Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)-based Quantitative Proteomics Study of a Thyroid Hormone-regulated Secretome in Human Hepatoma Cells

Chen, Cheng-Yi; Chi, Lang‐Ming; Chi, Hsiang-Cheng; Tsai, Ming-Ming; Tsai, Chung‐Ying; Tseng, Yi-Hsin; Lin, Yang-Hsiang; Chen, Wen‐Jone; Huang, Ya‐Hui; Lin, Kwang‐Huei

doi:10.1074/mcp.m111.011270

Cited by 33 publications

(36 citation statements)

References 73 publications

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“…T 3 /thyroid hormone receptor signaling promotes cell invasiveness involved in hepatoma progression through the direct or indirect upregulation of several genes, including furin (10), methionine adenosyltransferase 1A (9), plasminogen activator inhibitor-1 (18), and TNF-related apoptosis-inducing ligand (19). MiRNAs are small RNA molecules processed from endogenous precursor RNA with stem-loop structures that negatively regulate gene expression.…”

Section: Discussionmentioning

confidence: 99%

“…Animals were subjected to hyperthyroid, euthyroid, and hypothyroid conditions after injection (18,19). The hyperthyroid group of SCID mice injected with J7-TRa1 cells displayed higher miR-21 expression (Fig.…”

Section: Correlations Among Tr Mir-21 and Tiam1 In Vivomentioning

confidence: 99%

“…To investigate the correlation between T 3 /thyroid hormone receptor and miR-21 in T 3 -enhanced metastasis of hepatoma cells in vivo, J7-TRa1 xenografts of eu-, hypo-, and hyperthyroid severe combined immunodeficiency (SCID) mice generated in a previous study by our group (18,19) were used (Model II). Finally, to determine whether the negative correlation between the miR-21 and TIAM1 can be found in vivo, knockdown of miR-21 in SK-Hep1 cells was injected into SCID mice via the tail vein (Model III).…”

Section: Animal Modelmentioning

confidence: 99%

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Thyroid Hormone Regulation of miR-21 Enhances Migration and Invasion of Hepatoma

et al. 2013

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Thyroid hormone (T 3 ) signaling through the thyroid hormone receptor (TRa1) regulates hepatoma cell growth and pathophysiology, but the underlying mechanisms are unclear at present. Here, we have shown that the oncomir microRNA-21 (miR-21) is activated by T 3 through a native T 3 response element in the primary miR-21 promoter. Overexpression of miR-21 promoted hepatoma cell migration and invasion, similar to that observed with T 3 stimulation in hepatoma cells. In addition, anti-miR-21-induced suppression of cell migration was rescued by T 3 . The Rac-controlled regulator of invasion and metastasis, T-cell lymphoma invasion and metastasis 1 (TIAM1), was identified as a miR-21 target additionally downregulated by T 3 . Attenuation and overexpression of miR-21 induced upregulation and downregulation of TIAM1, respectively. TIAM1 attenuation, in turn, enhanced migration and invasion via the upregulation of b-catenin, vimentin, and matrix metalloproteinase-2 in hepatoma cells. Notably, correlations between TRa1, miR-21, and TIAM1 expression patterns in animal models paralleled those observed in vitro. In the clinic, we observed a positive correlation (P ¼ 0.005) between the tumor/nontumor ratios of TRa1 and miR-21 expression, whereas a negative correlation (P ¼ 0.019) was seen between miR-21 and TIAM1 expression in patients with hepatoma. Our findings collectively indicate that miR-21 stimulation by T 3 and subsequent TIAM1 suppression promotes hepatoma cell migration and invasion. Cancer Res; 73(8); 2505-17. Ó2013 AACR.

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Section: Discussionmentioning

confidence: 99%

Section: Correlations Among Tr Mir-21 and Tiam1 In Vivomentioning

confidence: 99%

Section: Animal Modelmentioning

confidence: 99%

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Thyroid Hormone Regulation of miR-21 Enhances Migration and Invasion of Hepatoma

et al. 2013

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“…[19][20][21][22][23][24][25] MS-based studies on the HepG2 secretome have been demonstrated, [26,25] including a recent report by Chen et al that investigated the thyroid-hormone regulated HepG2 secretome using SILAC. [27] In the current study, we harvested the SILAC-labeled HepG2 secretome and then spiked this sample into human plasma. LC-MS/MS analysis of the 'heavy secretome:plasma' sample (H/L) with and without albumin and IgG removal quantified 56 and 62 proteins, respectively.…”

Section: Improved For Comparative Lc-ms/ms Analysis Proteomicsmentioning

confidence: 99%

Intact stable isotope labeled plasma proteins from the SILAC‐labeled HepG2 secretome

et al. 2015

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The plasma proteome remains an attractive biospecimen for MS-based biomarker discovery studies. The success of these efforts relies on the continued development of quantitative MS-based proteomics approaches. Herein we report the use of the SILAC-labeled HepG2 secretome as a source for stable isotope labeled plasma proteins for quantitative LC-MS/MS measurements. The HepG2 liver cancer cell line secretes the major plasma proteins including serum albumin, apolipoproteins, protease inhibitors, coagulation factors, and transporters that represent some of the most abundant proteins in plasma. The SILAC-labeled HepG2 secretome was collected, spiked into human plasma (1:1 total protein), and then processed for LC-MS/MS analysis. A total of 62 and 56 plasma proteins were quantified (heavy:light (H/L) peptide pairs) from undepleted and depleted (serum albumin and IgG), respectively, with log2 H/L = ± 6. Major plasma proteins quantified included albumin, apolipoproteins (e.g., APOA1, APOA2, APOA4, APOB, APOC3, APOE, APOH, and APOM), protease inhibitors (e.g., A2M and SERPINs), coagulation factors (e.g., Factor V, Factor X, fibrinogen), and transport proteins (e.g., TTR). The average log2 H/L values for shared plasma proteins in both undepleted and depleted plasma samples were 0.43 and 0.44, respectively. This work further expands the SILAC strategy into MS-based biomarker discovery of clinical biospecimens.

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“…MS has certain advantages like prediction of molecular mass with the highest specificity and sensitivity with the use of smallest amount of sample [9][10][11]. Different mass spectrometry-based proteomic approaches have been used to identify biomarkers from various sources and are broadly classified into two categories: gel-based (2-DE and 2D-DIGE) and gel-free (SILAC, iTRAQ) techniques [12][13][14]. Detection of biomarkers through twodimensional gel electrophoresis (2-DE) is the most widely used gel-based approach [15].…”

Section: Lc-ms/ms or Maldi-ms/msmentioning

confidence: 99%

High Throughput Sequencing and Proteomics to Identify Immunogenic Proteins of a New Pathogen: The Dirty Genome Approach

2014

Omics in Clinical Practice

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Accurate diagnosis and proper monitoring of cancer patients remain a key obstacle for successful cancer treatment and prevention. Therein comes the need for biomarker discovery, which is crucial to the current oncological and other clinical practices having the potential to impact the diagnosis and prognosis. In fact, most of the biomarkers have been discovered utilizing the proteomicsbased approaches. Although high-throughput mass spectrometry-based proteomic approaches like SILAC, 2D-DIGE, and iTRAQ are filling up the pitfalls of the conventional techniques, still serum proteomics importunately poses hurdle in overcoming a wide range of protein concentrations, and also the availability of patient tissue samples is a limitation for the biomarker discovery. Thus, researchers have looked for alternatives, and profiling of candidate biomarkers through tissue culture of tumor cell lines comes up as a promising option. It is a rich source of tumor cell-derived proteins, thereby, representing a wide array of potential biomarkers. Interestingly, most of the clinical biomarkers in use today (CA 125, CA 15.3, CA 19.9, and PSA) were discovered through tissue culture-based system and tissue extracts. This paper tries to emphasize the tissue culture-based discovery of candidate biomarkers through various mass spectrometry-based proteomic approaches.

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Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)-based Quantitative Proteomics Study of a Thyroid Hormone-regulated Secretome in Human Hepatoma Cells

Cited by 33 publications

References 73 publications

Thyroid Hormone Regulation of miR-21 Enhances Migration and Invasion of Hepatoma

Thyroid Hormone Regulation of miR-21 Enhances Migration and Invasion of Hepatoma

Intact stable isotope labeled plasma proteins from the SILAC‐labeled HepG2 secretome

High Throughput Sequencing and Proteomics to Identify Immunogenic Proteins of a New Pathogen: The Dirty Genome Approach

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