Shufang Liang

Roles of microRNA on cancer cell metabolism

Advanced studies of microRNAs (miRNAs) have revealed their manifold biological functions, including control of cell proliferation, cell cycle and cell death. However, it seems that their roles as key regulators of metabolism have drawn more and more attention in the recent years. Cancer cells display increased metabolic autonomy in comparison to non-transformed cells, taking up nutrients and metabolizing them in pathways that support growth and proliferation. MiRNAs regulate cell metabolic processes through complicated mechanisms, including directly targeting key enzymes or transporters of metabolic processes and regulating transcription factors, oncogenes / tumor suppressors as well as multiple oncogenic signaling pathways. MiRNAs like miR-375, miR-143, miR-14 and miR-29b participate in controlling cancer cell metabolism by regulating the expression of genes whose protein products either directly regulate metabolic machinery or indirectly modulate the expression of metabolic enzymes, serving as master regulators, which will hopefully lead to a new therapeutic strategy for malignant cancer. This review focuses on miRNA regulations of cancer cell metabolism,including glucose uptake, glycolysis, tricarboxylic acid cycle and insulin production, lipid metabolism and amino acid biogenesis, as well as several oncogenic signaling pathways. Furthermore, the challenges of miRNA-based strategies for cancer diagnosis, prognosis and therapeutics have been discussed.

Mechanism of Cancer Cell Adaptation to Metabolic Stress Proteomics Identification of a Novel Thyroid Hormone-mediated Gastric Carcinogenic Signaling Pathway

Gastric cancer is the second most common cancer worldwide and has a poor prognosis. To determine the mechanism of adaptation to metabolic stress in cancer cells, we used gastric cancer as a model system to reveal the potential signaling pathways involved. Two-dimensional polyacrylamide gel electrophoresis coupled with ESI-Q-TOF MS/MS analysis was used to identify differentially expressed proteins between gastric tumor tissues and the corresponding noncancerous tissues. In total, 107 spots with significant alteration (±over 2-fold, p < 0.05) were positively identified by MS/MS analysis. Altered expression of representative proteins was validated by RT-PCR and Western blotting. Cluster analysis of the changed proteins revealed an interesting group of metabolic proteins, which suggested accumulation of triiodothyronine (T3; the major functional component of thyroid hormone) and overexpression of hypoxia-induced factor (HIF) in gastric carcinoma. These observations were further confirmed by electrochemiluminescence immunoassay and immunohistochemistry. T3-induced expression of HIF1-α and vascular endothelial growth factor was further verified using a gastric cancer cell line and in vivo mouse model. Because the early accumulation of HIF1-α was found to be independent of de novo transcription, we also found that the cytosolic cascade phosphatidylinositol 3-kinase/Akt pathway sensitive to T3 stimulus was involved. Furthermore we demonstrated that T3-induced overexpression of HIF1-α was mediated by fumarate accumulation and could be enhanced by fumarate hydratase inactivation but inhibited by 2-oxoglutarate. These results provide evidence for alteration of metabolic proteins and dysfunction of thyroid hormone regulation in gastric tumors, and a novel thyroid hormone-mediated tumorigenic signaling pathway is proposed. Our findings are considered a significant step toward a better understanding of adaptations to metabolic stress in gastric carcinogenesis.

Several Affinity Tags Commonly Used in Chromatographic Purification

Affinity tags have become powerful tools from basic biological research to structural and functional proteomics. They were widely used to facilitate the purification and detection of proteins of interest, as well as the separation of protein complexes. Here, we mainly discuss the benefits and drawbacks of several affinity or epitope tags frequently used, including hexahistidine tag, FLAG tag, Strep II tag, streptavidin-binding peptide (SBP) tag, calmodulin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), S-tag, HA tag, and c-Myc tag. In some cases, a large-size affinity tag, such as GST or MBP, can significantly impact on the structure and biological activity of the fusion partner protein. So it is usually necessary to excise the tag by protease. The most commonly used endopeptidases are enterokinase, factor Xa, thrombin, tobacco etch virus, and human rhinovirus 3C protease. The proteolysis features of these proteases are described in order to provide a general guidance on the proteolytic removal of the affinity tags.

MicroRNA-26a regulates glucose metabolism by direct targeting PDHX in colorectal cancer cells

BackgroundReprogramming energy metabolism has been an emerging hallmark of cancer cells. MicroRNAs play important roles in glucose metabolism.MethodsThe targets of microRNA-26a (miR-26a) were predicted by bioinformatics tools. The efficacy of miR-26a binding the 3′-untranslated region (UTR) of pyruvate dehydrogenase protein X component (PDHX) mRNA was evaluated using a dual-luciferase reporter assay. The PDHX expression at the mRNA and protein level in several colon cancer cell lines was quantified with real-time PCR and Western blot analysis respectively. The effects of miR-26a on glucose metabolism were determined by detecting the content of glucose consumption, production of lactate, pyruvate, and acetyl-coenzyme A.ResultsThe expression of miR-26a is inversely associated with the level of its targeting protein PDHX in several colon cancer cell lines with different malignancy potentials. MiR-26a inhibits PDHX expression by direct targeting the 3′-UTR of PDHX mRNA. The glucose consumption and lactate concentration were both greatly increased in colon cancer cells than the normal colon mucosal epithelia under physiological conditions. The overexpression of miR-26a in HCT116 cells efficiently improved the accumulation of pyruvate and decreased the production of acetyl coenzyme A. Meanwhile the inhibition of miR-26a expression induced inverse biological effects.ConclusionsMiR-26a regulates glucose metabolism of colorectal cancer cells by direct targeting the PDHX, which inhibits the conversion of pyruvate to acetyl coenzyme A in the citric acid cycle.

Comparative proteomics and correlated signaling network of rat hippocampus in the pilocarpine model of temporal lobe epilepsy

In temporal lobe epilepsy (TLE), the seizure origin typically involves the hippocampal formation. The pilocarpine‐induced TLE provides a model to investigate the molecular and functional characterization of epileptogenesis by mimicking the human epileptic condition. Here, we employed a 2‐D gel‐based proteomic technique to profile proteome changes in the rat hippocampus after pilocarpine treatment. Using MALDI MS and MS/MS, 57 differentially expressed proteins were identified, which were found either up‐regulated and/or down‐regulated at the two time points 12 h (acute period; Ap) and 72 h (silent period; Sp) compared with the control. These proteins can be related to underlying mechanism of pilocarpine‐induced TLE, indicating cytoskeleton modification, altered synaptic function, mitochondrial dysfunction, changed ion channel, and chaperone. Five of the identified proteins, synaptosomal‐associated protein 25 (SNAP25), synapsin‐2 (SYN2), homer protein homolog 2 (HOMER2), α‐internexin (INA), and voltage‐dependent anion channel 2 (VDAC2) were investigated by semiquantitative RT‐PCR, and SNAP25 and INA were further validated by Western blot and immunohistochemistry staining. Furthermore, association of these pilocarpine‐induced proteins with biological functions using the Ingenuity Pathway Analysis (IPA) tool showed that nucleic acid metabolism, system development, tissue and cell morphology were significantly altered. IPA of the canonical networks indicated that six membrane proteins (e.g., SNAP25, SYN2, and HOMER2) participated in three biological networks as starting proteins. Our results offer a clue to identify biomarkers for the development of pharmacological therapies targeted at epilepsy.

Quantitative proteomics for cancer biomarker discovery.

The mass spectrometry (MS)-based quantitative proteomics is powerful to discover disease biomarkers that can provide diagnostic, prognostic and therapeutic targets, and it also can address important problems in clinical and translational medical research. The current status of MS-based quantification strategy and technical advances of several main quantitative assays (two-dimensional (2-D) gel-based methods, stable isotope labeling with amino acids in cell culture (SILAC), isotope-coded affinity tag (ICAT), the isobaric tags for relative and absolute quantification (iTRAQ), ¹⁸O labeling, absolute quantitation and label-free quantitation) have been summarized and reviewed. At present, except 2-D gel-based methods, several stable isotope labeling quantitative techniques, including SILAC, ICAT and iTRAQ, etc, have been widely applied in identification of differential expression of proteins, post-translational modifications and protein-protein interactions in order to look for novel candidate cancer biomarkers from different physiological states of cells, body fluids or tissue samples. Also, the advantages and challenges of different quantitative proteomic approaches are discussed in identification and validation of candidate targets.

Quantitative secretome analysis reveals the interactions between epithelia and tumor cells by in vitro modulating colon cancer microenvironment

UNLABELLED In tumor microenvironment, interactions among multiple cell types are critical for cancer progression. Secreted proteins are responsible for crosstalk among these cells within tumor microenvironment. To elucidate the interactions of tumor and epithelia, we co-cultured colon cancer cell line HT29 with normal human colon mucosal epithelial cell line NCM460 to mimic tumor microenvironment in vitro and investigated the differential expression pattern of secretome. A quantitative proteomics approach based on stable isotope labeling by amino acids in cell culture (SILAC) and LC-mass spectrometry was used for secretome analysis. Totally 45 proteins were altered over 2-fold in co-cultured cellular supernatants between equal amounts of NCM460 and HT29 cells, compared with mono-cultured conditions. These differential secreted proteins involve in multiple tumor-associated biological functions. The secretion level and acting pattern of acrogranin, IGFBP6 and vimentin were changed along with different co-cultured cell number ratios between NCM460 and HT29 cells, simulating early, middle or advanced stage of colon cancer. Therefore, a quantitative secretome profiling based on a co-culture system can track secreted protein changes and their associated biological roles between tumor and epithelia, which gives a new insight on communications between tumor and epithelia as well as cancer biotherapy by inhibiting cell interactions. BIOLOGICAL SIGNIFICANCE Tumor microenvironment is a complex system and comprised of cancer cells and host stromal cells. The growth and progression of tumor have been recognized were affected by multidirectional interactions of secreted proteins (secretome), which were produced by the cells within tumor microenvironment. Focus on general secreted molecules of living cells via proteomic tools, is promising for investigating cell communication. Stable isotope labeling by amino acids in cell culture (SILAC) is a metabolic labeling strategy for quantitative analysis, which is gaining popularity because of its ease of implementation, the high quality of quantitative data obtained, robustness and compatibility with existing experimental workflows. Therefore, SILAC-based quantitative secretome analysis was employed for investigating interactions between epithelia and tumor by in vitro modulating colon cancer microenvironment with established co-culture system, which simplified the complexity of cancer microenvironment, also tracked secreted protein changes and their associated biological roles between epithelia and cancer cells. A series of tumor associated secreted proteins was quantitated and investigated in our study. So, the results give a new insight on communications between tumor and epithelia as well as cancer biotherapy by inhibiting interactions of them.

Quantitative protein expression profiling of 14-3-3 isoforms in human renal carcinoma shows 14-3-3 epsilon is involved in limitedly increasing renal cell proliferation.

14‐3‐3 proteins regulate many cellular processes that are implicated in cancer development, and the seven 14‐3‐3 isoforms have different expression level and isoform‐specific roles in different tumors. However, the biological functions of 14‐3‐3 proteins and their correlations with renal carcinoma have not been investigated so far. In our study, the expression profiles and functional characterization of 14‐3‐3 proteins were discovered by a sensitive stable isotope labeling with amino acids in cell culture based quantitative proteomics analysis in human renal carcinoma tissues. We found that 14‐3‐3ε was up‐regulated with 1.44‐fold changes in renal cancerous tissues compared with that in counterpart kidney tissues, and 14‐3‐3σ was almost not detected in both tissues due to its DNA highly methylated in our previous reports. The other five isoforms almost have similar expression level in two states of renal tissues. The following RT‐PCR, Western blot and immunohistochemistry analysis for specific 14‐3‐3 isoform expression were all consistent with the quantitative proteomic data. Furthermore, the overexpression of 14‐3‐3ε in vitro can limitedly prompt the abnormal growth of renal tumor cells.

Isoform-specific expression and characterization of 14-3-3 proteins in human glioma tissues discovered by stable isotope labeling with amino acids in cell culture-based proteomic analysis.

Human 14‐3‐3 proteins have isoform‐specific expression and functions in different kinds of normal or tumor cells and tissues. However, the expression profiling of 14‐3‐3 proteins and isoform‐specific biological functions are unclear in human glioma so far. In our study, the expression levels and characterization of 14‐3‐3 isoforms in human glioma tissues were investigated by a sensitive, accurate stable isotope labeling with amino acids in cell culture‐based quantitative proteomic strategy. As a result, except unexpressed 14‐3‐3σ, the other six isoforms, with different expression levels, were existed in glioma tissues and para‐cancerous brain tissues (PBTs). 14‐3‐3β and η were upregulated, whereas 14‐3‐3ζ was downregulated in glioma tissues compared with that in PBTs. And the other three isoforms 14‐3‐3ε, θ, and γ had similar expression levels in human glioma tissues and PBTs. Western blot and immunohistochemistry analysis were both consistent with the quantitative proteomic data. The loss of expression of 14‐3‐3σ was further discovered due to DNA high methylation in its coding region in glioma by methylation‐specific PCR analysis. These results indicated that the four isoforms, including 14‐3‐3β, η, ζ, and σ, may play important roles in tumorigenesis of human glioma, which is probably used as potential biomarkers for diagnosis and targets for treatment of human gliomas in future.

Quantitative proteomic analysis of HepG2 cells treated with quercetin suggests IQGAP1 involved in quercetin-induced regulation of cell proliferation and migration.

Quercetin, a wild distributed bioflavonoid, exhibits antitumor effects on murine models by inducing apoptosis and inhibiting growth of many cancer cell lines, while proteins involved in antitumor effects at proteomic level are still unclear. In our study, we used a quantitative proteomic strategy termed stable isotope labeling by amino acids in cell culture (SILAC)-mass spectrometry (MS) to study the differential proteomic profiling of HepG2 cells treated by quercetin. In all, there were 70 changed proteins among those quantified proteins in HepG2 cells treated by 50 microM quercetin for 48 h, and 14 proteins showed significant upregulation, whereas 56 proteins were downregulated. The functional classification of changed proteins includes signaling protein, protein synthesis, cytoskeleton, metabolism, etc. Of these, Ras GTPase-activating-like protein (IQGAP1) and beta-tubulin were found to be reduced at a large degree. The migration inhibition of HepG2 cells can be induced by quercetin, and the RNA and protein expression level of IQGAP1 and beta-tubulin were respectively decreased obviously in HepG2 cells exposed to quercetin for 48 h in the scratch migration assay. The downregulated expression of IQGAP1 and beta-tubulin by quercetin treatment correlated with cell migration ability, and quercetin probably inhibits HepG2 proliferation and migration through IQGAP1 and beta-tubulin expression changes and their interactions with other proteins.