To detect the expression of telomerase subunits (human telomerase reverse transcriptase, human telomerase associated protein 1 and human telomerase RNA) in gastric cancer and to examine the role that different telomerase subunits play in the gastric carcinogenesis, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect telomerase subunits messenger RNA in 24 samples of gastric cancer and corresponding non-cancerous tissue. The results showed that the positive rate of hTERT mRNA from gastric cancer and corresponding non-cancerous tissues was 100% and 25%, respectively. The former was significantly higher than the latter (chi2 = 26.4, P < 0.01). The positive rate of hTEP1 mRNA from gastric cancer and corresponding non-cancerous tissues was 100% and 91.7%, respectively and no significant difference was found between them (chi2 = 2.1, P > 0.05). The positive rates of hTR for gastric cancer and corresponding non-cancerous tissues were both 100% and no significant difference existed between them. It is concluded that in contrast to hTEP1 and hTR, the up-regulation of hTERT mRNA expression may play a more important role in the development of gastric cancer.
Carrier Proteins/biosynthesis ; Carrier Proteins/genetics ; RNA/biosynthesis ; RNA/genetics ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Stomach Neoplasms/*metabolism ; Telomerase/*biosynthesis ; Telomerase/genetics

RNA, Messenger ; biosynthesis ; genetics ; RNA, Viral ; chemistry ; genetics ; Ribonucleases ; biosynthesis ; genetics ; Virion ; chemistry ; genetics ; alpha-Fetoproteins ; biosynthesis ; genetics

Carcinoma, Hepatocellular ; metabolism ; Glypicans ; biosynthesis ; genetics ; Humans ; Liver Neoplasms ; metabolism ; RNA, Messenger ; biosynthesis ; genetics

To construct an immortalized rat astrocyte strain genetically modified by rat preprogalanin gene (IAST/GAL) and detect its galanin (GAL) expression and secretion, a cDNA fragment of rat GAL in plasmid of pBS KS(+)-GAL was inserted into eukaryotic expression vector pcDNA3.1 (+) by DNA recombinant technology, then the restriction enzyme digestion and DNA sequencing were carried out to evaluate the recombinant. The pcDNA3.1 (+)-GAL and pcDNA3.1 (+) construct were transfected into immortalized rat astrocyte strain (IAST) by lipofectamine and the population of cells which stably integrated the construct was selected with 600 microg/mL G418. Individual clones were screened and expanded into clonal cell strains. Detection of Neo gene was used to validate the success of the transfection. Immunocytochemical staining, RT-PCR and radioimmunoassay were used to detect the expression and secretion level of GAL. The recombinant had been successfully constructed by restriction enzyme digestion and DNA sequencing. Detection of Neo gene showed that the pcDNA3.1 (+)-GAL and pcDNA3.1 (+) have been successfully transfected into IAST. After selection by using G418, IAST/GAL and IAST/Neo cell strains were obtained. IAST/GAL, IAST/Neo and IAST were immunostained positively for GAL, but the GAL average optical density of IAST/GAL was significantly higher than that of IAST/Neo and IAST (P< 0.01). The level of GAL mRNA expression and the supernatant concentration of GAL in cultured IAST/GAL were significantly higher than those of IAST and IAST/Neo (P<0.01), but no significant differences were found between the IAST and IAST/Neo (P>0.05). It was concluded that IAST/GAL strain was constructed successfully and it might provide a basis for the further study of pain therapy.
Astrocytes/cytology ; Astrocytes/*metabolism ; Cell Line, Transformed ; Cells, Cultured ; Galanin/*biosynthesis ; Galanin/genetics ; Genetic Vectors ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Recombinant Proteins/biosynthesis ; Recombinant Proteins/genetics ; Transfection

OBJECTIVETo investigate inhibitory effect of short interfering RNA (siRNA) on the expression of lung resistance-related protein (LRP) in leukemia cells.

METHODSThe eukaryotic vectors of LRP, pcDNA3.0/LRP, were constructed. The transfection protocol of K562 cells grown in standard conditions consisted of different combinations of pcDNA3.0/LRP, pEGFP-C1 expressing mammalian enhanced green fluorescent protein (GFP), and their gene-specific siRNAs. RT-PCR and flow cytometry were employed to evaluate the mRNA and protein expression of LRP and fluoroscopy was performed for assay of GFP expression in the transfected cells.

RESULTSCompared with untreated K562 cells, pcDNA3.0/LRP-transfected cells showed increased LRP mRNA and protein expression and the positive cell percentage reached 30%. In the cells co-transfected with LRP gene-specific siRNA and pcDNA3.0/LRP, both LRP mRNA and protein expression decreased significantly to a level defined as negative results; the GFP expression showed no significant difference between the cells transfected with pEGFP-C1 and those co-transfected with LRP gene-specific siRNA and pEGFP-C1. LRP mRNA and protein expressions were also similar between the cells transfected with pcDNA3.0/LRP and those co-transfected with GFP gene-specific siRNA and pcDNA3.0/LRP.

CONCLUSIONSThe LRP gene-specific siRNA we designed is capable of degrading LRP mRNA and inhibiting the protein expression effectively and specifically, which shed light on the potential application of siRNA for gene-specific therapy to reverse LRP-induced multidrug resistance of leukemia cells.

Drug Resistance, Multiple ; genetics ; Genetic Therapy ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; K562 Cells ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Transfection ; Vault Ribonucleoprotein Particles ; biosynthesis ; genetics

Noncoding RNAs (ncRNAs) have played a critical role in cellular biological functions. Recently, some peptides or proteins originating from annotated ncRNAs were identified in organism development and various diseases. Here, we briefly review several novel peptides translated by annotated ncRNAs and related key functions. In addition, we summarize the potential mechanism of bifunctional ncRNAs and propose a specific "switch" triggering the transformation from the noncoding to the coding state under certain stimuli or cellular stress. The coding properties of ncRNAs and their peptide products may provide a novel horizon in proteomic research and can be regarded as a potential therapeutic target for the treatment of various diseases.
Animals ; Calcium/metabolism* ; Humans ; Open Reading Frames ; Protein Biosynthesis ; RNA, Messenger/genetics* ; RNA, Untranslated/physiology*

In order to investigate the relationship between the expression of heme oxygenase-2 (HO-2) mRNA and the pathogenesis of Hirschsprung's disease (HD), total ribonucleic acid (RNA) was extracted in the aganglionic and ganglionic segments of colon respectively from 15 cases of HD. The single-stranded cDNA of HO-2 was synthesized and further amplified by reverse transcription-polymerase chain reaction (RT-PCR). The expression of HO-2 mRNA was normal in ganglionic segments, but absent in aganglionic segments. It is concluded that the absence of HO-2 mRNA expression may be an important mechanism responsible for HD.
Colon/enzymology ; Heme Oxygenase (Decyclizing)/biosynthesis ; Heme Oxygenase (Decyclizing)/*genetics ; Hirschsprung Disease/*enzymology ; Hirschsprung Disease/etiology ; Hirschsprung Disease/genetics ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics

OBJECTIVESTo identify and clone human genes transactivated by HCV F protein by constructing a cDNA subtractive library using the suppression subtractive hybridization technique.

METHODSSuppression subtractive hybridization (SSH) and bioinformatics techniques were used for screening and cloning of the target genes transactivated by HCV F protein. The mRNA was isolated from HepG2 cells transfected with pcDNA3.1 (-)-F or with pcDNA3.1(-) empty vector as a control, and SSH method was employed to analyze the differentially expressed DNA sequence between the two groups. After restriction enzyme Rsa I digestion, small sized cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 or adaptor 2. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, it was then subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5 alpha. The cDNA was sequenced and analyzed in GenBank with blast search after PCR.

RESULTSThe subtractive library of genes transactivated by HCV F protein was constructed successfully. The amplified library contains 71 positive clones. Colony PCR shows that 56 clones contain 200-1000 bp inserts. Sequence analysis was performed on 28 clones randomly, and the full length sequences were obtained with using the bioinformatics method. Altogether 19 coding sequences were obtained, consisting of 17 known and 2 unknown.

CONCLUSIONSThe obtained sequences may be target genes transactivated by HCV F protein, and some gene coding proteins are those involved in cell cycle regulation, metabolism, and cell apoptosis.

Cloning, Molecular ; Hepacivirus ; genetics ; Humans ; Nucleic Acid Hybridization ; methods ; RNA, Messenger ; biosynthesis ; genetics ; Transcriptional Activation ; Viral Core Proteins ; biosynthesis ; genetics

OBJECTIVE: To determine the effects of exogenous RASSF1A gene on the proliferation and expression of P65 and subunit of NF-kappaB, in lung adenocarcinoma cell line A549. METHODS: pcDNA3.0-RASSF1A and pcDNA3. 0 were introduced into A549 cell line by lipofectin transfection, and the A549 cells stably expressing RASSF1A gene were established by G418 selection. The expression of RASSF1A was detected by Western blotting. The cytobiologic characterizations of the positive clone were analyzed by methythiazoletertraolium (MTT) assay and cytometry. The expressing of P65 was analyzed by RT-PCR and Western blotting. RESULTS: A549 cells stably expressing RASSF1A protein were established by lipofection mediated transfection and selected for further study. Compared with the nontransfected and vector transfected cells, the positive clone cells grew more slowly. Flow cytometric data showed that more positive clone cells went into phase G0/G1 and fewer cells went into phase S. The expression of P65 in nuclear protein in positive clone cells was lower than that of the control group while there was no obvious difference between the expression of p65 mRNA and P65 protein in total protein among the 3 groups. CONCLUSION RASSF1A gene might suppress the proliferation of A549 cells through blocking the activity of P65 protein.
Adenocarcinoma ; genetics ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Lung Neoplasms ; genetics ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; Transcription Factor RelA ; biosynthesis ; genetics ; Transfection ; Tumor Suppressor Proteins ; biosynthesis ; genetics

OBJECTIVETo screen specific small interfering RNA (siRNA) targeting mouse BMP9 gene and identify its function in BNLCL.2 fetal liver cells and C3H10 cells.

METHODSFour pairs of double-stranded DNA fragments for silencing mouse BMP9 were annealed in vitro and cloned into pSOS-BMP9 vector with BMP9 gene to construct pSOS-simBMP9 plasmid. The 4 pSOS-simBMP9 plasmids were separately transfected in HEK293 cells via Lipofectamine, and the gene silencing efficiency was assessed by GFP detection. BNLCL.2 fetal liver cells were infected with the constructed adenovirus simBMP9s, and their BMP9 expression was detected with RT-PCR and Western blotting. C3H10 cells were co-infected with Ad-simBMP9 and Ad-BMP9, and the inhibitory effect on BMP9-induced osteoblasts was evaluated by alkaline phosphatase (ALP) activity.

RESULTSGFP expression in the two simBMP9 groups was significantly decreased in HEK293 cells, and the endogenous expression of BMP9 was reduced by 50%-70% by adenovirus-mediated simBMP9 in the fetal liver cells. ALP activity in C3H10 cells was significantly higher in BMP9 group than in the control group (P<0.01), while the activity of the two Ad-simBMP9-infected groups was significantly lower than that in Ad-BMP9-infected group (P<0.01).

CONCLUSIONTwo specific siRNA targeting mouse BMP9 gene have been obtained, which can effectively inhibit both endogenous and exogenous expressions of BMP9 to facilitate the study of the mechanisms of BMP9 in liver cell differentiation.

Animals ; Cell Differentiation ; Cell Line ; Fetus ; Growth Differentiation Factor 2 ; biosynthesis ; genetics ; Hepatocytes ; metabolism ; Humans ; Mice ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Transfection