INHIBITION OF PROLIFERATION OF HUMAN BREAST CANCER MCF-7 CELLS BY SMALL INTERFERENCE RNA AGAINST LRP16 GENE

Objective: Our previous studies have firstly demonstrated that 17β -E2 up-regulates LRP16 gene expression in human breast cancer MCF-7 cells, and ectopic expression of the LRP16 gene promotes MCF-7 cells proliferation. Here, the effects of the LRP16 gene expression on growth of MCF-7 human breast cancer cells and the mechanism were further studied by establishing two stably LRP16-inhibitory MCR-7 cell lines. Methods: Hairpin small interference RNA (siRNA) strategy, by which hairpin siRNA was released by U6 promoter and was mediated by pLPC-based retroviral vector, was adopted to knockdown endogenous LRP16 level in MCF-7 cells. And the hairpin siRNA against green fluorescence protein (GFP) was used as the negative control. The suppressant efficiency of the LRP16 gene expression was confirmed by Nothern blot. Cell proliferation assay and soft agar colony formation assay were used to determine the status of the cells proliferation. Cell cycle checkpoints including cyclin E and cyclin D1 were examined by Western blot. Results: The results from cell proliferation assays suggested that down-regulation of LRP16 gene expression is capable of inhibiting MCF-7 breast cancer cell growth and down-regulation of the LRP16 gene expression is able to inhibit anchorage-independent growth of breast cancer cells in soft agar. We also demonstrated that cyclin E and cyclin D1 proteins were much lower in the LRP16-inhibitory cells than in the control cells. Conclusion: These data suggest that LRP16 gene play an important role in MCF-7 cells proliferation by regulating the pathway of the G1/S transition and may function as an important modulator in regulating the process of tumorigenesis in human breast.

RNA INTERFERENCE OF ANNEXIN II GENE IN PC3 CELLS BY USING SMALL INTERFERENCE RNA SYNTHESIZED WITH IN VITRO TRANSCRIPTION

Objective To silence annexin Ⅱ gene expression by using small interference RNA （siRNA） in prostate cancer cell line PC3. Methods For in vitro transcription, four sequences of 29-nucleotide DNA template oligonucleotides were designed, and one pair of the sequences were complementary to annexin Ⅱ gene. The other pair was negative control. The 8 nucleotides at the 3＇ end of each oligonucleotide were complementary to the T7 Promoter Primer. The sense and anti-sense siRNA templates were transcribed by T7 RNA polymerase and the resulting RNA transcripts were hybridized to create dsRNA. The siRNA was transfected into prostate cancer cell PC3. For assaying the efficiency of siRNA, confocal microscopy, Northern blotting, and Western blotting were employed to examine the expression of annexin Ⅱ protein and its mRNA. ^3H thymidine was used to measure DNA synthesis. Results The siRNA sequence specific to annexin Ⅱ gene was capable of inhibiting the expression of annexin Ⅱ protein and its mRNA. And cellular DNA synthesis was significantly reduced in siRNA transfected cells.Conclusions The protocol for the synthesis of siRNA by T7 RNA polymerase is feasible. Annexin Ⅱ might be involved in DNA synthesis.

RNA interference reveals chloride channel 7 gene helps short-term hypersalinity stress resistance in Hong Kong oyster Crassostrea hongkongensis

The chloride channel 7 gene(CLC 7)of the Hong Kong oyster Crassostrea hongkongensis was cloned and named ChCLC 7.The cDNA was 2572 bp in length,with a 5′non-coding region containing 25 bp,a 3′non-coding region containing 327 bp,and an open reading frame of 2298 bp.ChCLC 7 has 96.8%and 92.1%homology with CLC 7 of Crassostrea gigas and Crassostrea virginica,respectively,and it was clustered with CLC 7 of C.gigas and C.virginica.QRT-PCR showed that ChCLC 7 was expressed in all eight tissues,with the highest in adductor muscle and second in gill.The ChCLC 7 expression pattern in gill was altered significantly under high salinity stress with an overall upward and then downward trend.After RNA interference,the expression of ChCLC 7 and survival rate of oyster under high salinity stress was reduced significantly,and so did the concentration of hemolymph chloride ion in 48-96 h after RNA interference.We believed that ChCLC 7 could play an important role in osmoregulation of C.hongkongensis by regulating Cl^(-)transport.This study provided data for the analysis of molecular mechanism against oyster salinity stress.

Effects of RNA interference targeting transforming growth factor-beta 1 on immune hepatic fibrosis induced by Concanavalin A in mice

BACKGROUND: Previous studies have shown that transforming growth factor-beta 1 (TGF-beta 1) is the most potent means of stimulating liver fibrogenesis by myofibroblast-like cells derived from hepatic stellate cells. Thus, TGF-beta 1 could be a target for treating hepatic fibrosis. This study aimed to investigate the inhibitory effects of specific TGF-beta 1 small interference RNA (siRNA) on immune hepatic fibrosis induced by Concanavalin A (Con A) in mice. METHODS: Three short hairpin RNAs targeting different positions of TGF-beta 1 were designed and cloned to the plasmid pGenesil-1 to obtain three recombinant expression vectors (pGenesil-TGF-beta 1-ml, pGenesil-TGF-beta 1-m2 and pGenesil-TGF-beta 1-m3). Thirty male Kunming mice were randomly divided into 6 groups: normal, model, control, and three treatment groups. The immune hepatic fibrosis models were constructed by injecting Con A via the tail vein at 8 mg/kg per week for 6 weeks. At weeks 2, 4 and 6, pGenesil-TGF-beta 1-ml, pGenesil-TGF-beta 1-m2 or pGenesi1-TGF-beta 1-m3 was injected by a hydrodynamics-based transfection method via the tail vein at 0.8 ml/10 g within 24 hours after injection of Con A in each of the three treatment groups. The mice in the control group were injected with control plasmid pGenesil-HK at the same dose. All mice were sacrificed at week 7. The levels of hydroxyproline in liver tissue were determined by biochemistry. Liver histopathology was assessed by Van Gieson staining. The expression levels and localization of TGF-beta 1, Smad3, and Smad7 in liver tissue were detected by immunohistochemistry. The expression of TGF-beta 1, Smad3, Smad7 and alpha-smooth muscle actin (alpha-SMA) mRNAs in the liver were assessed by semi-quantitative RT-PCR. RESULTS: The levels of hydroxyproline in the liver tissue of the treatment groups were lower than those of the model group (P<0.01). Histopathologic assay showed that liver fibrogenesis was clearly improved in the treatment groups compared with the model group. The expression levels of TGF-beta 1 and Smad3 of liver tissue were also markedly lower in the treatment groups than in the model group (P<0.01), while the levels of Smad7 were higher in the treatment groups than in the model group (P<0.01). RT-PCR further showed that the expression of TGF-beta 1, Smad3 and alpha-SMA mRNA was significantly inhibited in the treatment groups compared with the model group, while the levels of Smad7 were increased. There was no difference in the above parameters among the three treatment groups or between the control and model groups (P>0.05), but the inhibitory effect of pGenesil-TGF-beta 1-ml was the highest among the treatment groups. CONCLUSIONS: Specific siRNA targeting of TGF-beta 1 markedly inhibited the fibrogenesis of immune hepatic fibrosis induced by Con A in mice. The anti-fibrosis mechanisms of siRNAs may be associated with the down-regulation of TGF-beta 1, Smad3 and alpha-SMA expression and up-regulation of Smad7 expression in liver tissue, which resulted in suppressing the activation of hepatic stellate cells. (Hepatobiliary Pancreat Dis Int 2009; 8: 300-308)

Development of Studies on RNA Interference

RNA interference （RNAi）, caused by endogenous or exogenous double- stranded RNA （dsRNA） homologous with target genes, refers to gene silencing widely existing in animals and plants. It was first found in plants, and now it has developed into a kind of biotechnology as well as an important approach in post- genome era. This paper is to summarize the achievements of studies on RNAi tech- nology in basic biology, medicine, pharmacy, botany and other fields.

RNA interference affects tumorigenicity and expression of insulin-like growth factor-1,insulin-like growth factor-1 receptor,and basic fibroblast growth factor-2 in rat C6 glioma cells

BACKGROUND： Human gliomas are more likely to express basic fibroblast growth factor-2 （FGF-2） insulin-like growth factor-1（IGF-1）, and IGF-1 receptor （IGF-1R） than normal brain tissue. These factors activate signal transduction systems of Ras/MAPK and PI3K/Akl, which promote glioma growth. OBJECTIVE： To utilize RNA interference （RNAi） technique to down-regulate FGF-2, IGF-1, and IGF-1R gene expression, and to investigate the effects of these genes on rat C6 glioma cells, as well as the feasibility of RNAi for treating glioma. DESIGN, TIME AND SETTING： This neurooncological, randomized, controlled, in vivo and in vitro experiment, which used RNAi methodology, was performed at the Laboratory of Molecular Biology, Institute of Biochemistry, Chinese Academy of Sciences between August 2005 and February 2008. MATERIALS： Rat C6 cell lines were purchased from Shanghai Institute of Cellular Biology Affiliated to Chinese Academy of Sciences. Small interfering RNA （siRNA） was synthesized by Shanghai GenePharma. Anti-IGF-1, anti-IGF-1R, anti-FGF-2, anti-mouse and anti-rabbit IgG G1-HRP antibodies were provided by Santa Cruz Biotechnology, USA. Four to six week-old BALB/c nude mice were purchased from the Laboratory Animal Center, Chinese Academy of Sciences. METHODS： C6 glioma cells were transfected with siRNA, which was chemically synthesized in vitro to correspond to endogenous FGF-2, IGF-1, and IGF-1R genes. The inhibition ratio of targeting mRNA expression was detected by semiquantitative RT-PCR, and protein expression was determined by Western blot analysis. C6 glioma cell proliferation was observed using a growth curve C6 glioma cell apoptosis rate and cell cycle were detected by flow cytometry. C6 glioma cell growth regression was observed by transwell migration assay. In addition, nude mouse subcutaneous tumor models were used in this study. For studying the anti-tumor effects of IGF-1 and IGF-1R siRNA, two blank control groups, with six mice each, were set up： A （2.5 μg siRNA was injected one week after C6 cells were inoculated, Le., when tumor volume reached 8 mm × 8 mm） and B （siRNA was injected at the same time with C6 cells were inoculated. To study the effects of FGF-2 siRNA, the groups consisted of a blank control group, negative control group, 2.6 μg siRNA group, 4 μg siRNA group, and 5.3 μg siRNA group, with six mice each. MAIN OUTCOME MEASURES： mRNA and protein inhibition ratio of FGF-2, IGF-1, and IGF-1 R; C6 glioma cell proliferation, apoptosis, and cycle growth arrest; C6 glioma cell growth regression and subcutaneous tumorigenicity rates. RESULTS： All siRNA constructs proved to be effective. After 48 hours, transfection of 200 nmol/L siRNA resulted in a FGF-2 or IGF-1R gene inhibition ratio 〉 80% and an IGF-1 gene inhibition ratio of approximately 70%. Protein expression levels for FGF-2, IGF-1, and IGF-1R decreased in a dose-dependent manner following siRNA transfection, with an inhibition rate 〉 85%, 60%, and 50%, respectively. C6 glioma cell proliferation and apoptosis rates increased in proportion to siRNA. The apoptosis rate of C6 glioma cells induced by FGF-2, IGF-1, and IGF-1R siRNA was 39.96%, 15.07% and 22.47%, respectively （P 〈 0.01）. Transfection of 200 nmol/L IGF or IGF-1R siRNA for 48 hours suppressed C6 glioma cell migration. At 30 days after intratumoral injection of 2.6, 4, and 5.3 tJg FGF-2 siRNA, tumor growth regression rate of FGF-2 siRNA was 56%, 67%, and 86%, respectively. The tumor growth regression rate was 71.88% and 45.71%, respectively, when IGF-1 or IGF-1R siRNA was intratumorally injected 1 week after C6 glioma cell transplantation. When IGF-1 or IGF-1 R siRNA was intratumorally injected during C6 glioma cell transplantation, the tumor growth regression rate was 78.13% and 74.29%, respectively. CONCLUSION： siRNA transfection downregulated gene expression of FGF-2, IGF-1, and IGF-1R In addition, siRNA treatment markedly suppressed glioma cell proliferation, growth, and migration, and concomitantly reduced subcutaneous tumorigenicity.

Induction of Silencing Effect of Swedish Mutant Amyloid Precursor Protein by RNA interference

Summary： Over-expression of APP and Swedish mutation could cause some familial early onset AD. In this study, a primary screening was conducted of effective small interference RNAs （siRNAs） targeted wild type APP （APPwt） and Swedish mutant APP （APPswe）. One siRNA targeting APPwt and the other siRNA targeting APPswe were designed, All these siRNAs were endogenously expressed by siRNAs expressing plasmids, COS-7 cells were transiently co-transfected with APP-GFP recombinant plasmids and siRNA expression vector, The silencing effect of each siRNA was quantitatively assessed by the level of expression of green fluorescent protein （GFP）. It was found that the siRNAs silenced APPwt and APPswe to different degrees, siRNA directed against APPswe was more effective in suppressing the expression of fusion gene of APPswe than that of APPwt. The silencing effect of siRNA directed against APPswe indicating allele-specific silencing property of the siRNAs. Therefore, siRNAs directed against APP play an important role both in the therapeutic study of Alzheimer disease and functional exploration ofAPP gene.

Rice Repetitive DNA Sequence RRD3:a Plant Promoter and Its Application to RNA Interference

Previously, a moderately repetitive DNA sequence （RRD3） was cloned from rice （Oryza sativa L.） by DNA renaturation kinetics. Sequence analysis revealed several conserved promoter motifs, including four TATA-boxes and a CAAT-box, and promoter activity was shown in Escherichia coli and mammalian expression systems. Here, we inserted the RRD3 fragment into the plant promoter-capture vector, pCAMBIA1391Z, and examined whether the RRD3 fragment has promoter activity in plants. Transgenic tobacco and rice calli both showed β-glucuronidase （GUS） activity, indicating that RRD3 can act as a promoter in both monocot and dicot plants. Based on the promoter characteristic of RRD3, we designed a plant universal binary vector, pCRiRRD3, which is suitable for performing researches on plant RNA interference. This vector has two multiple cloning sites to facilitate sense and antisense cloning of the target sequence, separated by an intron fragment of 200 bp. The efficiency of the vector for gene silencing was assayed by histochemical and quantitative fluorometric GUS assays in transgenic tobacco. These research results suggested that this plant RNAi vector pCRiRRD3 can effectively perform gene silencing researches on both monocot and dicot plants.

Interference RNA induction and drug target validation

The ability to knockdown the expression of an endogenous gene by RNAi has emerged as a powerful strategy for the rapid identification of specific gene functions. Vector-based constitutive expression of shRNA can result in stable and efficient knockdown of target genes. However, constitutive expression of shRNA imposes major limitations when analyzing the fimction of genes whose expression is vital for the survival of an organism. Inducible RNAi systems can circumvent this limitation by enabling the inhibition of expression of an essential gene only when the inducing agent is present, and the level of knockdown of the essential gene can be controlled and adjusted by the concentration of inducing agent. In this review, we briefly summarize the recent development of various inducible RNAi systems and their potential applications in drug target validation.

RNA interference mediated silencing of α-synuclein in MN9D cells and its effects on cell viability

Objective To silence the expression of α-synuclein in MN9D dopaminergic cells using vector mediated RNA interference （RNAi） and examined its effects on cell proliferation and viability. Methods We identified two 19-nucleotide stretches within the coding region of the α-synuclein gene and designed three sets of oligonucleotides to generate doublestranded （ds） oligos. The ds oligos were inserted into the pENTR^TM/Hl/TO vector and transfected into MN9D dopaminergic cells, α-Synuclein expression was detected by RT-PCR, real-time PCR, immunocytochemistry staining and Western blot. In addition, we measured cell proliferation using growth curves and cell viability by 3-（4, 5）-dimethylthiahiazo （-z-y 1）-3, 5-diphenytetrazoliumromide （M＇FF）. Results The mRNA and protein levels of α-synuclein gene were significantly down-regulated in pSH2/α-SYN-transfected cells compared with control MN9D and pSH/CON-transfected MN9D cells, while pSHI/α-SYN- transfected cells showed no significant difference. Silencing α-synuclein expression does not affect cell proliferation but may decrease cell viability. Conclusion Our results demonstrated pSH2/α-SYN is an effective small interfering RNA （siRNA） sequence and potent silencing of mouse α-synuclein expression in MN9D cells by vector-based RNAi, which provides the tools for studying the normal function of α-synuclein and examining its role in Parkinson＇s disease （PD） pathogenesis. α-Synuclein may be important for the viability of MN9D cells, and loss of α-synuclein may induce cell injury directly or indirectl

Lentivirus-mediated shRNA interference targeting STAT3 inhibits human pancreatic cancer cell invasion

AIM： To investigate RNA interference targeting signal transducer and activator of transcription-3 （STAT3） on invasion of human pancreatic cancer cells.METHODS： We constructed three plasmids of RNA interference targeting the STAT3 gene. After LV （lentivirus）-STAT3siRNA （STAT3 small interfering RNA） the vector was transfected into the human pancreatic cell line, SW1990 and cell proliferation was measured by the MTT assay. Flow cytometry was used to assess cell cycle. Vascular endothelial growth favor （VEGF） and matrix metalloproteinase-2 （MMP-2） mRNA and protein expression were examined by quantitative PCR and western blotting, respectively. The invasion ability of SW1990 cells was determined by cell invasion assay.RESULTS： We successfully constructed the LVSTAT3siRNA lentivirus vector and proved that it can suppress expression of STAT3 gene in SW1990 cells. RNA interference of STAT3 by the LV-STAT3siRNA construct significantly inhibited the growth of SW1990 cells, in addition to significantly decreasing both VEGF and MMP-2 mRNA and protein expression. Moreover, suppression of STAT3 by LV-STAT3siRNA decreased the invasion ability of SW1990 cells.CONCLUSION： The STAT3 signaling pathway may provide a novel therapeutic target for the treatment of pancreatic cancer since it inhibits the invasion ability of pancreatic cancer cells.

Inhibition of hepatitis B virus expression and replication by RNA interference in HepG2.2.15

AIM： To observe the inhibition of hepatitis B virus replication and expression by transfecting vector-based small interference RNA （siRNA） pGenesiI-HBV X targeting HBV X gene region into HepG2.2.15 cells. METHODS：pGenesil-HBV X was constructed and transfected into HepG2.2.15 cells via lipofection. HBV antigen secretion was determined 24, 48, and 72 h after transfection by time-resolved immunofluorometric assays （TRFIA）. HBV replication was examined by fluorescence quantitative PCR, and the expression of cytoplasmic viral proteins was determined by immunohistochemistry. RESULTS： The secretion of HBsAg and HBeAg into the supernatant was found to be inhibited by 28.5% and 32.2% （P 〈 0.01）, and by 38.67% （P 〈 0.05） and 42.86% （P 〈 0.01） at 48 h and 72 h after pGenesil-HBV X transfection, respectively. Immunohistochemical staining for cytoplasmic HBsAg showed a similar decline in HepG2.2.15 cells 48 h after transfection. The number of HBV genomes within culture supernatants was also significantly decreased 48 h and 72 h post-transfection as quantified by fluorescence PCR （P 〈 0.05）. CONCLUSION： In HepG2.2.15 cells, HBV replication and expression is inhibited by vector-based siRNA pGenesil- HBV X targeting the HBV X coding region.

Harnessing the RNA interference pathway to advance treatment and prevention of hepatocellular carcinoma

Primary liver cancer is the fifth most common malignan- cy in the world and is a leading cause of cancer-related mortality.Available treatment for hepatocellular carcino- ma(HCC),the commonest primary liver cancer,is rarely curative and there is a need to develop therapy that is more effective.Specific and powerful gene silencing that can be achieved by activating RNA interference(RNAi) has generated enthusiasm for exploiting this pathway for HCC therapy.Many studies have been carried out with the aim of silencing HCC-related cellular oncogenes or the hepatocarcinogenic hepatitis B virus(HBV)and hepatitis C virus(HCV).Proof of principle studies have demonstrated promising results,and an early clinical trial assessing RNAi-based HBV therapy is currently in progress.Although the data augur well,there are several significant hurdles that need to be overcome before the goal of RNAi-based therapy for HCC is realized.Particu- larly important are the efficient and safe delivery of RNAi effecters to target malignant tissue and the limitation of unintended harmful non-specific effects.

RNA interference and antiviral therapy

RNA interference (RNAi) is an evolutionally conserved gene silencing mechanism present in a variety of eukaryotic species. RNAi uses short double-stranded RNA (dsRNA) to trigger degradation or translation repression of homologous RNA targets in a sequence-specific manner. This system can be induced effectively in vitro and in vivo by direct application of small interfering RNAs (siRNAs), or by expression of short hairpin RNA (shRNA) with non-viral and viral vectors. To date, RNAi has been extensively used as a novel and effective tool for functional genomic studies, and has displayed great potential in treating human diseases, including human genetic and acquired disorders such as cancer and viral infections. In the present review, we focus on the recent development in the use of RNAi in the prevention and treatment of viral infections. The mechanisms, strategies, hurdles and prospects of employing RNAi in the pharmaceutical industry are also discussed.

Down-regulation of Bcl-X_L by RNA interference suppresses cell growth and induces apoptosis in human esophageal cancer cells

AIM： To determine the inhibitory effect of the vectorgenerated small interfering RNAs （siRNAs） on the expression of the BcI-XL gene in established human esophageal cancer cells, and to investigate the effect of the BcI-XL siRNAs on cell growth and apoptosis in esophageal cancer cells. METHODS： Three siRNA-expressing vectors targeting different sites of the Bcl-XL gene were constructed from pTZ-U6＋I vector. Cultured esophageal cancer cells were transfected with the siRNA-expressing vector （or the control vector） using lipofectamine 2000. BcI-XL gene expression was determined with semiquantitative RT- PCR assay and Western blotting. Among the three siRNA- expressing vectors, the most highly functional vector and its effect on cell growth and apoptosis in esophageal cancer ceils was further analyzed. RESULTS： Of the three siRNA-expressing vectors, siRNA- expressing vector No.1 was the most potent one which suppressed Bcl-XL mRNA production to 32.5% of that in the untreated esophageal cancer cells. Western blotting analysis showed that siRNA-expressing vector No.1 markedly down-regulated the expression of Bcl-XL in human esophageal cancer cells. Treatment of esophageal cancer cells with siRNA-expressing vector No.1 resulted in inhibition of cell growth and induction of apoptosis. CONCLUSION： Down-regulation of BcI-XL by vectorgenerated small interfering RNAs can suppress cell growth and induce apoptosis in human esophageal cancer cells.

Silencing of signal transducer and activator of transcription 3 expression by RNA interference suppresses growth of human hepatocellular carcinoma in tumor-bearing nude mice

AIM： To explore the effect of silencing of signal transducer and activator of transcription 3 （STAT3） expression by RNA interference （RNAi） on growth of human hepatocellular carcinoma （HCC） in tumorbearing nude mice in vivo.METHODS： To construct the recombinant plasmid of pSilencer 3.0-H1-STAT3-siRNA-GFP （pSHI-siRNA- STAT3） and establish the tumor-bearing nude mouse model of the HCC cell line SMMC7721, we used intratumoral injection together with electroblotting to transfect the recombinant plasmid pSHI-siRNA- STAT3 into the transplanted tumor. The weight of the nude mice and tumor volumes were recorded. STAT3 gene transcription was detected by semi-quantitative reverse transcription polymerase chain reaction （RT- PCR）. Level of protein expression and location of STAT3 were determined by Western blotting and immunohistochemical staining. STAT3-related genes such as survivin, c-myc, VEGF, p53 and caspase3 mRNA and protein expression were detected in tumor tissues at the same time. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） assay was used to detect apoptosis of tumor cells.RESULTS： The weight of the treated nude mice increased, and the tumor volume decreased markedly compared with those of the mock-treated and negative control groups （P 〈 0.01）. The results of RT-PCR and Western blotting showed that mRNA and protein levels of STAT3 declined markedly in the treated group. The change in STAT3-related gene expression in tumor tissues at the mRNA and protein level also varied, the expression of survivin, VEGF and c-myc were obviously reduced, and expression of p53 and caspase3 increased （P 〈 0.01）. Most of the tumor tissue ceils in the treated group developed apoptosis that was detected by TUNEL assay.CONCLUSION： Silencing of STAT3 expression by RNAi significantly inhibits expression of STAT3 mRNA and protein, and suppresses growth of human HCC in tumor-bearing nude mice. The mechanism may be related to down-regulation of survivin, VEGF and c-myc and up-regulation of p53 and caspase3 expression. Accordingly, the STAT3 gene may act as an important and effective target in gene therapy of HCC.

Preliminary experimental study of urethral reconstruction Nith tissue engineering and RNA interference techniques

This study investigated the feasibility of replacing urinary epithelial cells with oral keratinocytes and transforming growth factor-β1 （TGF-β1） small interfering RNA （siRNA）-transfected fibroblasts seeded on bladder acellular matrix graft （BAMG） in order to reconstruct tissue-engineered urethra. Constructed siRNAs, which expressed plasmids targeting TGF-β1, were transfected into rabbit fibroblasts. The effective siRNA was screened out by RT-PCR and was transfected into rabbit fibroblasts again. Synthesis of type I collagen in culture medium was measured by enzyme-linked immuno sorbent assay （ELISA）. Autologous oral keratinocytes and TGF-β1 siRNA-transfected fibroblasts were seeded onto BAMGs to obtain a tissue-engineered mucosa. The tissue-engineered mucosa was assessed morphologically and with the help of scanning electron microscopy. The TGF-β1 siRNA decreased the expression of fibroblasts synthesis type I collagen. Oral keratinocytes and TGF-β1 siRNA-transfected fibroblasts were seeded onto sterilized BAMG to obtain a tissue-engineered mucosa for urethral reconstruction. The compound graft was assessed using scanning electron microscope. Oral keratinocytes and TGF-β1 siRNA-transfected fibroblasts had a good compatibility with BAMG. The downregulation of fibroblasts synthesis type I collagen expression by constructed siRNA interfering TGF-β1 provided a potential basis for genetic therapy of urethral scar. Oral keratinocytes and TGF-β1 siRNA-transfected fibroblasts had good compatibility with BAMG and the compound graft could be a new choice for urethral reconstruction.

Down-Regulated Expression of RACK1 Gene by RNA Interference Enhances Drought Tolerance in Rice

The receptor for activated C-kinase 1 （RACK1） is a highly conserved scaffold protein with versatile functions, and plays important roles in the regulation of plant growth and development. Transgenic rice plants, in which the expression of RACK1 gene was inhibited by RNA interference （RNAi）, were studied to elucidate the possible functions of RACK1 in responses to drought stress in rice. Real-time PCR analysis showed that the expression of RACK1 in transgenic rice plants was inhibited by more than 50%. The tolerance to drought stress of the transgenic rice plants was higher as compared with the non-transgenic rice plants. The peroxidation of membrane and the production of malondialdehyde were significantly lower and the superoxide dismutase activity in transgenic rice plants was significantly higher than those in non-trangenic rice plants It is suggested that RACK1 negatively regulated the redox system-related tolerance to drought stress of rice plants.

Angiopoietin-1 targeted RNA interference suppresses angiogenesis and tumor growth of esophageal cancer

AIM: To determine the inhibitory effect of the adenovirus- based angiopoietin-1 (Ang-1) targeted small interfering RNA expression system (Ad/Ang-1si) on the expression of the Ang-1 gene, cell growth and apoptosis in human esophageal cancer cell line Eca109. METHODS: siRNA-expressing adenovirus targeting Ang-1 gene was constructed using the Ad Easy System. Cultured Eca109 cells were transfected with Ad/Ang-1si (Eca109/Ang-1si), and Ad/si was used to infect Eca109 cells as control (Eca109/si). Ang-1 gene expression and concentration was determined with RT-PCR and ELISA, respectively. Human umbilical vein endothelial cell (HUVEC) migration and proliferation were analyzed. After s.c. injection into athymic nu/nu mice, the tumor growth, vessel density and apoptosis of each group was also determined. RESULTS: HUVEC migration induced by conditioned medium from Ang-1si-transfected Eca109 cells was significantly less than that induced by conditioned medium from Eca109 cells and control adenovirus- transfected Eca109 cells. Furthermore, after s.c. injection into athymic nu/nu mice, the tumor growth and cell apoptosis of Ad/Ang-1si -expressing Eca109 cells was significantly lower than that of parental or control adenovirus-transfected cells. Vessel density assessed by CD31 immunohistochemical analysis and Ang-1 expression by RT-PCR were also decreased. CONCLUSION: The targeting Ang-1 may provide a therapeutic option for esophageal cancer.

Inhibition of peroxisome proliferator-activated receptor-γ in steroid-induced adipogenic differentiation of the bone marrow mesenchymal stem cells of rabbit using small interference RNA

Background Steroids inhibit osteogenic differentiation and decrease bone formation while concomitantly inducing adipose deposition in osteocytes.This leads to the fatty degeneration and necrosis of bone cells commonly seen in osteonecrosis of the femoral head.The peroxisome proliferator-activated receptor-γ （PPARγ） is an adipogenic transcription factor linked to the development of this disease and responsible for inducing adipogenesis over osteogenesis in bone marrow mesenchymal stem cells （BMSCs）.The aim of this study was to assess whether adipogenic differentiation could be suppressed,and thus osteogenic potential retained,by inhibiting PPARy expression in BMSCs.Methods Cells from the bone marrow of New Zealand rabbits were treated with 10-7 mol/L dexamethasone and infected with one of three small interference RNA （siRNA） adenovirus vectors （S1,S2,and S3） or non-targeting control siRNA （Con） and compared with dexamethasone-treated （model） and untreated （normal） cells.Cells were grown for 21 days and stained with Sudan Ⅲ for adipocyte formation.At various time points,cells were also assessed for changes in PPARγ,osteocalcin （OC）,Runx2,alkaline phosphatase （ALP） activity,and triglyceride （TG） content.Results Dexamethasone-treated model and control groups showed a significant increase in fatty acid-positive staining,which was inhibited in cells treated with PPARγ siRNA-treated,similar to normal untreated cells.All three siRNA groups significantly inhibited PPARγ mRNA and protein,adipocyte number,and TG content compared with the dexamethasonetreated model and control groups,matching that seen in normal cells.OC and Runx2 mRNA and protein,as well as ALP activity,were significantly higher in cells treated with siRNA against PPARγ,similar to that seen in the normal cells.These osteogenic markers were significantly lower in the dexamethasone-treated cell cultures.Conclusions The siRNA adenovirus vector targeting PPARγ can efficiently inhibit steroid-induced adipogenic differentiation in rabbit BMSCs and retain their osteogenic differentiation potential.