CORD-19-Sentences  

CORD-19:59c725e7aa0e91f3d055521d1786091179015003 JSONTXT 7 Projects

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Id Subject Object Predicate Lexical cue
TextSentencer_T1 0-122 Sentence denotes Human Papillomavirus Type 16 Mutant E7 Protein Induces Oncogenic Transformation via Up-regulation of Cyclin A and cdc25A *
TextSentencer_T2 124-132 Sentence denotes Abstract
TextSentencer_T3 133-343 Sentence denotes A new mutant human papillomavirus type 16 E7 gene, termed HPV16 HBE7, was isolated from cervical carcinoma biopsy samples from patients in an area with high incidence of cervical cancer (Hubei province, China).
TextSentencer_T4 344-506 Sentence denotes A previous study showed that the HPV16 HBE7 protein was primarily cytoplasmic while wild-type HPV16 E7 protein, termed HPV16 WE7, was concentrated in the nucleus.
TextSentencer_T5 507-747 Sentence denotes With the aim of studying the biological functions of HPV16 HBE7, the transforming potential of HPV16 HBE7 in NIH/3T3 cells was detected through observation of cell morphology, cell proliferation assay and anchorage-independent growth assay.
TextSentencer_T6 748-818 Sentence denotes The effect of HPV16 HBE7 on cell cycle was examined by flow cytometry.
TextSentencer_T7 819-994 Sentence denotes Dual-luciferase reporter assay and RT-PCR were used to investigate the influence of HPV16 HBE7 protein on the expression of regulation factors associated with G1/S checkpoint.
TextSentencer_T8 995-1098 Sentence denotes The results showed that HPV16 HBE7 protein, as well as HPV16 WE7 protein, held transformation activity.
TextSentencer_T9 1099-1234 Sentence denotes NIH/3T3 cells expressing HPV16 HBE7 could easily transition from G1 phase into S phase and expressed high level of cyclin A and cdc25A.
TextSentencer_T10 1235-1401 Sentence denotes These results indicated HPV16 mutant E7 protein, located in the cytoplasm, induces oncogenic transformation of NIH/3T3 cells via up-regulation of cyclin A and cdc25A.
TextSentencer_T11 1403-1496 Sentence denotes cervical cancer cells, and is vital to maintain the malignant phenotype of these cells (10) .
TextSentencer_T12 1497-1597 Sentence denotes Moreover, HPV16 E7 can transform a variety of cell lines, including NIH/3T3 mouse fibroblasts (27) .
TextSentencer_T13 1598-1840 Sentence denotes The most well-characterized biochemical property of HPV16 E7 is its ability to bind to the unphosphorylated retinoblastoma tumor suppressor protein (Rb) and disrupt Rb-E2F complexes, resulting in a loss of the G1/S checkpoint (1, 7, 35, 38) .
TextSentencer_T14 1841-1995 Sentence denotes The HPV16 E7 protein is a 98-amino-acid nuclear phosphoprotein and can be divided into three main domains -CR1, aa 1-15; CR2, aa 16-37; and CR3, aa 38-98.
TextSentencer_T15 1996-2087 Sentence denotes All 3 domains are essential for the manifestation of the biological properties of E7 (33) .
TextSentencer_T16 2088-2278 Sentence denotes CR1 and CR2 mediate binding of the viral oncoproteins to an overlapping set of cellular proteins, including the product of the retinoblastoma gene (pRB), cyclin A and cyclin E (18, 24, 36) .
TextSentencer_T17 2279-2361 Sentence denotes The CR3 domain contains two "CXXC" sequences participating in Zn binding (9, 23) .
TextSentencer_T18 2362-2507 Sentence denotes It was reported that zinc finger structure at the C-terminal was essential for HPV16 E7 to stabilize its structure and biological function (30) .
TextSentencer_T19 2508-2612 Sentence denotes Previous studies showed variants of HPV16 E7 gene were found in widely separated locations (5, 26, 31) .
TextSentencer_T20 2613-2724 Sentence denotes A mutant E7 (HPV16 HBE7) protein derived from a clinical HPV16 was isolated from Hubei province in China (16) .
TextSentencer_T21 2725-2805 Sentence denotes It was found that there were two mutations in HPV16 HBE7 ( GenBank accession no.
TextSentencer_T22 2806-2816 Sentence denotes AF393782).
TextSentencer_T23 2817-2977 Sentence denotes What is more, a non sense mutation was found in codon 43 which made HPV16 HBE7 into a truncated protein, corresponding to the first 43 amino acids of HPV16 WE7.
TextSentencer_T24 2978-3263 Sentence denotes Generally speaking, the great variance of gene structure in the virus will lead to changes of its immunological characteristics and its biological functions, for example, mutations leading to changes in the structure of coronaviruses produces changes in host range and virulence (27) .
TextSentencer_T25 3264-3440 Sentence denotes Previous research indicated that the HBE7 protein could induce mice to produce humoral immunity identical with the WE7 protein but with weaker specific cellular immunity (16) .
TextSentencer_T26 3441-3590 Sentence denotes The integrity of the C-terminal of the truncated HBE7 protein affects its intracellular localization (17) and may affect its transformation activity.
TextSentencer_T27 3591-3805 Sentence denotes This research shows that HPV16 HBE7 (the HPV16 E7 fragment), which is predominantly cytoplasmic, is also competent for transformation of NIH/3T3 cells and can efficiently activate expression of cyclin A and cdc25A.
TextSentencer_T28 3806-3985 Sentence denotes NIH/3T3 cells were purchased from the Cancer Research Institute of Zhongnan University (Hunan, China) and cultured in DMEM supplemented with 10% BCS at 37 ℃ in 5% CO 2 atmosphere.
TextSentencer_T29 3986-4111 Sentence denotes Recombinants of pcDNA3.1-HPV16HBE7 and pcDNA3.1-HPV16WE7 expressing HBE7 or WE7 respectively were generated previously (37) .
TextSentencer_T30 4112-4326 Sentence denotes To evaluate the transformation ability of HPV16 HBE7 and its potential to regulate the G1/S phase of cell cycle, NIH/3T3 cells (1.5 ×10 5 /well) were transfected with 0.8 µg plasmid DNA using lipofectamine TM 2000.
TextSentencer_T31 4327-4440 Sentence denotes Stable clones were selected using G418 (Wuhan TianYuan BioTechnologies co, China) at a concentration of 600µg/mL.
TextSentencer_T32 4441-4527 Sentence denotes Empty vector pcDNA3.1 (-) was also stably transfected into NIH/3T3 cells as a control.
TextSentencer_T33 4528-4650 Sentence denotes Expression of HPV16 E7 protein in stable clones was confirmed by reverse transcription-polymerase chain reaction (RT-PCR).
TextSentencer_T34 4651-4729 Sentence denotes Total RNAs of NIH/3T3 cells were extracted by Trizol reagent (MBI Fer-mentas).
TextSentencer_T35 4730-4834 Sentence denotes 5µg of total RNA was reverse-transcribed to cDNA with a first-strand cDNA synthesis kit (MBI Fermentas).
TextSentencer_T36 4835-5046 Sentence denotes The PCR was performed in a 50µL volume under the following conditions: initial denaturation step of 94 ℃ for 5 min, cycling step of denaturation at 94 ℃ for 60 s, annealing for 60s and extension at 72 ℃ for 60s.
TextSentencer_T37 5047-5104 Sentence denotes The mRNA levels of β-actin served as an internal control.
TextSentencer_T38 5105-5165 Sentence denotes The products of PCR were identified by agarose gel analysis.
TextSentencer_T39 5166-5321 Sentence denotes Changes of cell morphology in stable NIH/3T3 cell transfectants with pcDNA3.1-HPV16HBE7 were observed by transmission electron microscope (Olympus, Japan).
TextSentencer_T40 5322-5523 Sentence denotes In the process of culturing, the samples of 5 ×10 6 cells were collected and fixed with 2.0% glutaraldehyde, dehydrated, embedded, sectioned and finally visualized via transmission electron microscope.
TextSentencer_T41 5524-5591 Sentence denotes The cell morphology of parental NIH/3T3 cells was set as a control.
TextSentencer_T42 5592-5761 Sentence denotes The growth rate of NIH/3T3 cells stably expressing HBE7 in vitro was measured using the 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method (25) .
TextSentencer_T43 5762-5829 Sentence denotes Briefly, cells were seeded into 96-well plates (2×10 3 cells/well).
TextSentencer_T44 5830-5958 Sentence denotes On the day of harvest, 100μL of spent medium was replaced with an equal volume of fresh medium containing 10% MTT 5 mg/mL stock.
TextSentencer_T45 5959-6139 Sentence denotes Plates were incubated at 37 ℃ in a 5% CO 2 incubator for 4 h, then 100μL of dimethyl sulphoxide (DMSO) was added to each well and plates were shaken at room temperature for 10 min.
TextSentencer_T46 6141-6222 Sentence denotes Soft agarose assay was essentially performed according to previous methods (22) .
TextSentencer_T47 6223-6503 Sentence denotes The NIH/3T3 cells transfected with pcDNA3.1-HPV16HBE7 and parental NIH/3T3 cells were respectively suspended in 0.35% agarose with complete DMEM medium, plated at a density of 1×10 4 cells per well, previously coated with 0.7% agarose and maintained at 37˚C in 5% CO 2 atmosphere.
TextSentencer_T48 6504-6651 Sentence denotes The cultures were maintained for 3 weeks and colonies>150µm in diameter were counted under an inverted microscope at the magnification of 10 times.
TextSentencer_T49 6652-6754 Sentence denotes The distribution of cells in the cell cycle phases was determined by FACS analysis of the DNA content.
TextSentencer_T50 6756-6835 Sentence denotes The cyclinA promoter activity was determined by dual-luciferase reporter assay.
TextSentencer_T51 6836-6960 Sentence denotes NIH/3T3 cells were grown in 24 well plates to about 70%-80% confluence and were cotransfected with recombinants of pcDNA3.1-
TextSentencer_T52 6962-7052 Sentence denotes RT-PCR was performed in a semiquantitative manner with cyclinA, cdc25A, cyclin E and cdk2.
TextSentencer_T53 7053-7105 Sentence denotes Primers for amplification of each gene are listed in
TextSentencer_T54 7107-7177 Sentence denotes Data were presented as mean ± SD and analyzed with SPSS 11.0 software.
TextSentencer_T55 7178-7228 Sentence denotes P < 0.05 was considered statistically significant.
TextSentencer_T56 7229-7393 Sentence denotes A specific band could be seen in both NIH/3T3 cells stably transfected with pcDNA3.1-HPV16HBE7 and NIH/3T3 cells stably transfected with pcDNA3.1-HPV16WE7 (Fig.1) .
TextSentencer_T57 7394-7515 Sentence denotes It showed that NIH/3T3 cells stably expressing HBE7 (NIH/3T3-HBE7) and NIH/ 3T3 cells stably expressing WE7 (NIH/3T3-WE7)
TextSentencer_T58 7516-7543 Sentence denotes were successfully obtained.
TextSentencer_T59 7544-7659 Sentence denotes Transmission electron microscopy revealed that NIH/3T3 cells stably expressing HBE7 (NIH/3T3-HBE7) grew quite well.
TextSentencer_T60 7660-7883 Sentence denotes There were several bigger and abnormally shaped nucleoli in NIH/3T3-HBE7 cells and more pathologic nuclear phase and immature cell organs were found in NIH/3T3-HBE7 cells than in parental NIH/3T3 cells (Fig. 2) . (Fig. 3) .
TextSentencer_T61 7885-8023 Sentence denotes The tumorigenic potential of transformed cells was determined from their ability to grow as anchorageindependent colonies in soft agarose.
TextSentencer_T62 8024-8099 Sentence denotes Small colony formation could be seen in NIH/3T3-HBE7 cells two weeks later.
TextSentencer_T63 8100-8152 Sentence denotes At 15 to 21 days, obvious cell colonies were formed.
TextSentencer_T64 8153-8292 Sentence denotes On day 21, NIH/3T3-HBE7 cells could form some colonies in the soft agarose, while parental NIH/3T3 cells could not form colonies (Fig. 4) .
TextSentencer_T65 8293-8358 Sentence denotes These data suggested there was tumorigenesis in the HBE7 protein.
TextSentencer_T66 8359-8495 Sentence denotes The effect of the HBE7 gene on cell cycle distribution was determined to gain insights into the mechanism of its proliferative activity.
TextSentencer_T67 8496-8615 Sentence denotes As can be seen in Table 2 , the presence of the HBE7 gene resulted in The results showed that compared with pcDNA3.1(-)
TextSentencer_T68 8616-8734 Sentence denotes transfecting the cells, the activity of cyclin A promoter increased by 2 times in HBE7 transfecting cells (Fig. 5 ).
TextSentencer_T69 8735-8869 Sentence denotes These results demonstrate that HPV16HBE7 protein promotes the proliferation of NIH/3T3 cells and the HBE7 gene is a transforming gene.
TextSentencer_T70 8870-9045 Sentence denotes To further investigate the potential of HBE7 to regulate the G1/S phase of cell cycle, the expression level of regulation factors associated with G1/S checkpoint was examined.
TextSentencer_T71 9046-9103 Sentence denotes Semi-quantitative RT-PCR results are presented in Fig.6 .
TextSentencer_T72 9104-9176 Sentence denotes The relative mRNA expression levels of each gene are listed in Table 3 .