1.Overexpression, purification and helicase activity analysis of recombinant human PIF1 protein.
Chinese Journal of Biotechnology 2009;25(2):268-273
Pif1 subfamily helicase is conserved from yeast to humans with a lot of cellular functions. In order to elucidate the function of human PIF1 helicase from biochemical level, we cloned human PIF1 gene by PCR from HeLa cell cDNA library. We co-transformed a pMStRNA1 plasmid encoding rare tRNA codons and a plasmid encoding molecular chaperon to greatly enhance the overexpression of human PIF1 protein. Finally we purified full-length PIF1 helicase by column chromatograph carried out at 4 degrees C using fast protein liquid chromatograph (FPLC) system. The human PIF1 protein was purified in enough quantity for detailed biochemical analysis. Biochemical assay showed that PIF1 had ATPase activity and helicase activity. The purification and biochemical properties analysis of human PIF1 helicase will allow us to understand how, at the molecular and mechanistic level, this conserved helicase operates in the cell.
DNA Helicases
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biosynthesis
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genetics
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metabolism
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HeLa Cells
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Humans
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RNA, Transfer
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genetics
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Recombinant Proteins
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biosynthesis
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genetics
;
metabolism
2.Drosophila RecQ5 is required for efficient SSA repair and suppression of LOH in vivo.
Yixu CHEN ; Wen DUI ; Zhongsheng YU ; Changqing LI ; Jun MA ; Renjie JIAO
Protein & Cell 2010;1(5):478-490
RecQ5 in mammalian cells has been suggested to suppress inappropriate homologous recombination. However, the specific pathway(s) in which it is involved and the underlining mechanism(s) remain poorly understood. We took advantage of genetic tools in Drosophila to investigate how Drosophila RecQ5 (dRecQ5) functions in vivo in homologous recombination-mediated double strand break (DSB) repair. We generated null alleles of dRecQ5 using the targeted recombination technique. The mutant animals are homozygous viable, but with growth retardation during development. The mutants are sensitive to both exogenous DSB-inducing treatment, such as gamma-irradiation, and endogenously induced double strand breaks (DSBs) by I-Sce I endonuclease. In the absence of dRecQ5, single strand annealing (SSA)-mediated DSB repair is compromised with compensatory increases in either inter-homologous gene conversion, or non-homologous end joining (NHEJ) when inter-chromosomal homologous sequence is unavailable. Loss of function of dRecQ5 also leads to genome instability in loss of heterozygosity (LOH) assays. Together, our data demonstrate that dRecQ5 functions in SSA-mediated DSB repair to achieve its full efficiency and in suppression of LOH in Drosophila.
Animals
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DNA Repair
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genetics
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DNA, Single-Stranded
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genetics
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Drosophila Proteins
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genetics
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metabolism
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Drosophila melanogaster
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genetics
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metabolism
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Loss of Heterozygosity
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genetics
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RecQ Helicases
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genetics
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metabolism
3.The Tip60/Ep400 chromatin remodeling complex impacts basic cellular functions in cranial neural crest-derived tissue during early orofacial development.
Sebastian GEHLEN-BREITBACH ; Theresa SCHMID ; Franziska FRÖB ; Gabriele RODRIAN ; Matthias WEIDER ; Michael WEGNER ; Lina GÖLZ
International Journal of Oral Science 2023;15(1):16-16
The cranial neural crest plays a fundamental role in orofacial development and morphogenesis. Accordingly, mutations with impact on the cranial neural crest and its development lead to orofacial malformations such as cleft lip and palate. As a pluripotent and dynamic cell population, the cranial neural crest undergoes vast transcriptional and epigenomic alterations throughout the formation of facial structures pointing to an essential role of factors regulating chromatin state or transcription levels. Using CRISPR/Cas9-guided genome editing and conditional mutagenesis in the mouse, we here show that inactivation of Kat5 or Ep400 as the two essential enzymatic subunits of the Tip60/Ep400 chromatin remodeling complex severely affects carbohydrate and amino acid metabolism in cranial neural crest cells. The resulting decrease in protein synthesis, proliferation and survival leads to a drastic reduction of cranial neural crest cells early in fetal development and a loss of most facial structures in the absence of either protein. Following heterozygous loss of Kat5 in neural crest cells palatogenesis was impaired. These findings point to a decisive role of the Tip60/Ep400 chromatin remodeling complex in facial morphogenesis and lead us to conclude that the orofacial clefting observed in patients with heterozygous KAT5 missense mutations is at least in part due to disturbances in the cranial neural crest.
Animals
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Mice
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Chromatin Assembly and Disassembly
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Cleft Lip/genetics*
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Cleft Palate/genetics*
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DNA Helicases/metabolism*
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DNA-Binding Proteins
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Neural Crest/metabolism*
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Skull
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Transcription Factors/metabolism*
4.Small cell carcinoma of ovary, hypercalcemic type: analysis of clinicopathologic characteristics and the diagnostic utility of loss expression of SMARCA4 protein.
Lei WANG ; Cong TAN ; Xiaoyu TU ; Yongsheng ZHANG ; Xinxia LI ; Bin CHANG
Chinese Journal of Pathology 2015;44(12):859-863
OBJECTIVETo study the clinicopathologic features of small cell carcinoma of ovary, hypercalcemic type (SCCOHT) and to evaluate the diagnostic significance of loss of SMARCA4 expression.
METHODSThe clinicopathologic characteristics of 5 cases of SCCOHT were reviewed. The expression of SMARCA4 protein was detected by immunohistochemistry in the cases of SCCOHT and 240 cases of other primary malignant tumors of ovary and peritoneum.
RESULTSThe mean and medium age of these patients was 30 years and 28 years, respectively. The presenting symptoms included abdominal pain, distention and a pelvic mass. Hypercalcemia was found in 3 patients. The maximum diameter of tumors ranged from 13.5 to 22.0 cm. Extraovarian spread was demonstrated in all of the patients on presentation. Histologically, the tumors were composed of closely packed small round cells with scanty cytoplasm, hyperchromatic nuclei and irregular chromatin clumps. The tumor cells grew in sheets, nests, cords or trabecular pattern. Follicle-like spaces were observed in 4 cases. Three of the tumors contained large cells with abundant eosinophilic cytoplasm. Spindle cell morphology was found in 1 case. There were 2 cases with myxoid or hyaline stroma. Four out of five of SCCOHT cases showed loss of SMARCA4 protein while only 6.3% (15/240) of the other primary malignant tumors of ovary and peritoneum , including undifferentiated carcinoma (1/5), high-grade serous carcinoma (4.6%, 5/109), endometrioid carcinoma (7.7%, 2/26), clear cell carcinoma (1/9), mucinous carcinoma (1/5), mixed carcinoma (4.9%, 3/61), carcinosarcoma (1/9) and high-grade serous carcinoma of peritoneum (1/9), were negative.
CONCLUSIONSSCCOHT is a rare malignant tumor and often misdiagnosed as other types of ovarian small cell tumor. Loss expression of SMARCA4 protein is characteristic and facilitates the diagnosis and differential diagnosis of SCCOHT.
Adenocarcinoma, Mucinous ; Adult ; Carcinoma, Small Cell ; genetics ; metabolism ; pathology ; DNA Helicases ; genetics ; metabolism ; Female ; Humans ; Hypercalcemia ; pathology ; Immunohistochemistry ; Neoplasms, Glandular and Epithelial ; genetics ; metabolism ; pathology ; Nuclear Proteins ; genetics ; metabolism ; Ovarian Neoplasms ; genetics ; metabolism ; pathology ; Transcription Factors ; genetics ; metabolism
5.Gene expression of helicase antigen in patients with acute and chronic myeloid leukemia.
Qin CHEN ; Jiang LIN ; Jun QIAN ; Dong-Ming YAO ; Wei QIAN ; Yun LI ; Hai-Yan CHAI ; Jing YANG ; Cui-Zhu WANG ; Ming ZHANG ; Gao-Fei XIAO
Journal of Experimental Hematology 2011;19(5):1171-1175
The aim of this study was to investigate the expression status of the helicase antigen (HAGE) transcript and its clinical significance in patients with acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). The expression of HAGE cDNA in bone marrow mononuclear cells from AML and CML patients was detected by using real-time quantitative PCR. The results indicated that overexpression of HAGE transcript (117.12% - 9842.70%, median 434.96%) was detected in 14.8% (11/74) AML patients. AML patients with HAGE cDNA expression were significantly older than those HAGE-negative patients (median 67 and 45 years, respectively, p = 0.001). HAGE cDNA expression was more frequently present among the patients with acute monoblastic leukemia (M(4) and M(5), 7 of 20, 35.0%), compared to the patients with acute non-monoblastic leukemia (M(1), M(2), M(3) and M(6), 4 of 54, 7.4%) (p = 0.007). 28.6% (8/28) cases with normal karyotypes showed HAGE cDNA overexpression, significantly higher than 7.5% (3 of 40) in those with chromosomal abnormalities (p = 0.041). Overexpression of HAGE transcript was found in 9 (34.6%) CML cases and more frequently observed at accelerated phase and blast crisis (4/4, 100%) than that at chronic phase (5/22, 22.7%) (p = 0.008). It is concluded that HAGE cDNA expression is relevant to specific subtypes of AML and to the progression of CML.
Blast Crisis
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Bone Marrow Cells
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metabolism
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DEAD-box RNA Helicases
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genetics
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metabolism
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DNA, Complementary
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Gene Expression
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Humans
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Karyotype
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Leukemia, Myelogenous, Chronic, BCR-ABL Positive
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genetics
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metabolism
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Leukemia, Myeloid, Acute
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genetics
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metabolism
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Neoplasm Proteins
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genetics
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metabolism
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RNA, Messenger
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genetics
6.Differential stem cell aging kinetics in Hutchinson-Gilford progeria syndrome and Werner syndrome.
Zeming WU ; Weiqi ZHANG ; Moshi SONG ; Wei WANG ; Gang WEI ; Wei LI ; Jinghui LEI ; Yu HUANG ; Yanmei SANG ; Piu CHAN ; Chang CHEN ; Jing QU ; Keiichiro SUZUKI ; Juan Carlos Izpisua BELMONTE ; Guang-Hui LIU
Protein & Cell 2018;9(4):333-350
Hutchinson-Gilford progeria syndrome (HGPS) and Werner syndrome (WS) are two of the best characterized human progeroid syndromes. HGPS is caused by a point mutation in lamin A (LMNA) gene, resulting in the production of a truncated protein product-progerin. WS is caused by mutations in WRN gene, encoding a loss-of-function RecQ DNA helicase. Here, by gene editing we created isogenic human embryonic stem cells (ESCs) with heterozygous (G608G/+) or homozygous (G608G/G608G) LMNA mutation and biallelic WRN knockout, for modeling HGPS and WS pathogenesis, respectively. While ESCs and endothelial cells (ECs) did not present any features of premature senescence, HGPS- and WS-mesenchymal stem cells (MSCs) showed aging-associated phenotypes with different kinetics. WS-MSCs had early-onset mild premature aging phenotypes while HGPS-MSCs exhibited late-onset acute premature aging characterisitcs. Taken together, our study compares and contrasts the distinct pathologies underpinning the two premature aging disorders, and provides reliable stem-cell based models to identify new therapeutic strategies for pathological and physiological aging.
Aging
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genetics
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physiology
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DNA Helicases
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genetics
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Human Embryonic Stem Cells
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metabolism
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physiology
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Humans
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Kinetics
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Lamin Type A
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genetics
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Mesenchymal Stem Cells
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metabolism
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physiology
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Mutation
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Progeria
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genetics
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physiopathology
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Werner Syndrome
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genetics
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physiopathology
7.Non-Homologous End Joining Repair Mechanism-Mediated Deletion of CHD7 Gene in a Patient with Typical CHARGE Syndrome.
Seung Jun LEE ; Jong Hee CHAE ; Jung Ae LEE ; Sung Im CHO ; Soo Hyun SEO ; Hyunwoong PARK ; Moon Woo SEONG ; Sung Sup PARK
Annals of Laboratory Medicine 2015;35(1):141-145
CHARGE syndrome MIM #214800 is an autosomal dominant syndrome involving multiple congenital malformations. Clinical symptoms include coloboma, heart defects, choanal atresia, retardation of growth or development, genital hypoplasia, and ear anomalies or deafness. Mutations in the chromodomain helicase DNA binding protein 7 (CHD7) gene have been found in 65-70% of CHARGE syndrome patients. Here, we describe a 16-month-old boy with typical CHARGE syndrome, who was referred for CHD7 gene analysis. Sequence analysis and multiplex ligation-dependent probe amplification were performed. A heterozygous 38,304-bp deletion encompassing exon 3 with a 4-bp insertion was identified. There were no Alu sequences adjacent to the breakpoints, and no sequence microhomology was observed at the junction. Therefore, this large deletion may have been mediated by non-homologous end joining. The mechanism of the deletion in the current case differs from the previously suggested mechanisms underlying large deletions or complex genomic rearrangements in the CHD7 gene, and this is the first report of CHD7 deletion by this mechanism worldwide.
Alu Elements/genetics
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Base Sequence
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CHARGE Syndrome/diagnosis/*genetics
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DNA/chemistry/metabolism
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*DNA End-Joining Repair
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DNA Helicases/*genetics/metabolism
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DNA-Binding Proteins/*genetics/metabolism
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Exons
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Gene Dosage
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Heterozygote
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Humans
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Infant
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Male
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Multiplex Polymerase Chain Reaction
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Mutation
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Sequence Analysis, DNA
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*Sequence Deletion
8.Monoclonal antibody against G3BP: preparation, characterization and its application in analysis of human tumors.
Jun-yu NING ; Jiang-feng YOU ; Fei PEI ; Jie-liang WANG ; Xiang-lin CUI ; Jie ZHENG
Chinese Journal of Pathology 2005;34(4):215-219
OBJECTIVETo better understand the molecular mechanism of tumorigenesis and progression, the monoclonal antibody against G3BP (Ras-GAP SH3 binding protein), which serves as an important downstream effector of Ras signaling, was prepared, characterized and utilized in analysis of various human tumors.
METHODSBy using the prokaryotic expression vector pGEX-5X1, GST-G3BP fusion protein was expressed in E. coli BL21 under induction of IPTG. Purified GST-G3BP fusion protein was used to immunize BALB/c mice. The monoclonal antibody against G3BP was produced through conventional hybridoma method and characterized by ELISA, Western blot and immunohistochemical staining.
RESULTSA hybridoma cell line secreting anti-G3BP IgG1 subtype antibody was obtained. Western blot and competitive inhibition assay showed that the antibody was G3BP-specific. Immunohistochemical staining demonstrated that G3BP was over-expressed in formalin-fixed and paraffin-embedded tissues of some human tumors, such as lung cancer, colon cancer, gastric cancer and breast cancer. In breast cancer specimens, the degree of G3BP expression correlated positively with the presence of lymph node metastasis and c-erbB2 expression.
CONCLUSIONSThe G3BP-specific monoclonal antibody derived from recombination protein can be used in ELISA, Western blot and immunohistochemical assay. It may provide an important tool in analysis of G3BP in in vitro and in vivo experiments. Besides, G3BP may serve as another prognostic marker for breast cancer.
Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Biomarkers, Tumor ; Breast Neoplasms ; metabolism ; pathology ; Carrier Proteins ; genetics ; immunology ; metabolism ; DNA Helicases ; Female ; Genetic Vectors ; Humans ; Hybridomas ; secretion ; Lymphatic Metastasis ; Male ; Mice ; Mice, Inbred BALB C ; Poly-ADP-Ribose Binding Proteins ; RNA Helicases ; RNA Recognition Motif Proteins ; Receptor, ErbB-2 ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Tumor Cells, Cultured
9.Construction of RBM5 vector, establishment of stably transfected A549 cell line and preliminary research on the function of RBM5 gene.
Jian XIAO ; Nian LI ; Xiaowei XING ; Bixiu HE
Journal of Central South University(Medical Sciences) 2014;39(10):994-1000
OBJECTIVE:
To establish a stable A549 cell line transfected by RNA binding motif 5 (RBM5) expression vector, and to investigate the effect of RBM5 gene on proliferation of A549 cell line and the expression of DEAH box polypeptide 15 (DHX15).
METHODS:
The eukaryotic expression vector pcDNA3.1 (+)/RBM5 was constructed by a twostep PCR technique. Then, the recombinant plasmid pcDNA3.1 (+)/RBM5 was verified by DNA sequencing and transfected into the lung adenocarcinoma cell A549. The positive cells with overexpression of RBM5 gene were identified by Western blotting. Flow cytometry was used to analyze the cell cycles of the positive A549 cells [pcDNA3.1 (+)/RBM5-A549] and the negative controls [pcDNA3 .1 (+)- A549]. Finally, RT-PCR was used to detect the expression of DHX15, a splicing-related factor, in the positively transfected A549 cells and the negative controls.
RESULTS:
A pcDNA3.1 (+)/RBM5 eukaryotic expression vector has been constructed successfully, and the A549 cell line that stably transfected with RBM5 gene has been established. Compared with negative control cells, the percentage of G1 phase cells in the positive cells was increased, while the percentage of S phase was decreased (both P<0.01), and the expression of DHX15 is upregulated (P<0.01).
CONCLUSION
RBM5 gene can inhibit the cell cycle and upregulate the expression of DHX15 in A549 cells.
Cell Cycle
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Cell Cycle Proteins
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genetics
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Cell Line, Tumor
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Cell Proliferation
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DNA-Binding Proteins
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genetics
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Genetic Vectors
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Humans
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RNA Helicases
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metabolism
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RNA-Binding Proteins
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genetics
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Transfection
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Tumor Suppressor Proteins
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genetics
10.G3BP: a promising target for cancer therapy.
Acta Pharmaceutica Sinica 2010;45(8):945-951
G3BP (Ras-GTPase-activating protein SH3 domain binding protein), a protein which binds to RasGAP SH3 domain, belongs to RNA-binding protein family, implicating in the downstream of Ras signaling. G3BP harbors the activities of endoribonuclease and DNA helicase, and can induce stress granules formation. G3BP plays a general role in the signal pathways of cell proliferation, differentiation, apoptosis and RNA metabolism. It has been shown to be over-expressed in a number of human malignancies and has a close relationship with tumor invasion and metastasis. Given that it has been implicated in several pathways that are known to be involved in cancer biology, G3BP may provide a new target for cancer therapy.
Animals
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Carrier Proteins
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genetics
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metabolism
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DNA Helicases
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Drug Delivery Systems
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GTPase-Activating Proteins
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therapeutic use
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Humans
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Molecular Sequence Data
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Neoplasms
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drug therapy
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metabolism
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pathology
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Peptide Fragments
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therapeutic use
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Phosphorylation
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Poly-ADP-Ribose Binding Proteins
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RNA Helicases
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RNA Recognition Motif Proteins
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Signal Transduction
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ras GTPase-Activating Proteins
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metabolism
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src Homology Domains
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genetics