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1.
Oncol Rep ; 31(3): 1473-9, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24402374

ABSTRACT

Radioresistance is one of the main reasons for the failure of radiotherapy in lung cancer. The present study was conducted to identify the role of miR-511 in suppressing the growth of radioresistant lung adenocarcinoma cells. First, a radioresistant A549/R cell line was generated after prolonged exposure to X-rays for 68 Gy (2 Gy/day, 5 days/week) and the radioresistance was confirmed by wound healing assay. Next, oncogenic TRIB2 was found to be upregulated in the radioresistant A549/R cells when compared to that of the control A549 cells as determined by western blot analysis. As the upstream miRNA, quantitative PCR showed that miR-511 expression was decreased in the radioresistant A549/R cells. Overexpression of miR-511 in miR-511-transfected A549/R cells inhibited cell growth and increased the number of apoptotic cells when compared with the control treatment. Flow cytometric analysis further demonstrated that the growth suppressive effect of miR-511 on A549/R cells was mediated by regulation of the cell cycle, most likely due to a block in the G1-S transition. Finally, our results showed that the expression of BAX was lower in the radioresistant A549/R cells when compared with that in the control A549 cells. After downregulation of TRIB2 by miR-511 treatment, BAX expression was obviously increased in the miR-511-transfected apoptotic A549/R cells when compared to that in the NC-treated or control cultures. In summary, our results revealed that miR-511 regulates the growth of radioresistant A549/R cells by increasing BAX expression through TRIB2, which suggests that miR-511 may be a potential therapeutic molecule for the treatment of radioresistant lung adenocarcinoma.


Subject(s)
Apoptosis , MicroRNAs/physiology , bcl-2-Associated X Protein/metabolism , Adenocarcinoma , Adenocarcinoma of Lung , Calcium-Calmodulin-Dependent Protein Kinases , Cell Line, Tumor , Cell Movement , Cell Proliferation , Gene Expression , Gene Expression Regulation, Neoplastic , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Lung Neoplasms , RNA Interference , Radiation Tolerance , bcl-2-Associated X Protein/genetics
2.
Oncol Lett ; 6(4): 1025-1030, 2013 Oct.
Article in English | MEDLINE | ID: mdl-24137458

ABSTRACT

MicroRNAs (miRNAs) have significant roles in cell processes, including proliferation, apoptosis and stress responses. To investigate the involvement of miR-99 in the inhibition of HeLa cell proliferation, an miR-99 gene expression vector (pU6.1/miR-99), which overexpressed miR-99 in HeLa cells after transient transfection, was constructed. The expression of miR-99 was detected by qPCR. Cell proliferation and apoptosis were analyzed by cell viability, proliferation and apoptosis assays, as well as by electron microscopy. The results showed that overexpression of miR-99 in HeLa cells increased the HeLa cell mortality rate. Moreover, miR-99 overexpression was able to markedly inhibit HeLa cell proliferation according to the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cell apoptosis rate was significantly higher in pU6.1/miR-99-treated cells compared with that in the control cultures. Increases in intracellular electron density, as well as the proportion of nuclear plasma, blebbing phenomena and apoptotic bodies were observed in pU6.1/miR-99-treated cells compared with control cultures according to electron microscopy analysis. The Tribbles 2 (TRIB2) 3'-untranslated region was also observed to be targeted by miR-99 and the results further demonstrated that miR-99 was able to negatively regulate TRIB2 expression in HeLa cells The results indicate that miR-99 acts as a tumor suppressor gene in HeLa cells, establishing a theoretical basis for its application in cancer therapeutics.

3.
Oncol Rep ; 30(5): 2042-8, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24002681

ABSTRACT

Tumor angiogenesis, a pivotal process for cancer growth and metastasis, requires both upregulation of pro­angiogenic molecules and downregulation of anti­angiogenic molecules. Anti-angiogenesis therapy represents a promising way for cancer treatment. Tumstatin, a novel endogenous angiogenesis inhibitor, inhibits endothelial cell proliferation, pathological angiogenesis and tumor growth. Ornithine decarboxylase (ODC), overexpressed in various cancers, is associated with cell transformation, tumor invasion and angiogenesis. We found that the expression of tumstatin was suppressed in ODC-overexpressing human cancer cells and renal carcinoma tissues. We presumed that ODC overexpression may downregulate the expression of tumstatin. To be able to test this hypothesis, we generated HEK293 cells that overexpress ODC (ODC transfectants) and characterized the following experimental groups: PBS-treated group, mock transfectants, ODC transfectants, ODC transfectants transfected with pcDNA-ODCr (an antisense ODC-expressing plasmid) group and putrescine-treated group. The effect of ODC overexpression on tumstatin expression was examined by reverse transcriptase-polymerase chain reaction (RT-PCR), western blot analysis and dual luciferase reporter assay. ODC-overexpressing cells and putrescine-treated cells showed suppressed tumstatin mRNA and protein expression, and decreased tumstatin gene promoter activity. Thus, ODC overexpression suppresses the expression of tumstatin, which may provide fundamental evidence for the combination of anti-angiogenic therapy and conventional therapy for cancer treatment.


Subject(s)
Autoantigens/administration & dosage , Carcinoma, Renal Cell/enzymology , Cell Proliferation/drug effects , Collagen Type IV/administration & dosage , Ornithine Decarboxylase/biosynthesis , Angiogenesis Inhibitors/administration & dosage , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/pathology , Down-Regulation , Gene Expression Regulation, Neoplastic , HEK293 Cells , Humans , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/pathology , Ornithine Decarboxylase/genetics
4.
Biomed Pharmacother ; 67(2): 97-102, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23485110

ABSTRACT

miR-21 can act as an oncogene. MSH2 has been reported that it involved in the DNA mismatch repair (MMR) system and overexpression of MSH2 can induce cell apoptosis. We predicted that MSH2-3'-untranslated region (3'-UTR) was targeted by miR-21 using microRNA analysis softwares. To further explore the roles of miR-21 and MSH2 in A549 cells, we constructed pcDNA-GFP-msh-UTR vector (including MSH2-3'-UTR) to transfect A549 cells with miR-21, GFP positive cells were estimated under a fluorescence microscopy and by flow cytometry. We found miR-21 could obviously downregulate the expression of MSH2, which was further proved by western blotting. Moreover, we treated A549 cells with cisplatin and found that cisplatin could inhibit A549 cell growth in vitro and in vivo. We also found that cisplatin could downregulate miR-21 expression, while increase MSH2 expression in A549 cells. Our results demonstrated that cisplatin could upregulate the expression of MSH2 through downregulating miR-21 to inhibit A549 cell proliferation, which provides new gene targets for drug design or cancer therary.


Subject(s)
Cisplatin/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , MicroRNAs/genetics , MutS Homolog 2 Protein/genetics , 3' Untranslated Regions/drug effects , 3' Untranslated Regions/genetics , Cell Growth Processes/drug effects , Cell Growth Processes/genetics , Cell Line, Tumor , Down-Regulation/drug effects , Down-Regulation/genetics , Humans , MutS Homolog 2 Protein/metabolism , Up-Regulation/drug effects
5.
Mol Biol Rep ; 38(2): 949-55, 2011 Feb.
Article in English | MEDLINE | ID: mdl-20533091

ABSTRACT

Ornithine decarboxylase (ODC), the first rate-limiting enzyme of polyamine biosynthesis, was found to be associated with cell growth, proliferation and transformation. ODC gene expression in gastric cancer was increased and its level was positively correlated with the degree of malignity of gastric mucosa and development of gastric lesions. In order to evaluate the therapeutic effects of antisense RNA of ODC on gastric cancer, an antisense RNA of ODC expressing plasmid pcDNA-ODCr which delivered a 120 bp fragment complementary to the initiation codon of ODC gene was constructed and transfected to gastric cancer cells SGC7901 and MGC803. Expression of ODC in gastric cancer cells was determined by western blot. Cell proliferation was assessed by MTS assay. Cell cycle was analyzed by flow cytometry and Matrigel assay was performed to assess the ability of gastric cancer cell invasiveness. The results showed that the ODC gene expression in gastric cancer cells transfected with the pcDNA-ODCr was downregulated efficiently. Tumor cell proliferation was suppressed significantly, and cell cycle was arrested at G1 phase. Gastric cancer cells had reduced invasiveness after gene transfer. Our study suggested that antisense RNA of ODC expressing plasmid pcDNA-ODCr had antitumor activity by inhibiting the expression of ODC, and downregulation of ODC expression using a gene therapy approach might be a novel therapeutic strategy for gastric cancer.


Subject(s)
Gene Expression Regulation, Neoplastic , Ornithine Decarboxylase/biosynthesis , Stomach Neoplasms/metabolism , Stomach Neoplasms/therapy , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Collagen/chemistry , Drug Combinations , Flow Cytometry/methods , Gene Expression Profiling , Gene Transfer Techniques , Genetic Therapy/methods , Humans , Laminin/chemistry , Oligonucleotides, Antisense/genetics , Plasmids/metabolism , Proteoglycans/chemistry , RNA/metabolism
6.
Chin Med J (Engl) ; 123(16): 2269-73, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20819678

ABSTRACT

BACKGROUND: Tumstatin is a novel endogenous angiogenesis inhibitor which is widely studied using purified protein. The current study evaluates the antiangiogenic effects of tumstatin-overexpression plasmid in vitro, reveals the mechanism underlying the vascular endothelial cell growth inhibition and searches for a novel method administering tumstatin persistently. METHODS: The eukaryotic expression plasmid pcDNA-tumstatin encoding tumstatin gene was constructed and transfected to human umbilical vein endothelial cell ECV304 and human renal carcinoma cell ACHN. Expression of tumstatin in the two cell lines was determined by RT-PCR and Western blotting. Vascular endothelial cell proliferation was assessed by CCK-8 assay and cell cycle was analyzed by flow cytometry. To investigate the mechanism by which pcDNA-tumstatin inhibited vascular endothelial cell proliferation in vitro, cyclin D1 protein was detected by Western blotting. RESULTS: DNA sequence confirmed that pcDNA-tumstatin was successfully constructed. RT-PCR and Western blotting indicated that tumstatin could express in the two cell lines effectively. After tumstatin gene transfer, ECV304 cell growth was significantly inhibited and the cell cycle was arrested in G1 phase. And Western blotting showed that pcDNA-tumstatin decreased the level of cyclin D1 protein. CONCLUSIONS: Overexpression of tumstatin mediated by pcDNA 3.1 (+) specially inhibited vascular endothelial cells by arresting vascular endothelial cell in G1 phase resulting from downregulation of cyclin D1 and administration of tumstatin using a gene therapy might be a novel strategy for cancer therapy.


Subject(s)
Autoantigens/genetics , Autoantigens/metabolism , Collagen Type IV/genetics , Collagen Type IV/metabolism , Endothelial Cells/cytology , Endothelial Cells/metabolism , Blotting, Western , Cell Cycle/genetics , Cell Cycle/physiology , Cell Line , Cell Line, Tumor , Cell Proliferation , Flow Cytometry , Humans , Plasmids/genetics , Reverse Transcriptase Polymerase Chain Reaction
7.
Mol Biol Rep ; 37(5): 2273-7, 2010 Jun.
Article in English | MEDLINE | ID: mdl-19688274

ABSTRACT

Tumstatin is the 28 kDa NC1 domain of the alpha3 chain of type IV collagen that inhibits pathological angiogenesis and suppresses endothelial cell proliferation and tumor growth. In the present paper, we expressed and purified recombinant human tumstatin protein and then prepared the anti-tumstatin polyclonal antibody. To investigate the expression of tumstatin in renal carcinoma, tumstatin protein was detected by western blotting using the prepared anti-tumstatin antibody and tumstatin mRNA levels were assayed by RT-PCR. The results showed that the expression of tumstatin gene was down-regulated in renal carcinoma tissues and cells. Our study suggests that as a novel endogenous angiogenesis inhibitor, tumstatin gene expression may be a marker for diagnosis, therapy and prognosis of renal carcinoma.


Subject(s)
Autoantigens/genetics , Collagen Type IV/genetics , Down-Regulation/genetics , Kidney Neoplasms/genetics , Antibodies, Neoplasm/biosynthesis , Autoantigens/immunology , Autoantigens/isolation & purification , Autoantigens/metabolism , Cloning, Molecular , Collagen Type IV/immunology , Collagen Type IV/isolation & purification , Collagen Type IV/metabolism , Gene Expression Regulation, Neoplastic , Humans , Kidney/metabolism , Kidney/pathology , Kidney Neoplasms/immunology , Kidney Neoplasms/pathology
8.
Cancer Sci ; 100(11): 2126-32, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19686286

ABSTRACT

Spermidine/spermine N(1)-acetyltransferase (SSAT) is the rate-limiting step in polyamine catabolism. In a previous study, we constructed a recombinant adenovirus, Ad-SSAT, which can express human SSAT. In the present study, we investigated the effect of upregulated and downregulated SSAT on gastric cancer cells. We found that upregulated SSAT could inhibit the growth of MGC803 and SGC7901 cells, whereas adverse results were found with downregulated SSAT. We further analyzed cell cycle profiles and the expression levels of the major cell cycle regulatory proteins of S phase. The results showed that the growth inhibition was caused by S phase arrest. Ad-SSAT suppressed the expression of cyclin A and nuclear factor E2F1 in MGC803 and SGC7901 cells. We observed the E2F promoter activity caused by Ad-SSAT using a reporter gene assay. We also investigated the antitumorigenicity of upregulated SSAT by Ad-SSAT using a SGC7901 xenograft model in nude mice. Our results suggest that the upregulation of SSAT by Ad-SSAT infection inhibited the growth of gastric cancer in vitro and in vivo. Ad-SSAT arrested gastric cancer cells in S phase, which was mediated through downregulation of the cyclin A-E2F signaling pathway.


Subject(s)
Acetyltransferases/physiology , Adenoviridae/genetics , Stomach Neoplasms/prevention & control , Acetyltransferases/genetics , Animals , Biogenic Polyamines/analysis , Cell Line, Tumor , Cyclin A/antagonists & inhibitors , E2F1 Transcription Factor/antagonists & inhibitors , Genetic Vectors , Humans , Mice , Mice, Inbred BALB C , S Phase , Stomach Neoplasms/chemistry , Stomach Neoplasms/enzymology , Stomach Neoplasms/pathology , Up-Regulation
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