ABSTRACT
OBJECTIVES@#The effect of isoprenylcysteine carboxymethyltransferase (ICMT) silencing on the migration and invasion of tongue squamous cell carcinoma was investigated by constructing the small interfering RNA (siRNA) of ICMT.@*METHODS@#Through liposomal transfection, siRNA was transfected into human tongue squamous cell carcinoma CAL-27 and SCC-4 cells (ICMT-siRNA group) with a negative control group (transfected with NC-siRNA) and a blank control group (transfected with a transfection reagent but not with siRNA). Quantitative real-time polymerase chain reaction was performed to analyze the mRNA expression of ICMT and RhoA in each group of cells after transfection and to measure the silencing efficiency. Western blot was applied to examine the expression levels of ICMT, total RhoA, membrane RhoA, ROCK1, matrix metalloproteinase (MMP)-2, and MMP-9 proteins in each group. The migration and invasion abilities were evaluated via wound healing and Transwell motility assays.@*RESULTS@#After CAL-27 and SCC-4 cells were transfected with ICMT-siRNA, the expression levels of ICMT genes and proteins decreased significantly in the experimental group compared with those in the negative and blank control groups (@*CONCLUSIONS@#The migration and invasion abilities of CAL-27 and SCC-4 cells were reduced significantly after the transfection of ICMT-siRNA, and the involved mechanism might be related to the RhoA-ROCK signaling pathway.
Subject(s)
Humans , Carcinoma, Squamous Cell , Cell Line, Tumor , Cell Movement , Cell Proliferation , Neoplasm Invasiveness , Protein Methyltransferases , RNA, Small Interfering , Tongue , Tongue Neoplasms , Transfection , rho-Associated KinasesABSTRACT
OBJECTIVES@#This study aimed to explore the effects of silencing isoprenylcysteine carboxyl methyltransfe-rase (Icmt) through small interfering RNA (siRNA) interference on the proliferation and apoptosis of tongue squamous cell carcinoma (TSCC).@*METHODS@#Three siRNA were designed and constructed for the Icmt gene sequence and were then transfected into TSCC cells CAL-27 and SCC-4 to silence Icmt expression. The tested cells were divided as follows: RNA interference groups Icmt-siRNA-1, Icmt-siRNA-2, and Icmt-siRNA-3, negative control group, and blank control group. The transfection efficiency of siRNA was detected by the fluorescent group Cy3-labeled siRNA, and the expression of Icmt mRNA was screened by quantitive real-time polymerase chain reaction (qRT-PCR) selected the experimental group for subsequent experiments. The expression of Icmt, RhoA, Cyclin D1, p21, extracellular regulated protein kinases (ERK), and phospho-extracellular regulated protein kinases (p-ERK) were analyzed by Western blot. The proliferation abilities of TSCC cells were determined by cell counting kit-8 assay. The change in apoptosis was detected by AnnexinV-APC/propidium staining (PI) assay. Cell-cycle analysis was conducted by flow cytometry.@*RESULTS@#The expression of Icmt mRNA and protein in TSCC cells significantly decreased after Icmt-siRNA transfection (@*CONCLUSIONS@#Silencing Icmt can effectively downregulate its expression in TSCC cells, reduce the RhoA membrane targeting localization and cell proliferation, and induce apoptosis. Thus, Icmt may be a potential gene therapy target for TSCC.
Subject(s)
Humans , Apoptosis , Carcinoma, Squamous Cell , Cell Line, Tumor , Cell Proliferation , Protein Methyltransferases , RNA, Small Interfering , Tongue , Tongue NeoplasmsABSTRACT
<p><b>OBJECTIVE</b>To identify more effective and less toxic drugs to treat animal toxoplasmosis.</p><p><b>METHODS</b>Efficacy of seven kinds of sulfonamides against Toxoplasma gondii (T. gondii) in an acute murine model was evaluated. The mice used throughout the study were randomly assigned to many groups (10 mice each), which either remained uninfected or were infected intraperitoneally with tachyzoites of T. gondii (strains RH and CN). All groups were then treated with different sulfonamides and the optimal treatment protocol was determined candidates. Sulfadiazine-sodium (SD) was used for comparison.</p><p><b>RESULTS</b>The optimal therapy involved gavaging mice twice per day with 250 mg/kg bw of sulfachloropyrazine-sodium (SPZ) for five days. Using this protocol, the average survival time and the time-point of 50% fatalities were prolonged significantly compared with SD treatment. Treatment with SPZ protected 40% of mice from death, and the heart and kidney tissue of these animals was parasite-free, as determined by nested-PCR. SPZ showed excellent therapeutic effects in the treatment of T. gondii in an acute murine model and is therefore a promising drug candidate for the treatment and prevention of T. gondii in animals.</p><p><b>CONCLUSIONS</b>It can be concluded that the effective drug sulfachloropyrazine may be the new therapeutic options against animal toxoplasmosis.</p>
Subject(s)
Animals , Female , Mice , Administration, Oral , Antiprotozoal Agents , DNA, Protozoan , Disease Models, Animal , Heart , Parasitology , Kidney , Parasitology , Polymerase Chain Reaction , Sulfanilamides , Survival Analysis , Toxoplasma , Genetics , Toxoplasmosis , Drug Therapy , Treatment OutcomeABSTRACT
In order to clone and identify differentially expressed genes in the sporogony stage of Eimeria tenella, the cDNAs from unsporulated oocysts and sporulated oocysts of E. tenella were used as driver, respectively, the cDNAs from sporozoites of E. tenella was used tester, Two subtractive cDNA libraries of sporozoites were constructed by using the technique of suppression subtractive hybridization (SSH). the cDNAs from unsporulated oocysts was used driver, the cDNAs from sporulated ooceysts was used tester, one subtractive cDNA library of sporulated oocysts was constructed. PCR amplification revealed that the two subtractive cDNA libraries of sporozoites and one subtractive cDNA library of sporulated oocysts contained approximated 96%, 96% and 98% recombinant clones, respectively. Fifty positive clones were sequenced and analyzed in GenBank with Blast search from three subtractive cDNA libraries, respectively, thirteen unique sequences were found from the subtractive cDNA library of sporulated oocysts, eight ESTs shared significant identity with previously described. A total of forty unique sequences were obtained from the two subtractive cDNA libraries, nine ESTs shared significant identity with previously described, the other sequences represent novel genes of E. tenella with no significant homology to the proteins in Genbank. These results have provided the foundation for cloning new genes of E. tenella and further studying new approaches to control coccidiosis.
Subject(s)
Animals , Chickens , Parasitology , Coccidiosis , Parasitology , DNA, Protozoan , Genetics , Eimeria tenella , Genetics , Physiology , Gene Expression Regulation , Gene Library , Nucleic Acid Hybridization , Methods , Oocytes , Metabolism , Poultry Diseases , Parasitology , SporesABSTRACT
Two-dimensional electrophoresis (2-DE) was employed to compare the proteome of Diclazuril-resistance Eimeria tenella with that of sensitive strains for identifying unique proteins of these stains. 5 protein spots were found to express differentially. Four spots which remarkably were measured by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The data obtained from peptide mass fingerprinting were used in NCBInr database search, two protein spots in gel were identified as Eimeria tenella sporulated oocyst TA4 antigen protein, Heat shock 70kD protein, two protein spots were functional proteins of Eukaryote. These proteins are potentially basic work for finding molecular mechanism about drug-resistance of Eimeria tenella and new marker in the detection of resistance of Eimeria tenella.