Nuclear protein-1 (NUPR1) is a small nuclear protein that is responsive to various stress stimuli. Although NUPR1 has been associated with cancer development, its expression and roles in cholangiocarcinoma have not yet been described. In the present study, we found that NUPR1 was over-expressed in human cholangiocarcinoma tissues, using immunohistochemistry. The role of NUPR1 in cholangiocarcinoma was examined by its specific siRNA. NUPR1 siRNA decreased proliferation, migration and invasion of human cholangiocarcinoma cell lines (HuCCT1 and SNU1196 cells). From these results, we conclude that NUPR1 is over-expressed in cholangiocarcinoma and regulates the proliferation and motility of cancer cells.
Cell Line ; Cholangiocarcinoma ; Humans ; Immunohistochemistry ; Nuclear Proteins ; RNA, Small Interfering

Hepatitis C ; genetics ; Humans ; RNA, Catalytic ; genetics ; RNA, Small Nuclear ; genetics

RNA interference (RNAi) has been proved as a novel approach for gene therapy. However, RNAi mono-therapy only aims at single gene, it therefore may ultimately fail to cure cancers caused by polygene variation. To overcome the deficiency of RNAi mono-therapy, "combinatorial RNA interference" (coRNAi) was put forward as a new strategy. By co-expressing the inducers of RNAi triggering single or multiple targets directly and other RNA- or protein-based silencers, coRNAi keeps target genes silent, prevents carcinogenic progression and induces apoptosis of tumor cells. This paper mainly reviews the major strategies of coRNAi and their applications in cancer gene therapy.
Animals ; Apoptosis ; Genetic Therapy ; methods ; Humans ; MicroRNAs ; genetics ; Neoplasms ; genetics ; pathology ; therapy ; Oncogenes ; RNA Interference ; RNA, Small Interfering ; genetics ; RNA, Small Nuclear ; genetics

OBJECTIVETo modify the current PCR-based method for rapid and efficient preparation of small interfering RNA (siRNA) expression cassette to improve the efficiency of RNA interference.

METHODSThe U6 promoter sequence was amplified by PCR using the genomic DNA of K562 cells as the template, and cloned into pMD18-T vector which served as the template for further PCR amplification with the primers on the plasmid. The amplified product was directly used as the template for preparing siRNA expression cassette. The siRNA expression cassette targeting p53 gene was amplified, verified by sequencing, and transfected into SH-SY5Y cells. After a 48-hour transfection, the cells were harvested and the total RNA was for RT-PCR for evaluating the effect of RNA interference.

RESULTSThe sequencing result confirmed the correct U6 promoter sequence cloned from K562 cells. After transfection of SH-SY5Y cells for 48 h with siRNA expression cassette, the p53 gene expression was inhibited at the mRNA level in comparison with the control cells as demonstrated by RT-PCR detection.

CONCLUSIONThe siRNA expression cassette prepared using the established method described hereby can be well applicable in RNA interference research.

Gene Silencing ; Gene Targeting ; methods ; Genetic Vectors ; genetics ; Humans ; K562 Cells ; Polymerase Chain Reaction ; RNA, Small Interfering ; biosynthesis ; genetics ; RNA, Small Nuclear ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics

OBJECTIVETo study the cleavage activity on the HCV RNA of a chimeric recombinant of HCV specific ribozyme and U1 small nuclear RNA, which compartmentalizes within the nucleolus.

METHODSThe third stem-loop sequence of human U1 snRNA (position 95-116) within pBSIISK+ U1 was substituted by hammerhead ribozyme against HCV RNA by PCR and cloning methods, and the constructed plasmid was named pBSIISK+ (U1-Rz). Then the whole gene fragment of the chimeric ribozyme was cloned into a pGEM-T vector under the control of T7 promoter, and the constructed plasmid was named pGEM- (U1-Rz). The pGEM- (U1-Rz) and pGEM-Rz (containing the same ribozyme sequence as that in U1-Rz) transcripts as enzyme were transcribed in vitro. Also the (32)P-labeled pCMV/T7-NCRC luc (containing the gene sequence of the whole 5'-NCR and part core of HCV RNA) transcripts as target-RNAs were transcribed in vitro. The enzymes were incubated with the target RNAs under different conditions and autoradiographed after denaturing gel-electrophoresis.

RESULTSThe sequencing result showed that the construction of U1 snRNA chimeric ribozyme was correct. Compared with the ribozyme alone, both of them were active at 37 degree C and with Mg2+ (10 mmol/L) and TrisCl (10 mmol/L, pH7.9), and there was no remarkable difference between them. The cleavage activity of the chimeric ribozyme increased with the prolongation of reaction time and increment of enzyme concentration.

CONCLUSIONBoth ribozyme and U1 snRNA chimeric ribozyme exhibited specifically catalytic activity against HCV RNA in vitro. There was no remarkable difference between their cleavage efficiencies.

Chimera ; genetics ; Genetic Therapy ; Hepacivirus ; genetics ; Hepatitis C ; therapy ; RNA, Catalytic ; genetics ; metabolism ; RNA, Small Nuclear ; genetics ; pharmacology ; RNA, Viral ; genetics ; Recombinant Fusion Proteins ; pharmacology

PURPOSE: Prader-Willi Syndrome(PWS) is caused by absence of paternal contributions of the chromosome region 15q11-q13. To detact this region, high resolutional cytogenetic analysis, FISH with probe at PWS critical region or microsatellite polymorphism can be used. The gene for the small nuclear ribonucleoprotein polypeptide N(SNRPN) is not expressed in patients with PWS. We conducted molecular analysis with RT-PCR with SNRPN primers to find out more useful diagnostic tool in PWS. METHODS: Four patients with obesity and other characteristics of PWS were studied. The exprssion of SNRPN and control gene were studed by RT-PCR from peripheral lymphocytes. RESULTS :The SNRPN expression in reverse transcribed RNA from blood were easily detected in normal control but not in patients with suspected Parder-Willi Syndrome. CONCLUSION: We conclude that SNRPN expression study is a useful diagnostic method for detection of Prader-Willi Syndrome.
Cytogenetic Analysis ; Humans ; Lymphocytes ; Microsatellite Repeats ; Obesity ; Pathology, Molecular* ; Prader-Willi Syndrome* ; Ribonucleoproteins, Small Nuclear ; RNA ; snRNP Core Proteins*

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

It has been shown that CA repeats in the 3'-untranslated region (UTR) of bcl-2 mRNA contribute the constitutive decay of bcl-2 mRNA and that hnRNP L (heterogenous nuclear ribonucleoprotein L) interacts with CA repeats in the 3'-UTR of bcl-2 mRNA, both in vitro and in vivo. The aim of this study was to determine whether the alteration of hnRNP L affects the stability of bcl-2 mRNA in vivo. Human breast carcinoma MCF-7 cells were transfected with hnRNP L-specific shRNA or hnRNP L-expressing vector to decrease or increase hnRNP L levels, respectively, followed by an actinomycin D chase. An RT-PCR analysis showed that the rate of degradation of endogenous bcl-2 mRNA was not affected by the decrease or increase in the hnRNP L levels. Furthermore, during apoptosis or autophagy, in which bcl-2 expression has been reported to decrease, no difference in the degradation of bcl-2 mRNA was observed between control and hnRNP L-knock down MCF-7 Cells. On the other hand, the levels of AUF-1 and nucleolin, transacting factors for ARE in the 3'UTR of bcl-2 mRNA, were not significantly affected by the decrease in hnRNP L, suggesting that a disturbance in the quantitative balance between these transacting factors is not likely to interfere with the effect of hnRNP L. Collectively, the findings indicate that the decay of bcl-2 mRNA does not appear to be directly controlled by hnRNP L in vivo.
3' Untranslated Regions ; Apoptosis ; Autophagy ; Breast ; Dactinomycin ; Hand ; Heterogeneous-Nuclear Ribonucleoprotein L ; Heterogeneous-Nuclear Ribonucleoproteins ; Humans ; MCF-7 Cells ; Phosphoproteins ; Ribonucleoproteins ; RNA, Messenger ; RNA, Small Interfering ; RNA-Binding Proteins

OBJECTIVETo extend the use of vector-derived siRNA by generating multiple shRNAs in the same plasmid.

METHODSConstruct a vector that expresses shRNAs targeting on Ku70 and Ku80 in tandem. The gene silencing efficiency of each shRNA was verified previously. After identification by restriction digestion and DNA sequencing, the reconstructed plasmid, named psiRNAKus, was transfected into the human hepatoma cell line HepG2. The tandem-shRNA-induced silencing of targeted genes was determined by RT-PCR at RNA level and Western blot at protein level.

RESULTSThe shRNAs encoded by psiRNAKus down-regulated both the expression of Ku70 and Ku80.

CONCLUSIONThe vector-derived siRNA delivery system that allows multiple shRNA species to be expressed from the same vector may be of value in experimental and therapeutic applications.

Antigens, Nuclear ; genetics ; DNA-Binding Proteins ; genetics ; Gene Knockdown Techniques ; Hep G2 Cells ; Humans ; Ku Autoantigen ; Plasmids ; RNA ; genetics ; RNA, Small Interfering

OBJECTIVETo determine the advantage of U1 small nuclear RNA as a ribozyme vector (U1-Rz) to inhibit HCV replication in vivo.

METHODSThe 3rd stem-loop was substituted by HCV core specific ribozyme to construct an U1-Rz eucaryotic expression plasmid. Then it was co-transfected with pCMV/T7-NCRC Delta-luc into Huh7 cell line mediated by lipofectin. The cell lysis supernatant was subjected to Western blot and lumenometer to determine the luciferase levels.

RESULTSA U1 snRNA chimeric ribozyme was constructed successfully. Both Rz and U1-Rz inhibited luciferase expression in Huh7 by 48.64% and 87.46%, respectively.

CONCLUSIONRz has more efficacy in cells when using U1 snRNA delivery system. U1 can be an efficient vector for HCV specific ribozyme.

Base Sequence ; Genetic Therapy ; Genetic Vectors ; Hepacivirus ; physiology ; Humans ; Molecular Sequence Data ; RNA, Catalytic ; genetics ; RNA, Small Nuclear ; genetics ; Virus Replication ; genetics