Identification of plasma microRNA-22 as a marker for the diagnosis, prognosis, and chemosensitivity prediction of osteosarcoma.

OBJECTIVE: MicroRNA (miR)-22 plays crucial roles in malignant tumors and is involved in regulation of chemosensitivity. Additionally, altered expression of circulating miR-22 has been reported in various cancers. This study was designed to investigate plasma miR-22 expression in patients with osteosarcoma (OS) and determine its diagnostic, prognostic, and chemosensitivity prediction value. METHODS: Plasma miR-22 levels in 120 patients with OS and 120 healthy controls were detected by real-time quantitative reverse transcription PCR. Associations of plasma miR-22 expression with the patients' clinicopathological features and prognosis were then assessed. RESULTS: Plasma miR-22 levels in patients with OS were significantly lower than those in healthy controls. Low plasma miR-22 levels were correlated with large tumor size, advanced clinical stages, positive distant metastasis, and poor tumor response to preoperative chemotherapy. Plasma miR-22 could discriminate OS patients from controls and distinguish patients with a good response to therapy from those with a poor response to therapy. Multivariate analysis revealed that low plasma miR-22 expression was a significant independent predictor of unfavorable prognosis. CONCLUSIONS: Altered plasma levels of miR-22 might serve as a novel, noninvasive biomarker for OS diagnosis, prognosis, and chemosensitivity prediction.

[Regularities of Acupoint Combinations for Facial Paralysis in Chinese Ancient Times Analyzed in Accordance with Modern Complex Network Community Framework Partition].

OBJECTIVE: To analyze the characteristics of acupoint combinations (formula) for facial palsy in ancient lite-rature, so as to optimize the related acupoint prescriptions in clinical practice. METHODS: The database of ancient acupoint prescriptions for facial paralysis was established first after searching and collecting the related acu-moxibustion literature (178 categories as monographs, specific volumes of a book, etc.) by using key words of "facial palsy" or "deviation of the eye and mouth" including Chinese terms of "Diaoxianfeng ()" "Wopi ()"and "Kouwo()". The characteristics of acupoint selection were analyzed and acupoint prescriptions were optimized using bipartite network community structure partition. RESULTS: The results indicated that of the 56 acupoints for facial palsy collected in the literature, Shuigou (GV 26), Dicang (ST 4), Jiache (ST 6), Hegu(LI 4), Fengchi(GB 20), Zusanli (ST 36), Neiting (ST 44), Tinghui (GB 2), Chengjiang (CV 24) and Shangguan (GB 3) are the core acupoints in clinical application. The acupoints of Dicang (ST 4), Jiache (ST 6), Shuigou (GV 26), Fengchi(GB 20), Neiting (ST 44), Hegu (LI 4), Zusanli (ST 36), Lieque (LU 7), Shangguan (GB 3) and Erjian (LI 2) are most frequently selected for forming prescriptions.The optimized acupoint prescriptions are composed of Tinghui (GB 2), Fengchi (GB 20), Fengshi (GB 31), Xuanzhong (GB 39), Dicang (ST 4), Jiache (ST 6), Zusanli (ST 36), Dazhui (GV 14), Baihui (GV 20), Quchi (LI 11) and Jianyu (LI 15). Core acupoints were mainly distributed on the Stomach Meridian of Foot Yangming, Large Intestine Meridian of Hand Yangming and the Governor Vessel. Acupoints at the face and head are used more frequently than those in any other parts of the body. Specific acupoints are used more frequently than non-specific acupoints, among which Conf-luent Acupoints are most frequently used. CONCLUSIONS: In the treatment of facial paralysis in ancient China, selection of local acupoints is the basic principle and some specific acupoints are the core acupoints in ancient acupuncture prescriptions.

[Our considerations about research on regularities of acupoint combination based on bipartite network community structure partition].

Acupoint compatibility is not only a foundation for formulating acupoint recipes in clinical practice, but also a core of meridian and acupoint theory. Many studies have revealed that the application of multiple-acupoints combination in the treatment of different clinical conditions or illnesses may lead to three outcomes, i.e., synergistic effect, antagonistic effect and no effect. Therefore, the application of multiple acupoints combination is definitely not a simple result of 1 + 1 = 2, or 2 - 1 = 1 in the management of many clinical disorders, and rather, the reasonable combination of multiple acupoints is a non-liner, dynamic and complicated issue. It has been well documented that the complex biological network consists of modules/communities, a set of nodes that are more densely inter-connected among themselves than with the rest of the network, which can be detected by community detection according to their modularity. After analyzing the current status and development trends of acupoint comparability researches in both China and other countries of the world, the authors of the present paper put forward some ideas and methods for the research of correlation, composing characteristics and dynamically evolutionary regularities of acupoint combination from the macroscopic and local aspects, dynamic and static angles on the basis of the theory of community structure theory of complex networks.

Heat shock protein 70 interacts with aquaporin-2 and regulates its trafficking.

The trafficking of aquaporin-2 (AQP2) involves multiple complex pathways, including regulated, cAMP-, and cGMP-mediated pathways, as well as a constitutive recycling pathway. Although several accessory proteins have been indirectly implicated in AQP2 recycling, the direct protein-protein interactions that regulate this process remain largely unknown. Using yeast two-hybrid screening of a human kidney cDNA library, we have identified the 70-kDa heat shock proteins as AQP2-interacting proteins. Interaction was confirmed by mass spectrometry of proteins pulled down from rat kidney papilla extract using a GST-AQP2 C-terminal fusion protein (GST-A2C) as a bait, by co-immunoprecipitation (IP) assays, and by direct binding assays using purified hsc70 and the GST-A2C. The direct interaction of AQP2 with hsc70 is partially inhibited by ATP, and the Ser-256 residue in the AQP2 C terminus is important for this direct interaction. Vasopressin stimulation in cells enhances the interaction of hsc70 with AQP2 in IP assays, and vasopressin stimulation in vivo induces an increased co-localization of hsc70 and AQP2 on the apical membrane of principal cells in rat kidney collecting ducts. Functional knockdown of hsc70 activity in AQP2 expressing cells results in membrane accumulation of AQP2 and reduced endocytosis of rhodamine-transferrin. Our data also show that AQP2 interacts with hsp70 in multiple in vitro binding assays. Finally, in addition to hsc70 and hsp70, AQP2 interacts with several other key components of the endocytotic machinery in co-IP assays, including clathrin, dynamin, and AP2. To summarize, we have identified the 70-kDa heat shock proteins as a AQP2 interactors and have shown for hsc70 that this interaction is involved in AQP2 trafficking.

Alix (AIP1) is a vasopressin receptor (V2R)-interacting protein that increases lysosomal degradation of the V2R.

The vasopressin type 2 receptor (V2R) is a G protein-coupled receptor that plays a central role in renal water reabsorption. Termination of ligand (vasopressin) stimulation is an important physiological regulatory event, but few proteins that interact with the V2R during downregulation after vasopressin (VP) binding have been identified. Using yeast two-hybrid screening of a human kidney cDNA library, we show that a 100-kDa protein called ALG-2-interacting protein X (Alix) interacts with the last 29 amino acids of the V2R COOH terminus. This was confirmed by pull-down assays using a GST-V2R-COOH-tail fusion protein. Alix was immunolocalized in principal cells of the kidney, which also express the V2R. The function of the Alix-V2R interaction was studied by transfecting Alix into LLC-PK(1) epithelial cells expressing V2R-green fluorescent protein (GFP). Under basal conditions, V2R-GFP localized mainly at the plasma membrane. On VP treatment, V2R-GFP was internalized into perinuclear vesicles in the nontransfected cells. In contrast, V2R-GFP fluorescence was virtually undetectable 2 h after exposure to VP in cells that coexpressed Alix. Western blotting using an anti-GFP antibody showed marked degradation of the V2R after 2 h in the presence of VP and Alix, a time point at which little or no degradation was detected in the absence of Alix. In contrast, little or no degradation of the parathyroid hormone receptor was detectable in the presence or absence of Alix and/or the PTH ligand. The VP-induced disappearance of V2R-GFP was abolished by chloroquine, a lysosomal degradation inhibitor, but not by MG132, a proteosome inhibitor. These data suggest that Alix increases the rate of lysosomal degradation of V2R and may play an important regulatory role in the VP response by modulating V2R downregulation.

Inhibition of endocytosis causes phosphorylation (S256)-independent plasma membrane accumulation of AQP2.

Inhibition of clathrin-mediated endocytosis by expression of a GTPase-deficient dynamin mutant (dynamin-2/K44A) for 16 h results in an accumulation of plasma membrane aquaporin-2 (AQP2) in epithelial cells stably transfected with wild-type AQP2. We now show a similar effect of K44A dynamin in LLC-PK1 cells transfected with an S256 phosphorylation-deficient AQP2 mutant, AQP2(S256A), and in AQP2-transfected inner medullary collecting duct (IMCD) cells. More acute blockade of endocytosis in these cells with the cholesterol-depleting agent methyl-beta-cyclodextrin (mbetaCD; 10 mM) resulted in a rapid and extensive cell-surface accumulation of both wild-type AQP2 and AQP2 (S256A) within 15 min after treatment. This effect was similar to that induced by treatment of the cells with vasopressin. Blockade of endocytosis by mbetaCD was confirmed using quantitative analysis of FITC-dextran uptake and AQP2 membrane insertion was verified by cell-surface biotinylation. These data indicate that AQP2 recycles constitutively and rapidly between intracellular stores and the cell surface in LLC-PK1 and IMCD cells. The constitutive trafficking process is not dependent on phosphorylation of the serine-256 residue of AQP2, which is, however, an essential step for regulated vasopressin/cAMP-mediated translocation of AQP2. Our data show that rapid and extensive plasma membrane accumulation of AQP2 can occur in a vasopressin receptor (V2R)- and phosphorylation-independent manner, pointing to a potential means of bypassing the mutated V2R in X-linked nephrogenic diabetes insipidus to achieve cell surface expression of AQP2.

Aquaporin-2 localization in clathrin-coated pits: inhibition of endocytosis by dominant-negative dynamin.

Before the identification of aquaporin (AQP) proteins, vasopressin-regulated "water channels" were identified by freeze-fracture electron microscopy as aggregates or clusters of intramembraneous particles (IMPs) on hormonally stimulated target cell membranes. In the kidney collecting duct, these IMP clusters were subsequently identified as possible sites of clathrin-coated pit formation on the plasma membrane, and a clathrin-mediated mechanism for internalization of vasopressin-sensitive water channels was suggested. Using an antibody raised against the extracellular C loop of AQP2, we now provide direct evidence that AQP2 is concentrated in clathrin-coated pits on the apical surface of collecting duct principal cells. Furthermore, by using a fracture-label technique applied to LLC-PK(1) cells expressing an AQP2-c-myc construct, we show that AQP2 is located in IMP aggregates and is concentrated in shallow membrane invaginations on the surface of forskolin-stimulated cells. We also studied the functional role of clathrin-coated pits in AQP2 trafficking by using a GTPase-deficient dynamin mutation (K44A) to inhibit clathrin-mediated endocytosis. Immunofluorescence labeling and freeze-fracture electron microscopy showed that dominant-negative dynamin 1 and dynamin 2 mutants prevent the release of clathrin-coated pits from the plasma membrane and induce an accumulation of AQP2 on the plasma membrane of AQP2-transfected cells. These data provide the first direct evidence that AQP2 is located in clathrin-coated pits and show that AQP2 recycles between the plasma membrane and intracellular vesicles via a dynamin-dependent endocytotic pathway. We propose that the IMP clusters previously associated with vasopressin action represent sites of dynamin-dependent, clathrin-mediated endocytosis in which AQP2 is concentrated before internalization.

Functional role of the NPxxY motif in internalization of the type 2 vasopressin receptor in LLC-PK1 cells.

Interaction of the type 2 vasopressin receptor (V2R) with hormone causes desensitization and internalization. To study the role of the V2R NPxxY motif (which is involved in the clathrin-mediated endocytosis of several other receptors) in this process, we expressed FLAG-tagged wild-type V2R and a Y325F mutant V2R in LLC-PK1a epithelial cells that have low levels of endogenous V2R. Both proteins had a similar apical (35%) and basolateral (65%) membrane distribution. Substitution of Tyr325 with Phe325 prevented ligand-induced internalization of V2R determined by [3H]AVP binding and immunofluorescence but did not prevent ligand binding or signal transduction via adenylyl cyclase. Desensitization and resensitization of the V2R-Y325F mutation occurred independently of internalization. The involvement of clathrin in V2R downregulation was also shown by immunogold electron microscopy. We conclude that the NPxxY motif of the V2R is critically involved in receptor downregulation via clathrin-mediated internalization. However, this motif is not essential for the apical/basolateral sorting and polarized distribution of the V2R in LLC-PK1a cells or for adenylyl cyclase-mediated signal transduction.