Chun-Peng Zhao

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.

Cell compatibility of an eposimal vector mediated by the characteristic motifs of matrix attachment regions.

The characteristic sequence of β-interferon matrix attachment regions (MARs) can mediate transgene expression via episomal vectors in Chinese hamster ovary (CHO) cells. However, the host cells were from hamster ovaries, which are not suitable target cells for gene therapy. In this study, we aimed to evaluate the suitability of 12 different human cell lines as target cells for gene therapy. We transfected the cells with episomal vectors and obtained colonies stably expressing the vector products after G418 screening. Therefore the stably transfected cells were split into two and further cultured either in the presence or the absence of G418. Flow cytometry was used to observe the positive rate of cell transfection and level of green fluorescent protein (GFP) expression. Plasmid rescue assays, fluorescence in situ hybridization (FISH), and fluorescence quantitative polymerase chain reaction (PCR) were used to investigate the presence and gene copy numbers of plasmid in mammalian cells. The results showed that transfection efficiency and transgene expression levels in A375, Eca-109, and Changliver cells were high. In contrast, transgene silencing was observed in BJ, HSF, and A431 cells, and low expression of the transgene was observed in the other six cell lines. In addition, the plasmid was present in the episomal state in A375, Eca-109, and Chang-liver cells with relatively low copy numbers even under nonselective conditions. Thus, our results provide the first evidence showing transgene expression of an episomal vector mediated by the characteristic motifs of MARs for maintenance of the longterm stability of episomes in different types of cells.

Effect of β‑globin MAR characteristic elements on transgene expression

The aim of the present study was to investigate the effect of the characteristic elements of matrix attachment region (MAR) on transgene expression. Human β‑globin MAR was obtained by PCR amplification. A splicing MAR fragment containing all the characteristic elements of β‑globin MAR was artificially synthesized and then cloned into the eukaryotic expression vector. Following digestion and sequence identification, we transfected Chinese hamster ovary (CHO) cells with the two vectors, and then screened for the transformation of stable cells. The transgene expression level was analyzed by ELISA, and the copy numbers of the CAT gene were analyzed by real‑time fluorescent quantitative PCR. β‑globin MAR enhanced CAT reporter gene expression by 2.1489‑fold, whereas the β‑globin MAR characteristic elements did not enhance this expression. The real‑time fluorescent quantitative PCR analysis demonstrated that the relative copy numbers of the CAT gene of the β‑globin MAR expression vector were 1.2‑fold higher compared with those of the non‑MAR expression vector. MAR was able to improve the transgene expression level to a certain extent. The MAR characteristic elements did not improve the transgene expression alone. The transgenic expression levels were not linear with the transgene copy number; however, the enhancement of transgenic expression was relative to the increase in the gene copy number.

Identification of a potent MAR element from the human genome and assessment of its activity in stably transfected CHO cells

Low‐level and unstable transgene expression are common issues using the CHO cell expression system. Matrix attachment regions (MARs) enhance transgene expression levels, but additional research is needed to improve their function and to determine their mechanism of action. MAR‐6 from CHO chromosomes actively mediates high and consistent gene expression. In this study, we compared the effects of two new MARs and MAR‐6 on transgene expression in recombinant CHO cells and found one potent MAR element that can significantly increase transgene expression. Two MARs, including the human CSP‐B MAR element and DHFR intron MAR element from CHO cells, were cloned and inserted downstream of the poly(A) site in a eukaryotic vector. The constructs were transfected into CHO cells, and the expression levels and stability of eGFP were detected by flow cytometry. The three MAR sequences can be ranked in terms of overall eGFP expression, in decreasing order, as follows: human CSP‐B, DHFR intron MAR element and MAR‐6. Additionally, as expected, the three MAR‐containing vectors showed higher transfection efficiencies and transient transgene expression in comparison with those of the non‐MAR‐containing vector. Bioinformatics analysis indicated that the NFAT and VIBP elements within MAR sequences may contribute to the enhancement of eGFP expression. In conclusion, the human CSP‐B MAR element can improve transgene expression and its effects may be related to the NFAT and VIBP elements.