Genetically Expressed RNA Strand Displacement for Cellular Manipulation.

Nucleic acid strand displacement is a pivotal concept in dynamic nucleic acid nanotechnologies, which has been extensively investigated and applied across various fields. Compared with DNA systems, the genetically expressed RNA strand displacement technology offers unique advantages for construction of genetic circuits in living cells, where RNA expression and modulation may be seamlessly integrated into the genomic network for long-term and stable regulations of diversified biological functionalities. This Concept paper provides an overview of previous efforts on developments of synthetic gene circuits through utilization of RNA strand displacement, including our endeavors in this field. Moreover, future prospects, potential applications and challenges of the genetically expressed RNA strand displacement technology are also discussed.

Building RNA-Mediated Artificial Signaling Pathways between Endogenous Genes.

ConspectusSophisticated genetic networks play a pivotal role in orchestrating cellular responses through intricate signaling pathways across diverse environmental conditions. Beyond the inherent complexity of natural cellular signaling networks, the construction of artificial signaling pathways (ASPs) introduces a vast array of possibilities for reshaping cellular responses, enabling programmable control of living organisms. ASPs can be integrated with existing cellular networks and redirect output responses as desired, allowing seamless communication and coordination with other cellular processes, thereby achieving designable transduction within cells. Among diversified ASPs, establishing connections between originally independent endogenous genes is of particular significance in modifying the genetic networks, so that cells can be endowed with new capabilities to sense and deal with abnormal factors related to differentiated gene expression (i.e., solve the issues of the aberrant gene expression induced by either external or internal stimuli). In a typical scenario, the two genes X and Y in the cell are originally expressed independently. After the introduction of an ASP, changes in the expression of gene X may exert a designed impact on gene Y, subsequently inducing the cellular response related to gene Y. If X represents a disease signal and Y serves as a therapeutic module, the introduction of the ASP empowers cells with a new spontaneous defense system to handle potential risks, which holds great potential for both fundamental and translational studies.In this Account, we primarily review our endeavors in the construction of RNA-mediated ASPs between endogenous genes that can respond to differentiated RNA expression. In contrast to other molecules that may be restricted to specific pathways, synthetic RNA circuits can be easily utilized and expanded as a general platform for constructing ASPs with a high degree of programmability and tunability for diversified functionalities through predictable Watson-Crick base pairing. We first provide an overview of recent advancements in RNA-based genetic circuits, encompassing but not limited to utilization of RNA toehold switches, siRNA and CRISPR systems. Despite notable progress, most reported RNA circuits have to contain at least one exogenous RNA X as input or one engineered RNA Y as a target, which is not suitable for establishing endogenous gene connections. While exogenous RNAs can be engineered and controlled as desired, constructing a general and efficient platform for manipulation of naturally occurring RNAs poses a formidable challenge, especially for the mammalian system. With a focus on this goal, we are devoted to developing efficient strategies to manipulate cell responses by establishing RNA-mediated ASPs between endogenous genes, particularly in mammalian cells. Our step-by-step progress in engineering customized cell signaling circuits, from bacterial cells to mammalian cells, from gene expression regulation to phenotype control, and from small RNA to long mRNA of low abundance and more complex secondary structures, is systematically described. Finally, future perspectives and potential applications of these RNA-mediated ASPs between endogenous genes are also discussed.

Effects of Helicobacter pylori and Moluodan on the Wnt/ß-catenin signaling pathway in mice with precancerous gastric cancer lesions.

BACKGROUND: Helicobacter pylori (H. pylori) is the primary risk factor for gastric cancer (GC), the Wnt/ß-Catenin signaling pathway is closely linked to tumourigenesis. GC has a high mortality rate and treatment cost, and there are no drugs to prevent the progression of gastric precancerous lesions to GC. Therefore, it is necessary to find a novel drug that is inexpensive and preventive to against GC. AIM: To explore the effects of H. pylori and Moluodan on the Wnt/ß-Catenin signaling pathway and precancerous lesions of GC (PLGC). METHODS: Mice were divided into the control, N-methyl-N-nitrosourea (MNU), H. pylori + MNU, and Moluodan groups. We first created an H. pylori infection model in the H. pylori + MNU and Moluodan groups. A PLGC model was created in the remaining three groups except for the control group. Moluodan was fed to mice in the Moloudan group ad libitum. The general condition of mice were observed during the whole experiment period. Gastric tissues of mice were grossly and microscopically examined. Through quantitative real-time PCR (qRT-PCR) and Western blotting analysis, the expression of relevant genes were detected. RESULTS: Mice in the H. pylori + MNU group showed the worst performance in general condition, gastric tissue visual and microscopic observation, followed by the MNU group, Moluodan group and the control group. QRT-PCR and Western blotting analysis were used to detect the expression of relevant genes, the results showed that the H. pylori + MNU group had the highest expression, followed by the MNU group, Moluodan group and the control group. CONCLUSION: H. pylori can activate the Wnt/ß-catenin signaling pathway, thereby facilitating the development and progression of PLGC. Moluodan suppressed the activation of the Wnt/ß-catenin signaling pathway, thereby decreasing the progression of PLGC.

Harnessing Catalytic RNA Circuits for Construction of Artificial Signaling Pathways in Mammalian Cells.

Engineering of genetic networks with artificial signaling pathways (ASPs) can reprogram cellular responses and phenotypes under different circumstances for a variety of diagnostic and therapeutic purposes. However, construction of ASPs between originally independent endogenous genes in mammalian cells is highly challenging. Here we report an amplifiable RNA circuit that can theoretically build regulatory connections between any endogenous genes in mammalian cells. We harness the system of catalytic hairpin assembly with combination of controllable CRISPR-Cas9 function to transduce the signals from distinct messenger RNA expression of trigger genes into manipulation of target genes. Through introduction of these RNA-based genetic circuits, mammalian cells are endowed with autonomous capabilities to sense the changes of RNA expression either induced by ligand stimuli or from various cell types and control the cellular responses and fates via apoptosis-related ASPs. Our design provides a generalized platform for construction of ASPs inside the genetic networks of mammalian cells based on differentiated RNA expression.

Chemically Cross-Linked Hammerhead Ribozyme as an Efficient RNA Interference Tool.

RNA-cleaving ribozymes are promising candidates as general tools of RNA interference (RNAi) in gene manipulation. However, compared with other RNA systems, such as siRNA and CRISPR technologies, the ribozyme tools are still far from broad applications on RNAi due to their poor performance in the cellular context. In this work, we report an efficient RNAi tool based on chemically modified hammerhead ribozyme (HHR). By the introduction of an intramolecular linkage into the minimal HHR to reconstruct the distal interaction within the tertiary ribozyme structure, this cross-linked HHR exhibits efficient RNA substrate cleavage activities with almost no sequence constraint. Cellular experiments suggest that both exogenous and endogenous RNA expression can be dramatically knocked down by this HHR tool with levels comparable to those of siRNA. Unlike the widely applied protein-recruiting RNA systems (siRNA and CRISPR), this ribozyme tool functions solely on RNA itself with great simplicity, which may provide a new approach for gene manipulation in both fundamental and translational studies.

Establishing artificial gene connections through RNA displacement-assembly-controlled CRISPR/Cas9 function.

Construction of synthetic circuits that can reprogram genetic networks and signal pathways is a long-term goal for manipulation of biosystems. However, it is still highly challenging to build artificial genetic communications among endogenous RNA species due to their sequence independence and structural diversities. Here we report an RNA-based synthetic circuit that can establish regulatory linkages between expression of endogenous genes in both Escherichiacoli and mammalian cells. This design employs a displacement-assembly approach to modulate the activity of guide RNA for function control of CRISPR/Cas9. Our experiments demonstrate the great effectiveness of this RNA circuit for building artificial connections between expression of originally unrelated genes. Both exogenous and naturally occurring RNAs, including small/microRNAs and long mRNAs, are capable of controlling expression of another endogenous gene through this approach. Moreover, an artificial signal pathway inside mammalian cells is also successfully established to control cell apoptosis through our designed synthetic circuit. This study provides a general strategy for constructing synthetic RNA circuits, which can introduce artificial connections into the genetic networks of mammalian cells and alter the cellular phenotypes.

Controllable DNA hybridization by host-guest complexation-mediated ligand invasion.

Dynamic regulation of nucleic acid hybridization is fundamental for switchable nanostructures and controllable functionalities of nucleic acids in both material developments and biological regulations. In this work, we report a ligand-invasion pathway to regulate DNA hybridization based on host-guest interactions. We propose a concept of recognition handle as the ligand binding site to disrupt Watson-Crick base pairs and induce the direct dissociation of DNA duplex structures. Taking cucurbit[7]uril as the invading ligand and its guest molecules that are integrated into the nucleobase as recognition handles, we successfully achieve orthogonal and reversible manipulation of DNA duplex dissociation and recovery. Moreover, we further apply this approach of ligand-controlled nucleic acid hybridization for functional regulations of both the RNA-cleaving DNAzyme in test tubes and the antisense oligonucleotide in living cells. This ligand-invasion strategy establishes a general pathway toward dynamic control of nucleic acid structures and functionalities by supramolecular interactions.

Crosstalk between lncRNA DANCR and miR-125b-5p in HCC cell progression.

OBJECTIVE: To investigate the mechanism of long noncoding RNA (lncRNA) DANCR on the progression of hepatocellular carcinoma (HCC) cells. METHODS: The expression levels of DANCR and miR-125b-5p were measured in normal hepatocytes (LO2) and HCC cell lines by quantitative reverse transcription polymerase chain reaction. HepG2 and Huh-7 cells were transfected with sh-DANCR, the negative control (sh-NC), miR-125b-5p mimic, or mimic NC or cotransfected with sh-DANCR and miR-125b-5p inhibitor. HCC cell proliferation was assessed through CCK8 and plate colony formation assay. Western blot quantified the expression levels of Bcl-2, Bax, caspase-3, and cleaved-caspase-3. Apoptotic rate was detected as well as migratory and invasive capacities. The implication of the MAPK signal pathway was assessed by detecting the expression levels of p38, ERK1/2, JNK, p-p38, p-ERK1/2, and p-JNK. Interactions between DANCR and miR-125b-5p were detected by dual luciferase reporter assay. RESULTS: In HCC cells, DANCR was highly expressed and miR-125b-5p was decreased. sh-DANCR or miR-125b-5p mimic stimulation reduced HepG2 or Huh-7 cell progression while promoted cell apoptosis evidenced by increased apoptotic rate, elevated levels of Bax and cleaved-caspase-3, and decreased Bcl-2. Moreover, the migration rate and invasiveness of HCC cells were also inhibited by sh-DANCR and miR-125b-5p mimic. Levels of p-p38/p38, p-ERK1/2/ERK1/2, and p-JNK/JNK were suppressed by sh-DANCR and miR-125b-5p mimic. LncRNA DANCR negatively targeted and directly bound to miR-125b-5p. Knockdown of miR-125b-5p could reverse the inhibitory effects of sh-DANCR on HCC cells. CONCLUSION: In HCC cells, lncRNA DANCR sponges miR-125b-5p and activates MAPK pathway, thus facilitating HCC cell progression.

Vaginal dose of radical radiotherapy for cervical cancer in China: a multicenter study.

BACKGROUND: The posterior-inferior border of symphysis (PIBS) point system is a novel vaginal dose-reporting method and is a simple and reliable method proposed by the Medical University of Vienna proposed for both external-beam radiotherapy (EBRT) and brachytherapy (BT). In this multicenter study, we sought to first evaluate the vaginal radiation dose in Chinese cervical cancer patients according to the PIBS point system and then to analyze the factors influencing the dose distribution. METHODS: We collected data from the medical records of 936 cervical cancer patients who underwent concurrent radiochemotherapy at 13 different institutions in China. Radiation doses at points A, PIBS+ 2 cm, PIBS and PIBS-2 cm, International Commission on Radiation Units (ICRU)-R and ICRU-B were measured. RESULTS: The median total doses in EQD2α/ß = 3 at points PIBS+ 2 cm, PIBS and PIBS-2 cm were 82.5 (52.7-392.1) Gy, 56.2 (51.4-82.1) Gy and 2.6 (0.9-7.4) Gy, respectively. The median total doses in EQD2α/ß = 3 at ICRU-R and ICRU-B were 77.5 (54.8-132.4) Gy and 79.9 (60.7-133.7) Gy, respectively. The mean vaginal reference length (VRL) was 4.6 ± 1.0 cm (median, 4.5 cm). In patients with VRL ≤4.5 cm, the mean total doses in EQD2α/ß = 3 at points PIBS+ 2 cm, PIBS and PIBS-2 cm were 128.5, 60.7 and 0.8 Gy, respectively. In patients with VRL > 4.5 cm, the mean total doses at these three points were 68.9, 0.5 and 54.5 Gy, respectively. Classification of patients revealed significant differences (P < 0.05) between these two groups. CONCLUSIONS: With the PIBS point system, Chinese patients with a shorter VRL of < 4.5 cm received higher radiation doses at the PIBS+ 2 cm, PIBS and PIBS-2 cm points than European and American patients. Further studies are required to establish the dose-effect relationships with these points as references. The study was registered as a clinical trial (NCT03257475) on August 22, 2017.

Novel Metabolites from the Endophytic Fungus Chaetomium subaffine L01.

One natural p-terphenyl glycoside, gliocladinin C, and two furano-polyene derivatives, chaetominins A and B, were isolated from potato endophytic fungus Chaetomium subaffine. The absolute configurations of these compounds were elucidated by HR-ESI-MS, NMR, the DP4+ probabilities and electronic circular dichroism (ECD) spectra. Furthermore, gliocladinin C and chaetominin A showed cytotoxic activity against two selected human tumor cell lines (Hep-2 and HepG-2).

Association of Crohn's Disease with Aryl Hydrocarbon Receptor Gene Polymorphisms in Patients from Southeast China.

Aims The aryl hydrocarbon receptor (AhR) plays a pivotal role in regulating the innate and the acquired immune systems. The present study aimed to investigate the association of Crohn's disease (CD) with AhR polymorphisms in a cohort of patients from Southeast China. Methods An improved multiple ligase detection reaction technique was applied to examine the polymorphisms of rs2158041, rs2066853, and rs10249788 in 310 patients with CD and 573 controls. Results Compared to the controls, the variant allele (T) and genotype (CT+TT) of rs2158041 were less frequent in patients with CD (both p < 0.05). Similar conclusions were drawn from patients with ileal CD and with stricture CD as compared to the controls (all p < 0.0083). However, no significant differences were observed in allele and genotype frequencies of rs2066853 and rs10249788 between patients with CD and the controls (all p > 0.05). Although rs2158041 and rs10249788 were in complete linkage disequilibrium with rs2066853, respectively, only the frequency of haplotype (TG) formed by rs2158041 and rs2066853 was significantly lower in patients with CD than that in the controls (p < 0.05). Conclusions AhR (rs2158041) might be a susceptible locus for CD, especially for the two subtypes: ileal CD and stricture CD.

Association of ulcerative colitis with solute-linked carrier family 26 member A3 gene polymorphisms and its expression in colonic tissues in Chinese patients.

PURPOSE: Abnormalities of the solute-linked carrier family 26 member A3 (SLC26A3) are implicated in the pathogenesis of several diseases including ulcerative colitis (UC). The short communication aimed at investigating the associations of UC with SLC26A3 (rs17154444, rs7810937, rs7785539, rs2108225 and rs6951457) polymorphisms and its expression in colonic tissues. METHODS: The techniques of SNaPshot method, quantitative real-time PCR and immunohistochemical analysis were conducted. RESULTS AND CONCLUSION: We found that the rs2108225 variation in SLC26A3 might increase the risk of UC and affect its expression at both the mRNA and protein levels in colonic tissues of patients with UC. Moreover, the rs17154444 variation might influence the severity of UC.

Potentials of the elevated circulating miR-185 level as a biomarker for early diagnosis of HBV-related liver fibrosis.

Early diagnosis of liver fibrosis is critical for early intervention and prognosis of various chronic liver diseases. Conventional repeated histological assessment is impractical due to the associated invasiveness. In the current study, we evaluated circulating miR-185 as a potential biomarker to predict initiation and progression of liver fibrosis. We found that miR-185 was significantly up-regulated in blood specimens from patients with HBV-liver fibrosis and rats with liver fibrosis, the miR-185 levels were correlated with liver fibrosis progression, but not with the different viral loads in HBV-infected patients. miR-185 was observed in collagen deposition regions during advanced liver fibrosis. We found that differences in miR-185 levels facilitated the discrimination between early-staged or advanced-staged liver fibrosis and the healthy controls with high specificity, sensitivity, and likelihood ratio using receiver-operator characteristic analysis. miR-185 targeted SREBF1, and increased expression of COL1A1 and a-SMA genes that are hallmarks of liver fibrosis. Our data supported that circulating miR-185 levels could be used as potential biomarkers for the early diagnosis of liver fibrosis.

MiR-9a-5p regulates proliferation and migration of hepatic stellate cells under pressure through inhibition of Sirt1.

AIM: To reveal the functions of microRNAs (miRNAs) with respect to hepatic stellate cells (HSCs) in response to portal hypertension. METHODS: Primary rat HSCs were exposed to static water pressure (10 mmHg, 1 h) and the pressure-induced miRNA expression profile was detected by next-generation sequencing. Quantitative real-time polymerase chain reaction was used to verify the expression of miRNAs. A potential target of MiR-9a-5p was measured by a luciferase reporter assay and Western blot. CCK-8 assay and Transwell assay were used to detect the proliferation and migration of HSCs under pressure. RESULTS: According to the profile, the expression of miR-9a-5p was further confirmed to be significantly increased after pressure overload in HSCs (3.70 ± 0.61 vs 0.97 ± 0.15, P = 0.0226), which resulted in the proliferation, migration and activation of HSCs. In vivo, the up-regulation of miR-9a-5p (2.09 ± 0.91 vs 4.27 ± 1.74, P = 0.0025) and the down-regulation of Sirt1 (2.41 ± 0.51 vs 1.13 ± 0.11, P = 0.0006) were observed in rat fibrotic liver with portal hypertension. Sirt1 was a potential target gene of miR-9a-5p. Through restoring the expression of Sirt1 in miR-9a-5p transfected HSCs on pressure overload, we found that overexpression of Sirt1 could partially abrogate the miR-9a-5p mediated suppression of the proliferation, migration and activation of HSCs. CONCLUSION: Our results suggest that during liver fibrosis, portal hypertension may induce the proliferation, migration and activation of HSCs through the up-regulation of miR-9a-5p, which targets Sirt1.

Overexpression of miR-126 inhibits the activation and migration of HSCs through targeting CRK.

BACKGROUND & AIMS: MicroRNAs (miRNAs) have been shown to play essential roles in HSCs activation which contributes to hepatic fibrosis. Our previous miRNA microarray results suggested that miR-126 might be decreased during HSCs activation as other studies. The aim of this study is to investigate the role of miR-126 during HSCs activation. METHODS: In this study, the expression of miR-126 during HSCs activation was measured and confirmed by qRT-PCR. Then, miR-126 expression was restored by transfection of lentivirus vector encoding miR-126. Futhermore, cell proliferation was assayed by the cell counting kit-8 (CCK-8), cell migration was assayed by transwell assay, and the markers of activation of HSCs, α-SMA and collagen type I, were assayed by qRT-PCR, Western Blotting, Immunostaining and ELISA. Luciferase reporter assay was used to find the target of miR-126, and Western Blotting and Immunostaining was used to validate the target of miR-126. Then, the expression and the role of the target of miR-126 during HSCs activation was further assessed. RESULTS: The expression of miR-126 was confirmed to be significantly decreased during HSCs activation. Overexpression of miR-126 significantly inhibited HSCs migration but did not affect HSCs proliferation. The expression of α-SMA and collagen type I were both obviously decreased by miR-126 restoration. CRK was found to be the target of miR-126 and overexpression of miR-126 significantly inhibited CRK expression. And it was found that overexpression of CRK also significantly decreased miR-126 expression and promoted HSCs activation. CONCLUSIONS: Our study showed that overexpression of miR-126 significantly inhibited the activation and migration of HSCs through targeting CRK which can also decrease miR-126 expression and promote HSCs activation.

[Setting of logos on tobacco control information at outlets for retails and restaurants in 12 cities of China].

OBJECTIVE: To explore the setting of logos on tobacco control information at outlets for retails and restaurants in 12 selected cities of China. METHODS: For all the shops for retail of tobacco, alcohol, food and restaurants under survey in 333 blocks of 12 cities(Beijing, Tianjin, Shanghai, Qingdao, Hangzhou, Shaoxing, Suzhou, Nantong, Zhenjiang, Chengdu, Xining and Harbin), setting and contents of logos on tobacco control information, inside and outside them were examined. RESULTS: 45 700 objectives were included in the study. Among all types of retail shops, the identification rate of tobacco control information at the entrance and inside were 3.6% and 4.4%, with an overall identification rate as 7.0%. The overall rate at the entrance of all the restaurants was 4.6% which was larger than the ones at the retail shops. Our result showed that there were differences between cities and types of establishments and higher rates seen in the larger ones. Of all the places that having had placement of information on tobacco control, only 18.5% of them had put them both inside and outside. Slogans or images on "No Smoking" were the main forms of information but less than 10% of them would show signs as 'exclusive non-smoking'. CONCLUSION: Data from our survey showed that the identification rate of tobacco control information was at a low level in 12 cities, and differences were seen between cities, size of establishment, that called for improvement of the existing tobacco control policies in China.

Loss of expression of miR-335 is implicated in hepatic stellate cell migration and activation.

Activation and migration of resident stellate cells (HSCs) within the hepatic space of Disse play an important role in hepatic fibrosis, which accounts for the increased numbers of activated HSCs in areas of inflammation during hepatic fibrosis. Currently, microRNAs have been found to play essential roles in HSC differentiation, proliferation, apoptosis, fat accumulation and collagen production. However, little is known about microRNA mediated HSC activation and migration. In this study, the miRNA expression profiles of quiescent HSCs, partially activated HSCs and fully activated HSCs were compared in pairs. Gene ontology (GO) and GO-Map network analysis indicated that the activation of HSCs was regulated by microRNAs. Among them miR-335 was confirmed to be significantly reduced during HSC activation by qRT-PCR, and restoring expression of miR-335 inhibited HSC migration and reduced α-SMA and collagen type I. Previous study revealed that tenascin-C (TNC), an extracellular matrix glycoprotein involved in cell migration, might be a target of miR-335. Therefore, we further studied the TNC expression in miR-335 over-expressed HSCs. Our data showed that exogenous TNC could enhance HSC migration in vitro and miR-335 restoration resulted in a significant inhibition of TNC expression. These results demonstrated that miR-335 restoration inhibited HSC migration, at least in part, via downregulating the TNC expression.

The piggyBac transposon is an integrating non-viral gene transfer vector that enhances the efficiency of GDEPT.

Gene-directed enzyme prodrug therapy (GDEPT) is a strategy developed to selectively target cancer cells. However, the clinical benefit is limited due to its poor gene transfer efficiency. To overcome this obstacle, we took advantage of piggyBac (PB) transposon, a natural non-viral gene vector that can induce stable chromosomal integration and persistent gene expression in vertebrate cells, including human cells. To determine whether the vector can also mediate stable gene expression in ovarian cancer cells, we constructed a PB transposon system that simultaneously expresses the Herpes simplex virus thymidine kinase (HSV-tk) gene and the monomeric red fluorescent protein (mRFP1) reporter gene. The recombinant plasmid, pPB/TK, was transfected into ovarian adenocarcinoma cells SKOV3 with FuGENE HD reagent, and the efficiency was given by the percentage of mRFP1-positive cells detected by flow cytometry and confocal microscopy. The specific expression of HSV-tk in transfected cells was confirmed by RT-PCR and western blotting. The sensitivity of transfected cells to pro-drug ganciclovir (GCV) was determined by methylthiazoletetrazolium (MTT) assay. A total of 56.4 +/- 8.4% cells transfected with pPB/TK were mRFP1 positive, compared to no measurable mRFP1 expression in pORF-HSVtk-transfected cells. The expression level of HSV-tk in pPB/TK-transfected cells was 10 times higher than in pORF-HSVtk-transfected cells. The results show that pPB/TK transfection increases the sensitivity of cells to GCV in a dose-dependent manner. Our data indicate that the PB transposon system could enhance the anti-tumor efficiency of GDEPT in ovarian cancer.

Isolation and characterization of the human D-glyceric acidemia related glycerate kinase gene GLYCTK1 and its alternatively splicing variant GLYCTK2.

Deficiency of human glycerate kinase leads to D-glycerate acidemia/D-glyceric aciduria. Through PCR cloning assisted by in silico approach, we isolated the human glycerate kinase genes--Glycerate Kinase 1 (GLYCTK1) and its alternatively splicing variant--Glycerate Kinase 2 (GLYCTK2), which might be associated with D-glycerate acidemia/D-glyceric aciduria. The locus of GLYCTK gene is mapped to 3p21. PCR amplification in seventeen human tissue cDNAs revealed that both GLYCTK1 and GLYCTK2 are expressed widely almost in all these tissues. The expression of mouse Glyctk in various tissues was demonstrated by in situ hybridization. Both GLYCTK1 and GLYCTK2 proteins are localized in cytosol, and GLYCTK2 proteins are specifically localized in mitochondria. Present results revealed the characteristic expression pattern of murine Glyctk in neural system, skeleton muscle, supporting that glycerate kinase is implicated in D-glycerate acidemia/D-glyceric aciduria.

Blocking of N-acetylglucosaminyltransferase V induces cellular endoplasmic reticulum stress in human hepatocarcinoma 7,721 cells.

N-acetylglucosaminyltransferase V (GnT-V) is an important tumorigenesis and metastasis-associated enzyme. To study its biofunction, the GnT-V stably suppressed cell line (GnT-V-AS/7,721) was constructed from 7,721 hepatocarcinoma cells in previous study. In this study, cDNA array gene expression profiles were compared between GnT-V-AS/7,721 and parental 7,721 cells. The data indicated that GnT-V-AS/7,721 showed a characteristic expression pattern consistent with the ER stress. The molecular mechanism of the ER stress was explored in GnT-V-AS/7,721 by the analysis on key molecules in both two unfolded protein response (UPR) pathways. For ATF6 and Ire1/XBP-1 pathway, it was evidenced by the up-regulation of BIP at mRNA and protein level, and the appearance of the spliced form of XBP-1. As for PERK/eIF2alpha pathway, the activation of ER eIF2alpha kinase PERK was observed. To confirm the results from GnT-V-AS/7,721 cells, the key molecules in the UPR were examined again in 7,721 cells interfered with the GnT-V by the specific RNAi treatment. The results were similar with those from GnT-V-AS/7721, indicating that blocking of GnT-V can specifically activate ER stress in 7,721 cells. Rate of (3)H-Man incorporation corrected with rate of (3)H-Leu incorporation in GnT-V-AS/7,721 was down-regulated greatly compared with the control, which demonstrated the deficient function of the enzyme synthesizing N-glycans after GnT-V blocking. Moreover, the faster migrating form of chaperone GRP94 associated with the underglycosylation, and the extensively changed N-glycans structures of intracellular glycoproteins were also detected in GnT-V-AS/7,721. These results supported the mechanism that blocking of GnT-V expression impaired functions of chaperones and N-glycan-synthesizing enzymes, which caused UPR in vivo.