stability in transfected CHO cells. The promoters and enhancer were cloned or synthesized, and mutation at C-404 in the CMV promoter was generated; then all elements were transfected into CHO cells. Stably transfected CHO cells were identified via screening under the selection pressure of G418. Flow cytometry, qPCR, and qRT-PCR were used to explore eGFP expression levels, gene copy number, and mRNA expression levels, respectively. Furthermore, the erythropoietin (EPO) gene was used to test the selected strong promoter. Of the six promoters, the CHEF-1α promoter yielded the highest transgene expression levels, whereas the CMV promoter maintained transgene expression more stably during long-term culture of cells. We conclude that CHEF-1α promoter conferred higher level of EPO expression in CHO cells, but the CMV promoter with its high levels of stability performs best in this vector system.

Treating Colon Cancer Cells with FK228 Reveals a Link between Histone Lysine Acetylation and Extensive Changes in the Cellular Proteome

The therapeutic value of FK228 as a cancer treatment option is well known, and various types of cancer have been shown to respond to this drug. However, the complete mechanism of FK228 and the affect it has on histone lysine acetylation and the colon cancer cell proteome are largely unknown. In the present study, we used stable isotope labeling by amino acids in cell culture (SILAC) and affinity enrichment followed by high-resolution liquid chromatograph-mass spectrometer (LC-MS)/MS analysis to quantitate the changes in the lysine acetylome in HCT-8 cells after FK228 treatment. A total of 1,194 lysine acetylation sites in 751 proteins were quantified, with 115 of the sites in 85 proteins being significantly upregulated and 38 of the sites in 32 proteins being significantly downregulated in response to FK228 treatment. Interestingly, 47 histone lysine acetylation sites were identified in the core histone proteins. We also found a novel lysine acetylation site on H2BK121. These significantly altered proteins are involved in multiple biological functions as well as a myriad of metabolic and enzyme-regulated pathways. Taken together, the link between FK228 function and the downstream changes in the HCT-8 cell proteome observed in response to FK228 treatment is established.

Episomal Lentiviral Vector-Mediated miR-145 Overexpression Inhibits Proliferation and Induces Apoptosis of Human Esophageal Carcinomas Cells.

BACKGROUND Gene therapy is a promising approach for the treatment of various cancers. However, most viral vectors used for this purpose carry risks, including potential integration into the host genome. OBJECTIVE We addressed this issue in the present study by constructing an episomal lentiviral vector using the .-interferon matrix attachment region to express the microRNA -145(miR-145), and examining the effect of miR-145 overexpression on human esophageal carcinomas (EC) cells. Some recent relevant patents are also discussed. METHOD Expression levels of miR-145 and the marker protein enhanced green fluorescent protein (EGFP) in infected ECA109 and EC9706 human esophageal carcinoma cells were detected by quantitative PCR and flow cytometry, respectively. Cell proliferation and apoptosis were assessed by Cell Counting Kit-8 and flow cytometry, respectively. Plasmid rescue experiments and fluorescence in situ hybridization were used to determine the episomal status of the transfected vector. RESULTS We found that EGFP and miR-145 were highly expressed in EC cells, and miR-145 overexpression inhibited cell proliferation and induced apoptosis. Moreover, the lentiviral vector did not integrate into the host genome, but was maintained episomally at lower copy numbers. CONCLUSION Taken together, our results demonstrate that miR-145-expressing episomal lentiviral vectors are a promising tool for gene therapy in the treatment of EC.