Improved PCR-Based Subtractive Hybridization Strategy for Cloning Differentially Expressed Genes

Zhu, Feng; Wang, Yan; Zhao, Zhongliang; Chai, Yongrong; Liu, Fan; Wang, Q.; Peng, Weidan; Yang, Angang; Wang, C.J.

doi:10.2144/00292st06

Cited by 34 publications

(33 citation statements)

References 7 publications

(4 reference statements)

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“…Although the first MAGE family gene was discovered in 1991, MAGEs have been well Table I studied for >20 years in melanomas (20), yet their functions still remain unclear. Based on our previous study, Apr-1 protein was found to be localized in the nucleus and is believed to be an apoptosis-related gene since its transcripts were upregulated during all-trans-retinoic acid-induced apoptosis in human promyelocytic leukaemia cells (5,14). In situ hybridization assay on tissue microarrays showed that Apr-1 is expressed in esophageal carcinoma, normal hepatic tissue and hepatic tissue adjacent to hepatocellular carcinoma, but is absent in normal esophageal mucosa and hepatocellular carcinoma (data not shown).…”

Section: Discussionmentioning

confidence: 99%

“…Apoptosis-related protein-1 (Apr-1), also known as melanoma-associated antigen (MAGE)-H1 and restin, was first cloned by our group from the apoptotic tumor cell line HL-60 when induced by all-trans retinoic acid (ATRA) (5). It belongs to the MAGE gene superfamily based on the bioinformatic analysis of its gene structure compared with other homologues in the GenBank (6).…”

Section: Introductionmentioning

confidence: 99%

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Apoptosis-related protein-1 acts as a tumor suppressor in cholangiocarcinoma cells by inducing cell cycle arrest via downregulation of cyclin-dependent kinase subunits

Zheng

Wang

et al. 2015

Oncology Reports

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Abstract. Cholangiocarcinoma, a malignancy arising from the biliary tract, is associated with high mortality due to the late diagnosis and lack of effective therapeutic approaches. Our knowledge of the molecular alterations during the carcinogenesis of cholangiocarcinoma is limited. Previous study suggests that apoptosis-related protein-1 (Apr-1) is involved in cancer cell proliferation and survival. In the present study, we first detected the expression pattern of Apr-1 in human cholangiocarcinoma tissues and the effects of forced Apr-1 expression on cell proliferation and cell cycle progression. Cell cycle gene array analysis was used to identify downstream molecules that were regulated by Apr-1, and their expression levels were further evaluated in human cholangiocarcinoma tissues. We showed that Apr-1 expression was downregulated in human cholangiocarcinoma tissues. Forced expression of Apr-1 inhibited cell proliferation of cholangiocarcinoma cell line QBC939 and induced G2/M phase arrest. Downregulation of cell cycle-related genes cyclin-dependent kinase (Cdk) 2, and cyclin-dependent kinase subunits (Cks) 1 and 2 was involved in Apr-1-induced cell cycle arrest. Furthermore, we found that Cdk2 and Cks1/2 expression levels were elevated in human cholangiocarcinoma tissues. Taken together, our data showed that Apr-1 plays a crucial role in cell proliferation by controlling cell cycle progression, implying a tumor-suppressor function of Apr-1 in cholangiocarcinoma carcinogenesis. Thus, the present study provides a rationale to further study the underlying mechanisms of Apr-1 downregulation in cholangiocarcinoma for exploring potential diagnostic and therapeutic targets.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Apoptosis-related protein-1 acts as a tumor suppressor in cholangiocarcinoma cells by inducing cell cycle arrest via downregulation of cyclin-dependent kinase subunits

Zheng

Wang

et al. 2015

Oncology Reports

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“…The advantages of this cell line for genetic screening include: 1) each gene is present as a single copy, enabling gene inactivation (except those genes on chromosome 8); and 2) KBM7 cells are human and of the hematopoietic lineage, increasing the likelihood that any genes we identify could be relevant to the natural context for N-BPs, the bones of human patients 26 . Using this haploid approach, we identified a poorly characterized gene, ATRAID, a.k.a., APR3/C2orf28 27 , as the gene most significantly enriched for insertions in alendronate resistant cells compared to untreated cells (Fig. 1b, and Supplementary Table 1a).…”

mentioning

confidence: 94%

“…1i, j), we focused our further characterization on ATRAID. ATRAID was originally identified as a gene whose mRNA expression is strongly induced by the ligand, all-trans retinoic acid 27 . ATRAID is conserved in chordates and contains a signal peptide, Toll-like-receptor leucine rich repeat, EGF-like domain, and a transmembrane domain ( Fig.…”

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confidence: 99%

ATRAIDregulates the action of nitrogen-containing bisphosphonates on bone

Surface

Burrow²,

Park³

et al. 2018

Preprint

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. CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/338350 doi: bioRxiv preprint first posted online Jun. 4, 2018; 3 Nitrogen-containing bisphosphonates (N-BPs) are the standard treatment for osteoporosis and several other bone diseases 11,12 . Certain N-BPs (pamidronate (Aredia®), zoledronate (Zometa®)) are also routinely prescribed to prevent skeletal complications in patients with multiple myeloma and with bone metastases from other malignancies, including breast and prostate cancer 13 . However, because N-BPs cause rare yet serious side-effects, such as atypical fractures and osteonecrosis of the jaw, many patients avoid taking them [5][6][7]11 , causing the number of prescriptions to plummet over 50% in the last decade 7,14 . Thus, there is a sizeable and growing need to better understand the genetic factors that might underlie the onand off-target clinical effects of the N-BPs.A goal of personalized medicine is to identify biomarkers that underlie drug responsiveness. In the case of the N-BPs, it can be said that there are limited personalization options owing to the limited number of genes implicated in the mechanism of action of N-BPs on bone. was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/338350 doi: bioRxiv preprint first posted online Jun. 4, 2018; 4 understanding of the molecular mechanisms by which N-BPs regulate the major bone cell types would be improved by further studies.To provide insight into the mechanism(s) of N-BPs action, we performed a genetic screen to identify human genes required for the anti-proliferative effects of N-BPs (Fig. 1a) Other genes identified in our alendronate haploid screen (SNTG1, PLCL1, and EPHB1) have been previously connected to N-BP action on bone cells and/or human bone diseases [28][29][30] . To systematically evaluate all "hits" we identified (i.e., the 185 genes with FDR p-value < 0.05, see Fig. 1b), we developed a computational approach utilizing a molecular biology-optimized version of PubMed (currently at ~27M records) to assess to what extent our haploid screen identified genes were cited as relevant to human studies that focus on bone (see "PubMed citation analysis" in the Methods section for details). We asked whether our alendronate haploid screen hits were mentioned in publications co-occurring with the term "bone" vs. other tissues along with identifiers of genome-wide screening, namely the strings, "GWAS" or "genome-wide" (Fig. 1c). Indeed, our alendronate screen hits were enriched in publications co-mentioning "bone" and genome-wide studies compared to control gene sets (Fig. 1d, Supplementary Table 1b). This . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed...

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“…Several new techniques were adopted on the basis of routine subtractive hybridization, such as the cap-finder method, long-distance PCR (LD-PCR), streptavidin magnetic bead-mediated subtraction and spin column chromatography. This modified method was named improved subtractive hybridization (Zhu et al, 2000). In this procedure, differentially expressed cDNA fragments were selectively amplified and amplification of nondifferentially expressed cDNAs was simultaneously suppressed.…”

Section: Introductionmentioning

confidence: 99%

Identification of Up-Regulated Genes After Complete Spinal Cord Transection in Adult Rats

Ma¹,

Liu²,

Li³

et al. 2006

Cell Mol Neurobiol

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Spinal cord injury (SCI) initiates a cascade of events and these responses to injury are likely to be mediated and reflected by changes in mRNA concentrations. As a step towards understanding the complex mechanisms underlying repair and regeneration after SCI, the gene expression pattern was examined 4.5 days after complete transection at T8-9 level of rat spinal cord. Improved subtractive hybridization was used to establish a subtracted cDNA library using cDNAs from normal rat spinal cord as driver and cDNAs from injured spinal cord as tester. By expressed sequence tag (EST) sequencing, we obtained 73 EST fragments from this library, representing 40 differentially expressed genes. Among them, 32 were known genes and 8 were novel genes. Functions of all annotated genes were scattered in almost every important field of cell life such as DNA repair, detoxification, mRNA quality control, cell cycle control, and signaling, which reflected the complexity of SCI and regeneration. Then we verified subtraction results with semiquantitative RT-PCR for eight genes. These analyses confirmed, to a large extent, that the subtraction results accurately reflected the molecular changes occurring at 4.5 days post-SCI. The current study identified a number of genes that may shed new light on SCI-related inflammation, neuroprotection, neurite-outgrowth, synaptogenesis, and astrogliosis. In conclusion, the identification of molecular changes using improved subtractive hybridization may lead to a better understanding of molecular mechanisms responsible for repair and regeneration after SCI.

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Improved PCR-Based Subtractive Hybridization Strategy for Cloning Differentially Expressed Genes

Cited by 34 publications

References 7 publications

Apoptosis-related protein-1 acts as a tumor suppressor in cholangiocarcinoma cells by inducing cell cycle arrest via downregulation of cyclin-dependent kinase subunits

Apoptosis-related protein-1 acts as a tumor suppressor in cholangiocarcinoma cells by inducing cell cycle arrest via downregulation of cyclin-dependent kinase subunits

ATRAIDregulates the action of nitrogen-containing bisphosphonates on bone

Identification of Up-Regulated Genes After Complete Spinal Cord Transection in Adult Rats

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