1.Effect of total flavonoids of Scutellaria baicalensis Georgi on expression of influenza A virus nucleoprotein in HeLa cells.
Qing ZHANG ; Bin YANG ; Nongrong WANG ; Linjian DUAN ; Shiqin HE ; Jian SUN
Journal of Southern Medical University 2012;32(7):966-969
OBJECTIVETo investigate the effect of total flavonoids of Scutellaria baicalensis Georgi (TFSB) on exogenous expression of influenza A virus nucleoprotein (NP) in HeLa cells.
METHODSHeLa cells were transiently transfected with the empty vector pcDNA3.1(+) or pcDNA3.1(+)/NP vector harboring influenza A virus NP. The pcDNA3.1(+)/NP-transfected cells were treated with TSFB and the expression of influenza A virus NP in the cell supernatant was measured using colloidal gold immunochromatography 48 h after the transfection; fluorescent quantitative RT-PCR was performed to measure the starting copy number of NP gene.
RESULTSThe cells transfected with pcDNA3.1 (+)/NP with and without TFSB treatment were positive for NP expression. Fluorescent quantitative RT-PCR showed that the starting copy number of NP gene in pcDNA3.1(+)/NP-transfected cells was (8.90±2.53)×10⁶ copies/µl, showing no significant difference from that of (6.15±1.49)×10⁶ copies/µl in pcDNA3.1(+)/NP-transfected cells with subsequent TFSB treatment (P>0.05).
CONCLUSIONTFSB treatment does not obviously affect exogenous influenza A virus NP gene expression or its protein synthesis in HeLa cells.
Flavonoids ; pharmacology ; Gene Expression ; HeLa Cells ; Humans ; RNA-Binding Proteins ; biosynthesis ; genetics ; Scutellaria baicalensis ; Transfection ; Viral Core Proteins ; biosynthesis ; genetics
2.Targeted inhibition of osteoclastogenesis reveals the pathogenesis and therapeutics of bone loss under sympathetic neurostress.
Bingdong SUI ; Jin LIU ; Chenxi ZHENG ; Lei DANG ; Ji CHEN ; Yuan CAO ; Kaichao ZHANG ; Lu LIU ; Minyan DANG ; Liqiang ZHANG ; Nan CHEN ; Tao HE ; Kun XUAN ; Fang JIN ; Ge ZHANG ; Yan JIN ; Chenghu HU
International Journal of Oral Science 2022;14(1):39-39
Sympathetic cues via the adrenergic signaling critically regulate bone homeostasis and contribute to neurostress-induced bone loss, but the mechanisms and therapeutics remain incompletely elucidated. Here, we reveal an osteoclastogenesis-centered functionally important osteopenic pathogenesis under sympatho-adrenergic activation with characterized microRNA response and efficient therapeutics. We discovered that osteoclastic miR-21 was tightly regulated by sympatho-adrenergic cues downstream the β2-adrenergic receptor (β2AR) signaling, critically modulated osteoclastogenesis in vivo by inhibiting programmed cell death 4 (Pdcd4), and mediated detrimental effects of both isoproterenol (ISO) and chronic variable stress (CVS) on bone. Intriguingly, without affecting osteoblastic bone formation, bone protection against ISO and CVS was sufficiently achieved by a (D-Asp8)-lipid nanoparticle-mediated targeted inhibition of osteoclastic miR-21 or by clinically relevant drugs to suppress osteoclastogenesis. Collectively, these results unravel a previously underdetermined molecular and functional paradigm that osteoclastogenesis crucially contributes to sympatho-adrenergic regulation of bone and establish multiple targeted therapeutic strategies to counteract osteopenias under stresses.
Adrenergic Agents/pharmacology*
;
Apoptosis Regulatory Proteins/pharmacology*
;
Bone Diseases, Metabolic/metabolism*
;
Humans
;
Liposomes
;
MicroRNAs/genetics*
;
Nanoparticles
;
Osteoclasts
;
Osteogenesis/physiology*
;
RNA-Binding Proteins/pharmacology*
3.Hammerhead ribozyme against human telomerase catalytic subunit (hTERT) induced apoptosis of liver cancer cells.
Dong-po SONG ; Ju-sheng LIN ; Gui-lian FU ; Xue-mei SUN ; Xin-juan KONG ; Pei-yuan LI ; Xin MA
Chinese Journal of Hepatology 2004;12(10):616-619
OBJECTIVETo construct vector pEGFP-C1-hTERT-ribozyme (pGTRz-U6) and its mutant (pGTmRz-U6) against hTERT containing U6 promoter, then transfect them into human liver cancer cell line SMMC7721 to observe the action of the human telomerase catalytic subunit (hTERT) hammerhead ribozyme on proliferation and apoptosis of human liver cancer cell SMMC7721.
METHODSEukaryotic expressing vector pGTRz-U6 and mutant pGTmRz-U6 were constructed and transfected into SMMC7721 using Lipofectamine2000 Reagent, with pEGFP-C1 as the control group. After strict screening by G418, positive clones were cultured; the amount of expression of ribozyme and hTERT was detected by RT-PCR; cell proliferation by MTT; telomerase activity by TRAP and silver staining assay; cell apoptosis by FCM.
RESULTSWe found that the two ribozymes were expressed persistently in SMMC7721; different expression levels (P < 0.01) of hTERT among SMMC7721-Rz, SMMC7721-mRz and SMMC7721-pEGFP-C1 was exhibited by the analysis of variance with SPSS software. The difference between SMMC7721-Rz and the others is significant in t-test (P < 0.01), while there was no difference between SMMC7721-mRz and SMMC7721-pEGFP-C1 (P > 0.05). With the advance of cell division, telomerase activities of the cells treated by SMMC7721-Rz and SMMC7721-mRz decreased gradually, and the percentage of apoptosis of the cells transfected with Rz and mRz increased gradually. The apoptosis percentage of 7PDS SMMC7721-Rz was 29.86%, while those of SMMC7721-mRz and SMMC7721-pEGFP-C1 were 9.87% and 3.36%, respectively.
CONCLUSIONThe apoptosis level of SMMC7721 induced by hTERT ribozyme increases as cells divide, and this ribozyme maybe a potential approach for liver cancer gene therapy.
Apoptosis ; physiology ; Cell Line, Tumor ; DNA-Binding Proteins ; genetics ; pharmacology ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Mutation ; RNA, Catalytic ; genetics ; pharmacology ; Telomerase ; genetics ; pharmacology ; Transfection
4.Antisense ERCC1 RNA decreases the repair capability of damaged DNA in lung cancer cells induced by benzoapyrene.
Xiao-ming WU ; Yi-kai ZHOU ; Shun-qing XU ; Qiao-ling HAO ; Shu REN
Chinese Journal of Preventive Medicine 2003;37(3):167-170
OBJECTIVETo investigate the effect of ERCC1 gene on the repair capability of damaged DNA in lung cancer A549 cells induced by benzo[a]pyrene.
METHODSRecombinant plasmid expressing ERCC1 antisense RNA was constructed and transfected into A549 cells by Lipofectin reagent. The stable-transfected cell colonies were selected by hygromycin. Cell viability was determined by the MTT assay. The level of ERCC1 mRNA was measured by Northern Blot analysis. Single cell gel electrophoresis assay was applied to determine the cellular DNA damage and fifty cells for each group were counted.
RESULTSSeven positive colonies expressing ERCC1 antisense RNA were screened. There was no growth rate difference between the antisense-transfected cells and the parental cells. The endogenous mRNA level in transfected colonies decreased in varied degrees, i.e. 12% approximately 86% of that of the parental cells in Northern Blot assay. After 24 h treatment of 10 micro mol/l benzo[a]pyrene, the repair capability for DNA damage in transfected colonies was reduced to 29% approximately 71% of that of the parental cells. Also, a statistically significant correlation was observed between expression of ERCC1 mRNA and repair capability (r = 0.84).
CONCLUSIONAntisense ERCC1 RNA decreased the repair capability for damaged DNA in lung cancer cells induced by benzo[a]pyrene.
Benzo(a)pyrene ; toxicity ; Cell Line, Tumor ; DNA Damage ; drug effects ; DNA Repair ; drug effects ; DNA-Binding Proteins ; genetics ; metabolism ; pharmacology ; Endonucleases ; genetics ; metabolism ; pharmacology ; Humans ; Lung Neoplasms ; pathology ; Plasmids ; RNA, Antisense ; pharmacology ; RNA, Messenger ; metabolism ; Repressor Proteins ; Transfection
5.Study on the sensitivity of leukemic cells to arsenic trioxide enhanced by targeted suppression of mIRNA-21.
Yu-min LI ; Jing-yi GU ; Xue-jiao ZHU
Chinese Journal of Integrated Traditional and Western Medicine 2010;30(2):170-173
OBJECTIVETo study the effect of antisense oligonucleotide targeted on miRNA-21 (AMO-miR-21) for enhancing the arsenic trioxide (As2O3) sensitivity of leukemic K562 cells and its possible acting mechanism.
METHODSChemosynthetic AMO-miR-21 was transfected to K562 cells using Lipofectamine TM 2000. The inhibitory effects of As2O3 and AMO-miR-21, used singly or in combining, on cell proliferation were detected by MTT, their inhibition rate and IC50 were calculated. Cell cycle and apoptosis were assessed with PI stain; expression of miRNA-21 in cells was detected quantitatively by real-time PCR, and the potential target gene PDCD, protein expression was detected by immuno-fluorimetry.
RESULTSUsed in combining with AMO-miR-21, the IC50 of As2O3, could be lowered from 2.1 micromol/L to 1.23 micromol/L, and the sensitivity of cells to As2O3 increased to 1.78-fold; with the amount of apoptotic cells increased significantly. Transfection with AMO-miR-21 alone could downregulate the expression of miRNA-21 in cells (P < 0.01), and up-regulate PDCD, protein expression level significantly.
CONCLUSIONSCombined use of AMO-miR-21 and As2O3 could increase the sensitivity of K562 cells to As2O3, which provides a novel potential approach for treatment of leukemia. AMO-miR-21 realizes it anti-tumor action by way of targeted inhibition on miRNA-21, and further up-regulates the expression of anti-tumor gene PDCD4.
Antineoplastic Agents ; pharmacology ; Apoptosis Regulatory Proteins ; metabolism ; Arsenicals ; pharmacology ; Gene Expression Regulation, Leukemic ; Humans ; K562 Cells ; MicroRNAs ; drug effects ; Oligonucleotides, Antisense ; pharmacology ; Oxides ; pharmacology ; RNA-Binding Proteins ; metabolism ; Transfection
6.Cholera toxin mediated regulation of the expression of Gq alpha and G11 alpha GTP binding proteins.
Experimental & Molecular Medicine 1999;31(2):89-94
Previously it has been shown that persistent activation of the stimulatory adenylyl cyclase pathway with cholera toxin (CT) downregulates the Gs alpha polypeptide (80%) in a cAMP-independent manner in C6 glioma cells (Shah, 1997). This study was conducted to examine the short and long term effects of CT on the regulation of pertussis toxin-sensitive and -insensitive G proteins and their transcripts in C6 glioma cells. Treatment of C6 cells with CT (100 ng/ml) up to 16 h had no effect on either Gi or Gq/11 alpha proteins. However, prolonged exposure (24-48 h) caused increased expression of Gi (20-30%) and Gq/11 alpha proteins (40%). Urea gradient gels, which can separate Gq alpha and G11 alpha proteins, revealed that prolonged CT treatment increased the expression of both of these G proteins. The CT-mediated enhanced expression of Gq alpha and G11 alpha proteins was accompanied by increased mRNA levels of these proteins as determined by RT/PCR. Cyclic-AMP elevating agents like forskolin (10 microM) and db-cAMP (1 mM) mimicked the effect of CT on Gi but not Gq/11 alpha proteins. These studies show long term cAMP-dependent regulation of Gi and cAMP-independent expression of Gq/11 alpha proteins in C6 glioma cells.
Animal
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Blotting, Western
;
Bucladesine/pharmacology
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Cholera Toxin/pharmacology*
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Cyclic AMP-Dependent Protein Kinases/metabolism
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Forskolin/pharmacology
;
GTP-Binding Proteins/genetics*
;
GTP-Binding Proteins/biosynthesis
;
Gene Expression Regulation*
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Glioma
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Membrane Proteins/analysis
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RNA, Messenger/metabolism
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RNA, Messenger/genetics
;
Rats
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Reverse Transcriptase Polymerase Chain Reaction
7.2-DG enhances TRAIL-induced apoptosis of leukemia HL-60 cells.
Su-Rong ZHAO ; Hai-Feng DUAN ; Pei ZHANG ; Hao LIU ; Chen-Chen JIANG ; Zhi-Wen JIANG
Journal of Experimental Hematology 2013;21(2):351-355
This study was purposed to investigate the effects of 2-deoxy-D-glucose (2-DG) on sensitizing HL-60 cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis and its possible mechanism. The proliferative inhibition of HL-60 cells treated with different concentrations of 2-DG and TRAIL was measured by MTT assay. The cells were treated with 2-DG, TRAIL, and 2-DG combined with TRAIL at the concentration < IC50 value, i.e. 10 mmol/L for 2-DG and 100 ng/ml for TRAIL. Apoptosis was analyzed by flow cytometry with PI staining; the expression of RIP1, GRP78, and PARP was analyzed by Western blot; the activity of caspase-3 was detected by special detection kit. The results showed that the combined treatment of HL-60 cells for 48 h induced an apoptotic rate of (45.1 ± 4.3)%, which was significantly higher than that of treated with 2-DG or TRAIL alone; at the same time, the combined treatment potentiated the expression of GRP78 and caspase-3 activity, and down-regulated the expression of RIP1. It is concluded that 2-DG can sensitize HL-60 cells to TRAIL-induced apoptosis, which may be correlated with excessive endoplasmic reticulum stress response, down-regulation of RIP1, and increase of caspase-3 activity.
Apoptosis
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drug effects
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Caspase 3
;
metabolism
;
Deoxyglucose
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pharmacology
;
HL-60 Cells
;
Heat-Shock Proteins
;
metabolism
;
Humans
;
Nuclear Pore Complex Proteins
;
metabolism
;
RNA-Binding Proteins
;
metabolism
;
TNF-Related Apoptosis-Inducing Ligand
;
metabolism
;
pharmacology
8.Effects of xbp-1 gene silencing on bortezomib-induced apoptosis in human multiple myeloma cells.
Yang YANG ; Hong-Juan DONG ; Guang-Xun GAO ; Yi-Wei WANG ; Hong-Tao GU ; Mi-Mi SHU ; Hua-Feng ZHU ; Xie-Qun CHEN
Journal of Experimental Hematology 2010;18(5):1177-1180
This study was purposed to investigate the effect of xbp-1 gene silencing on bortezomib-induced apoptosis in multiple myeloma cell line NCI-H929 (H929). After xbp-1 gene expression was interfered by small hairpin RNA, the cell apoptosis was assayed by flow cytometry with Annexin V-FITC/PI staining, and the expression level of XBP-1 protein was detected by Western blot. The results showed that XBP-1 protein level of H929 cells was inhibited effectively by the PLL3.7 lentiviral vector mediated expression xbp-1 shRNA. The apoptosis rate was significantly higher in xbp-1 shRNA-expressing cells than in untreated control group [(10.13±0.61)% vs (2.5±0.2)%, p<0.05]. After treatment with bortezomib, the apoptosis rate of XBP-1 protein functionally deficient H929 cells was significantly higher than those in vector control group [(45.07±1)% vs (19.53±0.8)%, p<0.05]. It is concluded that xbp-1 gene silencing can significantly enhance the pro-apoptotic activity of bortezomib in multiple myeloma cells.
Apoptosis
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drug effects
;
Boronic Acids
;
pharmacology
;
Bortezomib
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Cell Line, Tumor
;
DNA-Binding Proteins
;
genetics
;
Gene Silencing
;
Humans
;
Multiple Myeloma
;
genetics
;
Pyrazines
;
pharmacology
;
RNA, Small Interfering
;
genetics
;
Regulatory Factor X Transcription Factors
;
Transcription Factors
;
genetics
;
X-Box Binding Protein 1
9.hMRE11 plays an important role in U937 cellular response to DNA double-strand breaks following etoposide.
Ling-Bo LIU ; Lei TIAN ; Wei-Ming LI ; Lei LI ; Li WANG ; Ping ZOU
Journal of Experimental Hematology 2007;15(1):10-15
MRE11 plays an important role in the signal transduction of DNA damage response, therefore this study was purposed to explore the relationship between hMRE11 focus formation and DNA double-strand breaks (DSBs) caused by etoposide (VP-16) in human promonocytic cells U937. After U937 cells were treated with VP-16, the drug-induced DSBs were assessed by pulsed-field gel electrophoresis (PFGE), the gene transcription levels of hMRE11 were evaluated by RT-PCR, the nuclear focus formation of hMRE11 protein was examined using immunofluorescence technique, the cell cycle in parallel was analyzed by flow cytometry. The results showed that the percentage of U937 cells with DSBs induced by VP-16 raised from 13.0 +/- 2.3% in VP-16 2 microg/ml to 32.0 +/- 4.3% in VP-16 20 microg/ml (P < 0.01) along with increase of VP-16 dose. No difference of the hMRE11 mRNA level in U937 cells following the treatment with 100 microg/ml VP-16 at different times was discovered (P > 0.05). The hMRE11 protein was abundantly and uniformly distributed in the nuclei of untreated U937 cells outside of nucleoli, however, it formed discrete nuclear foci following VP-16 treatment. The mean value of nuclear foci increased by 5 to 20 times following the drug dosing (P < 0.01). An average of 5 nuclear foci per positive nucleus were observed at a dose of 2 microg/ml, and it was increased to an average of over 14 nuclear foci per positive nucleus after treating with VP-16 20 microg/ml. The percentage of nuclei containing hMRE11 nuclear foci also increased from less than 10% after treatment wiht VP-16 2 microg/ml to over 50% after VP-16 20 microg/ml (P < 0.01) following treatment with VP-16. After U937 cells were treated with 100 microg/ml VP-16 for 2 hours and fixed at 4, 8, 12 and 24 hours, the percentage of nuclei with hMRE11 nuclear foci increased to 61.54 +/- 3.6% (the control U937 cells: 0.47 +/- 1.17%, P < 0.01) at 8 hours, with a subsequent decrease in the percentage of nuclear foci-positive cells by 24 hours. The ratio of S-phase U937 cells at 8 hours after being treated with 100 microg/ml VP-16 for 2 hours was 47.55 +/- 2.35%, and that without 100 microg/ml VP-16 was 21.95 +/- 2.91% (P < 0.05). It is concluded that the nuclear focus formation of hMRE11 protein may be a response to DNA damage induced by topoisomerase II inhibitor VP-16 in human promonocytic cell line U937.
Antineoplastic Agents, Phytogenic
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pharmacology
;
DNA Damage
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DNA Repair
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genetics
;
physiology
;
DNA-Binding Proteins
;
biosynthesis
;
genetics
;
metabolism
;
physiology
;
Dose-Response Relationship, Drug
;
Etoposide
;
pharmacology
;
Humans
;
MRE11 Homologue Protein
;
Protein Binding
;
RNA, Messenger
;
biosynthesis
;
genetics
;
Signal Transduction
;
Topoisomerase II Inhibitors
;
U937 Cells
10.Inhibiting effect of arsenic trioxide on telomerase activity of NB4 and Jurkat cell lines.
Yun-Fang WANG ; Hong-Yan SUN ; Quan-Li WANG ; Xin-Quan LI
Journal of Experimental Hematology 2003;11(4):359-362
To investigate the inhibiting effect of arsenic trioxide (As(2)O(3)) on the telomerase activity of leukemia cell lines NB4 and Jurkat cells, MTT assay, electrophoresis of genomic DNA, protein/DNA dual parameter flow cytometry as well as a semi-quantitative telomeric repeat amplification protocol (TRAP) assay and RT-PCR were used to examine the effect of As(2)O(3) on cell proliferation, telomerase activity and expression of cell cycle regulatory proteins. The results showed that cell proliferation and telomerase activity were significantly inhibited and apoptosis was induced in these cells after exposure to As(2)O(3). Furthermore, the expression of some cell cycle and apoptosis related proteins, such as Bcl-2, Rb, P16, caspase-3, cyclin A and cyclin E, was altered in As(2)O(3) treated NB4 cells. Cell cycle was arrested at G(1) and G(2)/M phases in both cells. It is concluded that the change of cell cycle regulatory proteins plays an important role in decline of the telomerase activity during the proliferation inhibition and apoptosis of NB4 and Jurkat cells induced by As(2)O(3).
Antineoplastic Agents
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pharmacology
;
Apoptosis
;
drug effects
;
Arsenicals
;
pharmacology
;
Cell Cycle
;
drug effects
;
Cell Division
;
drug effects
;
DNA-Binding Proteins
;
Enzyme Inhibitors
;
pharmacology
;
Humans
;
Jurkat Cells
;
Oxides
;
pharmacology
;
RNA, Messenger
;
analysis
;
Telomerase
;
antagonists & inhibitors
;
genetics