Small nuclear RNAs (snRNAs) refer to a class of highly abundant and functionally important non-coding small RNAs that are localized in the eukaryotic nucleus. These snRNAs are highly conserved in different eukaryotes during evolution and form complexes with specific chaperones to fulfill critical biological functions, including precursor messenger RNA (pre-mRNA) splicing and ribosomal RNA (rRNA) modification. Consequently, the regulation of snRNA gene expression is a crucial biological process for plants. In plants, the transcription and processing of snRNAs are regulated by RNA polymerase (Pol), snRNA-activating protein complex (SNAPc), defective in snRNA processing (DSP), and specific cis-elements in the snRNA promoter regions. Proper regulation of snRNA expression is essential for normal plant growth, development, and stress responses. This review summarizes the classification, structures, transcriptional regulation, and biological functions of plant snRNA genes, while outlining future research directions for snRNAs.
RNA, Small Nuclear/physiology* ; Gene Expression Regulation, Plant ; Transcription, Genetic ; Plants/metabolism* ; RNA, Plant/genetics*

OBJECTIVE: To explore the role of SIRT1 in the occurrence of epithelial-mesenchymal transition (EMT) in EC-9706 and Eca-109 cells and the possible mechanism. METHODS: Three chemically synthesized siRNA targeting SIRT1 were transfected into EC-9706 and Eca-109 cells with the non-transfected cells and cells transfected with the negative siRNAs as controls. Real-time PCR and Western blotting were used to detect the expressions of SIRT1, E-cadherin, vimentin, Snail, Twist1 and ZEB in the cells. Transwell invasion assay and wounding healing assay were used to examine the changes in the invasion and metastasis abilities of the cells after transfection. RESULTS: EC-9706 and Eca-109 cells transfected with SIRT1 siRNA1 and SIRT1 siRNA3 showed significantly decreased mRNA and protein expressions of SIRT1 ( < 0.05). Transwell invasion assay and wounding healing assay showed that transfection with SIRT1 siRNA1 and SIRT1 siRNA3 caused significantly lowered invasion and metastasis abilities in EC-9706 and Eca-109 cells ( < 0.05). In EC-9706 and Eca-109 cells transfected with SIRT1 siRNA1 and SIRT1 siRNA3, the expression level of E-cadherin was significantly increased while the expressions of vimentin, Snail and Twist were significantly lowered ( < 0.05). CONCLUSIONS SIRT1 participates in the invasion and metastasis of EC-9706 and Eca- 109 cells probably by inducing EMT via regulating the expression of Snail.
Antigens, CD ; metabolism ; Cadherins ; metabolism ; Cell Line, Tumor ; Cell Movement ; Epithelial-Mesenchymal Transition ; physiology ; Humans ; Neoplasm Invasiveness ; Nuclear Proteins ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; metabolism ; Sirtuin 1 ; genetics ; metabolism ; Snail Family Transcription Factors ; metabolism ; Transfection ; Twist-Related Protein 1 ; metabolism ; Vimentin ; metabolism ; Zinc Finger E-box-Binding Homeobox 1 ; metabolism

OBJECTIVETo investigate the role of wild-type p53-induced gene 1 (WIG-1) on the regulation of multi-drug resistance in small cell lung cancer.

METHODSThe expressions of WIG-1 protein and gene were detected by Western blot and real-time PCR (RT-PCR) in both the drug-sensitive H69 and drug-resistant H69AR cell lines, respectively. Meanwhile, the differential expression of WIG-1 was also detected in peripheral blood samples of responders and non-responder patients. Furthermore, the WIG-1 expression was inhibited by siRNA in H69AR cells, then the drug-sensitivities of H69AR cells to chemotherapy agents such as ADM, DDP, VP-16 were detected by CCK8 assay, and apoptosis rate was detected by flow cytometry. The possible association of WIG-1 with clinical parameters was evaluated.

RESULTSThe expression of WIG-1 was significantly increased in H69AR cells (5.965 ± 0.890) than that in the H69 cells (1.023 ± 0.127) (P = 0.007). The expression of WIG-1 was significantly increased in the non-responder patients (4.169 ± 0. 970) than in the H69 cells and responders (1.673 ± 0.127) (P < 0.001). The drug-sensitivities of H69AR cells to chemotherapeutic drugs were increased when the expression of the WIG-1 was down-regulated. The apoptosis rate was significantly decreased in the H69AR cells (1.037 ± 0.049)% compared with that in the H69 cells [(7.963 ± 0.097)%, (P < 0.01)]. The apoptosis rate was increased in the H69AR-Si-WIG-1 cells (20.915 ± 0.890)% than that of (1.037 ± 0.049)% in the H69AR and H69AR-NC group (2.025 ± 0.097)% (P < 0.01). The expression of WIG-1 was not significantly associated with gender, and age (P > 0.05), but significantly correlated with chemosensitivity, overall survival and clinical stage (P < 0.001 for all).

CONCLUSIONSOur results suggest that WIG-1 is involved in the regulation of the multidrug resistance mechanism in small cell lung cancer. Selective silencing of the WIG-1 gene may reverse the multidrug resistance of SCLC via increasing cell apoptosis.

Antineoplastic Agents ; Apoptosis ; DNA-Binding Proteins ; metabolism ; Down-Regulation ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; physiology ; Etoposide ; Humans ; Lung Neoplasms ; metabolism ; Nuclear Proteins ; metabolism ; RNA, Small Interfering ; Small Cell Lung Carcinoma ; metabolism

DNA double-strand break (DSB) is the most severe form of DNA damage, which is repaired mainly through high-fidelity homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). Defects in the DNA damage response lead to genomic instability and ultimately predispose organs to cancer. Nicotinamide phosphoribosyltransferase (Nampt), which is involved in nicotinamide adenine dinucleotide metabolism, is overexpressed in a variety of tumors. In this report, we found that Nampt physically associated with CtIP and DNA-PKcs/Ku80, which are key factors in HR and NHEJ, respectively. Depletion of Nampt by small interfering RNA (siRNA) led to defective NHEJ-mediated DSB repair and enhanced HR-mediated repair. Furthermore, the inhibition of Nampt expression promoted proliferation of cancer cells and normal human fibroblasts and decreased β-galactosidase staining, indicating a delay in the onset of cellular senescence in normal human fibroblasts. Taken together, our results suggest that Nampt is a suppressor of HR-mediated DSB repair and an enhancer of NHEJ-mediated DSB repair, contributing to the acceleration of cellular senescence.
Antigen-Antibody Complex ; metabolism ; Antigens, Nuclear ; genetics ; metabolism ; Carrier Proteins ; genetics ; metabolism ; Cell Line ; Cell Proliferation ; Cellular Senescence ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; DNA Repair ; DNA-Activated Protein Kinase ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Fibroblasts ; cytology ; HeLa Cells ; Homologous Recombination ; genetics ; physiology ; Humans ; Ku Autoantigen ; Nicotinamide Phosphoribosyltransferase ; genetics ; metabolism ; physiology ; Nuclear Proteins ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; beta-Galactosidase ; metabolism

OBJECTIVE: To explore the effects of nuclear protein-like transcription factor nuclear receptor subfamily 2 group E member 1 (NR2E1) on the growth, division, and proliferation of neuroblastoma cell line IMR32. METHODS: A NR2E1 shiRNA plasmid vector was constructed and transfected into neuroblastoma cell line IMR32 using lipofedamine™2000. Subsequent cell growth was measured by cell counting and the protein expression of somatic nuclear division was examined by immunofluorescent staining. RESULTS: At 48 h after the neuroblastoma cells IMR32 were transfected with NR2E1-shiRNA vector, the related nuclear division protein and the proliferation of the transfected cells IMR32 were remarkably depressed. CONCLUSION Cells division and proliferation of neuroblastoma cell line IMR32 is inhibited through transfection with the NR2E1-shiRNA plasmid vector.
Cell Division ; genetics ; physiology ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Neuroblastoma ; pathology ; RNA, Small Interfering ; genetics ; Receptors, Cytoplasmic and Nuclear ; genetics ; metabolism ; Transfection

Antineoplastic Agents ; therapeutic use ; BRCA2 Protein ; metabolism ; Breast Neoplasms ; drug therapy ; metabolism ; pathology ; Cell Cycle Proteins ; metabolism ; physiology ; Centrosome ; metabolism ; Drug Screening Assays, Antitumor ; Female ; Humans ; Microtubule-Associated Proteins ; metabolism ; Neoplasm Invasiveness ; Nuclear Proteins ; metabolism ; Phosphorylation ; Prognosis ; Protein-Serine-Threonine Kinases ; metabolism ; physiology ; Proto-Oncogene Proteins ; metabolism ; physiology ; RNA, Small Interfering ; pharmacology ; Tumor Suppressor Protein p53 ; metabolism

BACKGROUND AND OBJECTIVEPrevious studies have shown that Bmi-1 is overexpressed in a variety of tumors, suggesting that Bmi-1 plays an important role in tumorigenesis. In this study, we investigated the effect of Bim-1 siRNA on cell proliferation, cell cycle, cell apoptosis and migration of human esophageal carcinoma EC9706 cells, and explored its potential mechanisms.

METHODSBmi-1 small interfering RNA (siRNA) was transferred into EC9706 cells. Then, cell proliferation was measured using cell counting kit-8 (CCK-8), cell cycle and cell apoptosis were analyzed by flow cytometry, cell migration ability was detected using Boyden chamber assay, and the mRNA and protein expression levels of Bmi-1, p16, Bcl-2, Bax, and MMP-2 were determined using real-time polymerase chain reaction (PCR) and Western blot analysis, respectively.

RESULTSBmi-1 siRNA treatment significantly inhibited the expression of Bmi-1 at both mRNA and protein levels in EC9706 cells. Cell proliferation rate decreased dramatically in the Bmi-1 siRNA treated group than in the untreated group and in the scrambled siRNA treated group (both P < 0.001). In Bmi-1 treated group, the percentage of cells at G(0)/G(1) stage was 71.93%, which was higher than that in the untreated group (47.36%) or scramble siRNA treated group (48.47%) (both P < 0.001). Early cell apoptosis rate also increased significantly in the Bmi-1 siRNA treated group (both 17.32%) than in the untreated group (2.61%) and in the scramble siRNA treated group (2.73%) (both P < 0.001). Further experiment suggested that downregulation of Bmi-1 led to less cell migration. In EC9706 cells transfected by Bmi-1 siRNA, the expression levels of p16 and Bax increased, while the expression level of Bcl-2 decreased.

CONCLUSIONSBmi-1 downregulation in esophageal carcinoma cells inhibits cell proliferation, cell cycle, and cell migration, while increases cell apoptosis. These results suggest that Bmi-1 is a potential molecular target of treating esophageal cancer.

Apoptosis ; Cell Cycle ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Down-Regulation ; Esophageal Neoplasms ; metabolism ; pathology ; Gene Expression Regulation, Neoplastic ; Humans ; Nuclear Proteins ; genetics ; metabolism ; physiology ; Polycomb Repressive Complex 1 ; Proto-Oncogene Proteins ; genetics ; metabolism ; physiology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Repressor Proteins ; genetics ; metabolism ; physiology ; Transfection ; bcl-2-Associated X Protein ; metabolism

OBJECTIVEConstruction of a small interfering RNA (siRNA) eukaryotic expression vector specific to mouse myocardin gene and study on the role of myocardin-siRNA on differentiation of mouse bone mesenchymal stem cells (MSCs) into smooth muscle-like cells induced by PDGF-BB in vitro.

METHODSMouse MSCs were isolated from bone marrow and cultured with 50 mg/L PDGF-BB and fetal bovine serum (20%). Specific myocardin-siRNA sequence was cloned into a plasmid pGenesil-1.0 vector, which contained U6 promoter. The recombinant plasmid and control plasmid were transfected into MSCs which had been cultured with PDGF-BB for 6 days beforehand. The expression of myocardin mRNA was detected by RT-PCR 48 hours after the transfection. Immunohistochemistry was used to detect the SM-MHC and to identify the smooth muscle-like cells.

RESULTSThe recombinant plasmids carrying myocardin-siRNA sequences were constructed successfully and the myocardin mRNA was reduced 42.86% by pGen-myo-shRNA in comparing with that of the controls (P<0.01); and the expression of SM-MHC protein was down-regulated (P<0.01).

CONCLUSIONSubset of mouse MSCs have the potential to differentiate into smooth muscle-like cells, a possible cell source responsible for atherosclerotic plaque formation, and myocardin expression may play an important role during this process.

Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; Cells, Cultured ; Down-Regulation ; Gene Silencing ; Genetic Vectors ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Mice ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; metabolism ; Myosin Heavy Chains ; metabolism ; Nuclear Proteins ; biosynthesis ; genetics ; physiology ; Plaque, Atherosclerotic ; pathology ; Plasmids ; Platelet-Derived Growth Factor ; pharmacology ; Proto-Oncogene Proteins c-sis ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; Trans-Activators ; biosynthesis ; genetics ; physiology ; Transfection

hUBEW, a newly identified class I ubiquitin conjugating enzyme, probably plays an important role in tumorigenesis and DNA repair processes. RNA interference (RNAi) is a process in cells to degrade specific homologous mRNA by forming duplex RNA and has been developed into a powerful tool to study gene functions. In this study, the H1-U6 dual promoter RNAi plasmid was constructed and the target sequence for hUbe2w could be transcribed from both strands and form a double stranded RNA with two 5'Uridine overhangs, which closely resembles endogenous functional siRNA. The hUbe2w cDNA was amplified from reverse transcription of the 293FT total RNA by RT-PCR, and then cloned into the pGL3-Control, pCMV-myc and pDsRed-express-C1 plasmids respectively, which were selected as report vectors to detect the RNAi effects. The plasmids were co-transfected into HEK293FT cells, and then the luciferase activity and hUBE2W protein expression were measured respectively. The Resulted reduction of mRNA and protein level demonstrate that the targets of 125 and 259 could significantly inhibit the hUbe2w expression.
Base Sequence ; Humans ; Molecular Sequence Data ; Plasmids ; genetics ; Promoter Regions, Genetic ; genetics ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; physiology ; RNA, Small Nuclear ; genetics ; Ubiquitin-Conjugating Enzymes ; genetics ; metabolism

In the experiment, we designed and synthesized two siRNAs based on the sequence of nuclear receptor-related factor 1 (Nurr1) mRNA. They were separately subcloned into the plasmid of pSilenCircle (pSC) containing U6 promoter. The pSC-Nurr1 vectors (pSC-N1 and pSC-N2) specific to Nurr1 gene and the negative control vector of short-hairpin RNA (shRNA) eukaryotic expression vector were constructed. We cultured the dopaminergic cell line MN9D and the verified vectors were transfected with LipofectamineTM 2000 in vitro. The positive cell clones transfected with pSC were obtained after being screened with 500 mug/ml G418. After that, the silencing effects of Nurr1 and TH mRNA or protein were detected by real time RT-PCR and Western blot. The neurite extension of MN9D cells was observed and photographed by inverted microscope. The results showed that Nurr1 mRNA expression in MN9D cells was specifically down-regulated by the vectors of pSC-N1 and pSC-N2, and the silencing effects were 62.3% and 45.6%, respectively. The dopaminergic phenotype of TH mRNA was also suppressed significantly and the silencing effects were 76.3% and 62.6%, respectively. Meanwhile, the expressions of Nurr1 and TH proteins were also significantly suppressed, and the silencing effects of Nurr1 and TH protein were 57.4%, 72.0% and 79.1%, 70.1% respectively. The negative control and liposome groups had no effect on the two genes. In conclusion, Nurr1 shRNA expressing vectors can inhibit the expressions of Nurr1 and TH mRNA or protein in MN9D cells, and Nurr1 might play a role in neurite extension of MN9D cells. Nurr1 shRNA expressing vector may provide a novel applicable strategy for the study on the function of the genes associated with Parkinson disease and the development of dopaminergic neuron.
Cell Line ; Dopaminergic Neurons ; cytology ; metabolism ; Down-Regulation ; Fetus ; Humans ; Mesencephalon ; cytology ; Neurites ; physiology ; Nuclear Receptor Subfamily 4, Group A, Member 2 ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Transfection ; Tyrosine 3-Monooxygenase ; genetics ; metabolism