A female patient with 33 ages suffered the ischiorectal lesion that experienced incision without removal of lesion. The features suggested the ischiorectal endometria included solid organ caused pain in the ischiorectal region, no fever, increased pain and bigger organ during menstruation. The total removal operation of this organ and microscopic tests was carried out to treat and affirm the ischiorectal endometria
Endometrium ; Microscopy, Interference

Acanthamoeba cysts are resistant to unfavorable physiological conditions and various disinfectants. Acanthamoeba cysts have 2 walls containing various sugar moieties, and in particular, one third of the inner wall is composed of cellulose. In this study, it has been shown that down-regulation of cellulose synthase by small interfering RNA (siRNA) significantly inhibits the formation of mature Acanthamoeba castellanii cysts. Calcofluor white staining and transmission electron microscopy revealed that siRNA transfected amoeba failed to form an inner wall during encystation and thus are likely to be more vulnerable. In addition, the expression of xylose isomerase, which is involved in cyst wall formation, was not altered in cellulose synthase down-regulated amoeba, indicating that cellulose synthase is a crucial factor for inner wall formation by Acanthamoeba during encystation.
Acanthamoeba castellanii/*enzymology/genetics/metabolism ; Aldose-Ketose Isomerases/*biosynthesis ; Amebiasis/*pathology ; Benzenesulfonates ; Cell Wall/chemistry/genetics/*metabolism ; Cellulose/biosynthesis ; Down-Regulation ; Encephalitis/parasitology ; Glucosyltransferases/*biosynthesis/genetics ; Keratitis/parasitology ; Microscopy, Electron, Transmission ; RNA Interference ; RNA, Small Interfering

BACKGROUNDThe discovery of water channel aquaporins (AQPs) has greatly expanded the understanding of the regulation of the water permeability of biological membranes. Aquaporin-1 (AQP1) may be involved in fluid transport in numerous pathological conditions. The objective of the present study was to examine whether AQP1 is present in cultured rat pleural mesothelial cells (PMCs) and to investigate the specific inhibitory effect of RNA interference (RNAi) on AQP1 expression in PMCs, which may provide a new method for the further studies on the relation between expression of AQP1 in PMCs and pleural fluid removal in vivo.

METHODSPMCs were isolated and cultured from rat pleura. The expression of AQP1 in PMCs was confirmed by immunocytochemical staining and reverse transcriptase-polymerase chain reaction (RT-PCR). Two eukaryotic expression plasmid vectors of short hairpin RNA (shRNA) specific for the AQP1 gene of rat sapien were designed and constructed. The recombinant plasmid vectors were transfected into cultured rat PMCs by cation liposomes. Flow cytometry was used to screen the most effective shRNA at 48 hours after transfection. The expressions of AQP1 mRNA and protein were detected by RT-PCR and Western blotting method at 48 hours after transfection.

RESULTSRT-PCR and immunostaining revealed that AQP1 mRNA and protein were present in cultured rat PMCs. Two effective eukaryotic expression plasmid vectors of shRNA specific for the AQP1 gene were constructed successfully. The levels of the expression of AQP1 were inhibited by 83.45%, 90.93%, respectively, at mRNA level and 41.24%, 67.60%, respectively at protein level by two recombinant plasmids at 48 hours after transfection. The expression of AQP1 in PMCs transfected with plasmid was significantly lower than that of the cells transfected with the control plasmid HK and that of the untransfected cells (P < 0.01). There was no significant difference in AQP1 expression between the control group and the group transfected with AQP1 nonspecific shRNAs (P < 0.05).

CONCLUSIONSThe expression of AQP1 was present in rat PMCs. The application of shRNA-AQP1 could markedly inhibit the expression of AQP1 in cultured rat PMCs. The use of RNAi is a promising tool for future research into the mechanisms of pleural fluid in vivo.

Animals ; Aquaporin 1 ; antagonists & inhibitors ; genetics ; Cells, Cultured ; Epithelial Cells ; metabolism ; Flow Cytometry ; Male ; Microscopy, Fluorescence ; Pleura ; cytology ; metabolism ; Pleural Effusion ; therapy ; RNA Interference ; RNA, Small Interfering ; genetics ; Rats

OBJECTIVETo investigate whether short interfering RNAs(siRNAs) of beta-site APP cleaving enzyme (BACE) can inhibit the expression of BACE in mammalian cells.

METHODSThe gene of EGFP, U6 promoter and beta-secretase targeting siRNA were cloned by PCR, respectively. The PCR products were inserted into plasmid pLXSN. The interfering vector pLXSN/EGFP-U6-siBACE was transferred into SK-N-SH cells to express BACE. The inhibition effect of BACE siRNA on BACE expression was investigated by fluoroscopy and immunohistochemistry method.

RESULTThe interfering vector pLXSN/EGFP-U6-siBACE was constructed successfully. The BACE siRNA inhibited the expression of BACE in the SK-N-SH cells specifically and effectively, and the production of A beta was reduced.

CONCLUSIONBACE siRNA can inhibit the expression of BACE gene of mammalian cells.

Amyloid Precursor Protein Secretases ; genetics ; metabolism ; Animals ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Immunohistochemistry ; Mice ; Microscopy, Fluorescence ; NIH 3T3 Cells ; Neuroblastoma ; genetics ; metabolism ; pathology ; Plasmids ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Tumor Cells, Cultured

Chloroquine ; pharmacology ; Down-Regulation ; Gene Expression ; drug effects ; HEK293 Cells ; Humans ; Leupeptins ; pharmacology ; Membrane Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Microscopy, Fluorescence ; Protein Binding ; Protein Subunits ; genetics ; metabolism ; RNA Interference ; RNA, Small Interfering ; metabolism ; Shab Potassium Channels ; genetics ; metabolism

The vitamin K epoxide reductase complex subunit 1 (VKORC1), the rate-limiting enzyme for vitamin K recycling, is significantly down-regulated in the kidneys of urolithiasis patients. This study searched for direct evidence to define the inhibitory activity of VKORC1 against calcium oxalate (CaOx) crystal formation. In the experiment of VKORC1 overexpression, HK-2 cells were transfected with the pFLAG-CMV-7.1-VKORC1 plasmid as a pFLAG-CMV-7.1-VKORC1 transfection group or the pFLAG-CMV-7.1 plasmid as a pFLAG-CMV-7.1 control group. In the experiment of VKORC1 knockdown, HK-2 cells were transfected with the PGPU6/GFP/Neo-VKORC1shRNA-2 as a PGPU6/GFP/Neo-VKORC1shRNA-2 transfection group or the PGPU6/GFP/Neo-shRNA-NC plasmid as a PGPU6/GFP/Neo-shRNA-NC control group. The expression of VKORC1 in HK-2 cells was detected by real-time quantitative PCR and Western blotting. The CaOx crystal formation was observed under the laser-scanning confocal microscope. It was found that the expression levels of VKORC1 mRNA and protein were significantly higher in the pFLAG-CMV-7.1-VKORC1 transfection group than in the pFLAG-CMV-7.1 control group (P<0.01). The number of CaOx crystals in HK-2 cells incubated in fluorescently labeled CaOx monohydrate (COM) crystal medium for 48 h was 14±4 per field (100×) in the pFLAG-CMV-7.1-VKORC1 transfection group and 26±5 per field (100×) in the pFLAG-CMV-7.1 control group respectively under the laser-scanning confocal microscope. The amount of CaOx crystal aggregation and formation in the pFLAG-CMV-7.1-VKORC1 transfection group was significantly reduced as compared with the pFLAG-CMV-7.1 control group (P<0.05). The expression levels of VKORC1 mRNA and protein were significantly lower in the PGPU6/GFP/Neo-VKORC1shRNA-2 transfection group than in the PGPU6/GFP/Neo-shRNA-NC control group (P<0.05). The number of CaOx crystals in HK-2 cells incubated in fluorescently labeled COM crystal medium was 65±11 per field (100×) in the PGPU6/GFP/Neo-VKORC1shRNA-2 transfection group and 24±6 per field (100×) in the PGPU6/GFP/Neo-shRNA-NC control group respectively under the laser-scanning confocal microscope. The amount of CaOx crystal aggregation and formation in the PGPU6/GFP/Neo-VKORC1shRNA-2 transfection group was significantly increased as compared with the PGPU6/GFP/Neo-shRNA-NC control group (P<0.05). These findings suggested that the VKORC1 protein could inhibit CaOx salt crystallization, adhesion and aggregation. This research would help us to understand the mechanisms involving the interaction between crystallization and epithelial cells and the formation of CaOx.
Apoptosis ; drug effects ; Blotting, Western ; Calcium Oxalate ; chemistry ; metabolism ; pharmacology ; Cell Line ; Crystallization ; Dose-Response Relationship, Drug ; Flow Cytometry ; Gene Expression ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Microscopy, Confocal ; RNA Interference ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors ; Transfection ; Vitamin K Epoxide Reductases ; genetics ; metabolism

This study was aimed to construct mouse Fas-targeting si RNA-expressing recombinant retroviral vector in order to explore the therapeutic potential of Fas inhibition by siRNA in the treatment of aplastic anemia and to provide a basia for extensive development of RNA interference techninque. The U6(+) 27 promoter cassette and siFas sequence were obtained by PCR method. The U6-siFas fragment was cloned into the multiple restriction site of pLXSN-EGFP and directly downstream of EGFP gene. The resultant retroviral vector pLXSN/EGFP-siFas was packaged using PA317 cell line and tittered using NIH3T3 cell line. P815 cells were infected by the retroviral vector. EGFP expression in P815 was observed under fluorescent microscope and Fas inhibition effect was detected by immunohistochemistry. The results indicated that successfully constructed retrovirus vector pLXSN/EGFP-siFas was could not only deliver siRNA into mammalian cells efficiently and inhibit Fas expression in P815 cells, but also could express EGFP as marker and neomycine resistance gene to allow antibiotic selection. It is concluded that the successful construction of this retroviral vector would greatly facilitate the application of RNA interference and lay the foundation for therapeutic study of Fas inhibition in the treatment of aplastic anemia.
Anemia, Aplastic ; genetics ; pathology ; therapy ; Animals ; Cell Line ; Cell Line, Tumor ; Cloning, Molecular ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Immunohistochemistry ; Mice ; Microscopy, Fluorescence ; NIH 3T3 Cells ; RNA Interference ; RNA, Small Interfering ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Retroviridae ; genetics ; Transfection ; fas Receptor ; genetics ; metabolism

OBJECTIVETo construct the specific stable expression and high efficiency small interfering RNA(siRNA) expression vector that can block DNMT1 gene function.

METHODSUsing vector-based RNA interference technique, the authors constructed a vector to transcribe functional short interfering RNA (RNAi). After transfection by lipofectmine (TM) reagent, the treated cells were selected by G418. The expression levels of RNA and protein of DNMT1 were analyzed by reverse transcription polymerase chain reaction(RT-PCR) and Western blotting. The status of methylation of E-cadherin was analyzed by methylation-specific PCR(MSP).

RESULTSThe expression level of endogenous DNMT1 mRNA in transfected SMMC-7721 cell lines with DNMT1 RNAi construct was 43% less than that in control cell 7721-pSU cell lines. The protein level in the former was about 10% less than that in the latter. The efficiency of the siRNA of DNMT1 was found to be higher than 90%. Demethylation of promoter of E-cadherin was obtained due to the inhibition of DNMT1.

CONCLUSIONDNMT1 siRNA stable expressing vector was obtained by gene-recombined technology. There was no complete sameness between the levels of protein and RNA in gene silenced cell lines. The efficiency of the siRNA should be confirmed by Western-blotting.

Blotting, Western ; Cell Line, Tumor ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; genetics ; metabolism ; DNA Methylation ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Microscopy, Fluorescence ; RNA Interference ; RNA, Small Interfering ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

OBJECTIVETo study the role of inhibition FAK expression by FAK siRNA in liver cancer cell (MHCC97-H) adhesion, invasion and cytoskeleton rearrangement.

METHODSFAK siRNA was transfected into MHCC97-H cell by Lipofectamine 2000; then, FAK expression was detected by Western blot analysis. The change of cell adhesive and invasive ability after RNAi was checked by cell adhesive assay and cell invasive assay respectively. Meanwhile, matrix metalloproteinase-2 secretion was checked by gelatin zymography. Cytoskeleton rearrangement labeled by immunofluorescence antibody was examined by confocal laser scanning microscope.

RESULTSFAK expression in MHCC97-H cell was obviously inhibited by specific FAK siRNA; However, it was not inhibited by negative siRNA. Adhesion rate between MHCC97-H cell and extracellular matrix decreased from 57.3% to 35.8% after RNA interference (P < 0.05). Compared with untreated group, the number of cell penetrating matrigel also decreased from 31.3 +/- 2.6 to 14.5 +/- 3.1 after transfection (P < 0.05). Besides, matrix metalloproteinase-2 secretion was significantly reduced for FAK expression inhibited by FAK siRNA. FAK inhibition influenced Vinculin rearrangement, blocked the formation of lamellipodium, delayed the time of focal adhesion formation.

CONCLUSIONDown-regulation the expression of FAK can reduce adhesive rate and invasive number of MHCC97-H cell by influencing cytoskeleton rearrangement and decreasing matrix metalloproteinase-2 secretion.

Blotting, Western ; Carcinoma, Hepatocellular ; enzymology ; genetics ; pathology ; Cell Adhesion ; Cell Line, Tumor ; Cell Movement ; Cytoskeleton ; metabolism ; Fluorescent Antibody Technique ; Focal Adhesion Kinase 1 ; genetics ; metabolism ; Humans ; Liver Neoplasms ; enzymology ; genetics ; pathology ; Matrix Metalloproteinase 2 ; metabolism ; Microscopy, Confocal ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection ; methods

OBJECTIVETo construct a recombinant retroviral vector for RNA interference targeting human telomerase reverse transcriptase (hTERT).

METHODSThe sequences coding for enhanced fluorescence protein (EGFP), U6 promoter and a small interfering RNA (siRNA) targeting hTERT were amplified by PCR, respectively, and sub-cloned sequentially into the retroviral shuttle plasmid pLXSN to construct the plasmid pLXSN-EGFP-U6-siTERT. The recombinant expression plasmid was identified by restriction enzyme digestion and sequencing. Fluorescence microscopy and flow cytometry were employed to analyze EGFP expression in NIH3T3 transfected with the recombinant plasmid, and MMT assay was performed to evaluate the growth inhibition of Hela cells resulting from RNA interference mediated by the plasmid.

RESULTSSequence analysis and restriction enzyme digestion showed that the recombinant expression plasmid pLXSN-EGFP-U6-siTERT was constructed successfully. Twenty-four hours after transfection of NIH3T3 cells with the recombinant plasmid, the expression rate of EGFP reached 24.1% as shown by flow cytometry. MTT assay demonstrated a cell death rate of 53.2% 72 h after transfection of Hela cells with the plasmid.

CONCLUSIONThe successful construction of the recombinant retroviral plasmid mediating potent cell growth inhibition suggests the great potential of RNA interference technique in suppressing hTERT expression in mammalian tumor cells.

Animals ; Cloning, Molecular ; Flow Cytometry ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; HeLa Cells ; Humans ; Mice ; Microscopy, Fluorescence ; NIH 3T3 Cells ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Retroviridae ; genetics ; Telomerase ; biosynthesis ; genetics