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.

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.

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.

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.

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.

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 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

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.

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.

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.

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.

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-regulation of IL-8 expression in human airway epithelial cells through helper-dependent adenoviral-mediated RNA interference

Interleukin (IL)-8 is a potent neutrophil chemotactic factor and a crucial mediator in neutrophil-dependent inflammation.Various cell types produce IL-8, either in response to external stimuli such as cytokines or bacterial infection, or aftermalignant transformation. Anti-IL-8 strategies have been considered for anti-inflammatory therapy. In this paper wedemonstrate that the RNA interference technique can be used to efficiently down-regulate IL-8 protein expression inairway epithelial cells. We used a helper-dependent adenoviral vector to express a small hairpin (sh)RNA targetinghuman IL-8 in cultured airway epithelial cells (IB3-1, Cftr-/-; C38, Cftr-corrected) stimulated with TNF-α, IL-1β orheat-inactivated Burkholderia cenocepacia. Stimulated IL-8 expression in IB3-1 and C38 cells was significantly reducedby shRNA expression. The shRNA targeting IL-8 had no effect on the activation of NF-κB, or on the protein levels ofIκB or IL-6, suggesting that this anti-IL-8 strategy was highly specific, and therefore may offer potential for thetreatment of inflammatory diseases.

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.

RNA interference-mediated gene silencing of vascular endothelial growth factor in colon cancer cells

AIM： TO inhibit the expression of vascular endothelial growth factor （VEGF） in colon cancer cell line by RNA interference （RNAi）.METHODS： Followed the service of E-RNAi, we designed and constructed two kinds of shRNA expression vectors aiming at the VEGF gene, then transfected them into colon cancer HT29 cells by lipofectamineTM 2000. The level of VEGF mRNA was investigated by RT-PCR and Northern blotting. The protein expression of VEGF was observed by immunofluoresence staining and Western blotting.RESULTS： We got two kinds of VEGF specific shRNA expression vectors which could efficiently inhibit the expression of VEGF in HT29 cells. RT-PCR, Northern blotting, immunofluoresence staining and Western blotting showed that inhibition rate for VEGF expression was up to 42%, 89%, 73% and 82%, respectively.CONCLUSION： The expression of VEGF can be inhibited by RNA interference in HT29 cells.

Downregulation of NFAT5 by RNA interference reduces monoclonal antibody productivity of hybridoma cells

Hybridoma cells display an increase in antibody productivity following exposure to hypertonic conditions. However, the underlying mechanism is not well understood. In the present study, we hypothesize that the nuclear factor of activated T cells 5 （NFAT5）/tonicity enhancer binding protein （TonEBP） functions to increase the antibody productivity of hybridoma cells. NFAT5 is an osmosensitive mammalian transcription factor. However, its ubiquitous expression in various organs that are not bathed in hypertonic milieu suggests that NFAT5 may also regulate cell growth and function under isotonic conditions. In this study, we examined the expression of NFAT5 in hybridoma cells by Western blot analysis, and found that it increased significantly in hypertonic medium. To further define the function of NFAT5 in hybridoma cells, RNA interference technique was used to downregulate the expression of NFAT5 in SGB-8 cells （a hybridoma cell line）. In isotonic medium, antibody productivity ofhybridoma cells was reduced by downregulation of NFAT5 while cell proliferation was not influenced. The results presented here demonstrate that NFAT5 not only plays an important role in osmotic stress response pathway in hybridoma cells but also is essential for optimal antibody productivity.

Phosphoinositide-3-kinase,catalytic,alpha polypeptide RNA interference inhibits growth of colon cancer cell SW948

AIM:To investigate the gene knock-down effect by the phosphoinositide-3-kinase,catalytic,alpha polypeptide(PIK3CA)-targeted double-stranded RNA(dsRNA) and its effect on cell proliferation and cycle distribution in SW948.METHODS:Two PIK3CA-targeted dsRNAs were constructed and transfected into SW948 cells.Transfections were performed using lipofectamine TM 2000.The transfection effectiveness was calculated basing on the rate of fluorescence cell of SW948 at 6 h after transfection.Total messenger RNA was extracted from these cells using the RNeasy kit,and semiquantitative reverse transcription polymerase chain reaction was performed to detect the down-regulation of PIK3CA,AKT1,MYC,and CCND1 gene expression.Cells were harvested,proteins were resolved,and western blot was employed to detect the expression levels of PIK3CA,AKT1,MYC,and CCND1 gene.Cell proliferation was assessed by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide assay and the inhibition rate was calculated.Soft agar colony formation assay was performed basing on colonies greater than 60 μm in diameter at ×100 magnification.The effect on cell cycle distribution and apoptosis was assessed by flow cytometry.All experiments were performed in triplicate.RESULTS:Green fluorescence was observed in SW948 cell transfected with plasmid Pgenesil-1,and the transfection effectiveness was about 65%.Forty-eight hours post-transfection,mRNA expression of PIK3CA in SW948 cells was 0.51 ± 0.04 vs 0.49 ± 0.03 vs 0.92 ± 0.01 vs 0.93 ± 0.03(P = 0.001) in Pgenesil-CA1,Pgenesil-CA2,negative and blank group respectively.mRNA expression of AKT1 was 0.50 ± 0.03 vs 0.48 ± 0.01 vs 0.93 ± 0.04 vs 0.92 ± 0.02(P = 0.000) in Pgenesil-CA1,Pgenesil-CA2,negative and blank group respectively.mRNA expression of MYC was 0.49 ± 0.01 vs 0.50 ± 0.04 vs 0.90 ± 0.02 vs 0.91 ± 0.03(P = 0.001) in the four groups respectively.mRNA expression of CCND1 was 0.45 ± 0.02 vs 0.51 ± 0.01 vs 0.96 ± 0.03 vs 0.98 ± 0.01(P = 0.001) in the four groups respectively.The protein level of PIK3CA was 0.53 ± 0.01 vs 0.54 ± 0.02 vs 0.92 ± 0.03 vs 0.91 ± 0.02(P = 0.001) in Pgenesil-CA1,Pgenesil-CA2,negative and blank group respectively.The protein level of AKT1 in the four groups was 0.49 ± 0.02 vs 0.55 ± 0.03 vs 0.94 ± 0.03 vs 0.95 ± 0.04,P = 0.000).The protein level of MYC in the four groups was 0.51 ± 0.03 vs 0.52 ± 0.04 vs 0.92 ± 0.02 vs 0.95 ± 0.01(P = 0.000).The protein level of CCND1 in the four groups was 0.54 ± 0.04 vs 0.56 ± 0.03 vs 0.93 ± 0.01 vs 0.93 ± 0.03(P = 0.000).Both Pgenesil-CA1 and Pgenesil-CA2 plasmids significantly suppressed the growth of SW948 cells when compared with the negative or blank group at 48 h after transfec-tion(29% vs 25% vs 17% vs 14%,P = 0.001),60 h after transfection(38% vs 34% vs 19% vs 16%,P = 0.001),and 72 h after transfection(53% vs 48% vs 20% vs 17%,P = 0.000).Numbers of colonies in negative,blank,CA1,and CA2 groups were 42 ± 4,45 ± 5,8 ± 2,and 10 ± 3,respectively(P = 0.000).There were more than 4.5 times colonies in the blank and negative control groups as there were in the CA1 and CA2 groups.In addition,the colonies in blank and negative control groups were also larger than those in the CA1 and CA2 groups.The percentage of cells in the CA1 and CA2 groups was significantly higher in G 0 /G 1 phase,but lower in S and G 2 /M phase when compared with the negative and control groups.Moreover,cell apoptosis rates in the CA1 and CA2 groups were 5.11 ± 0.32 and 4.73 ± 0.32,which were significantly higher than those in negative(0.95 ± 0.11,P = 0.000) and blank groups(0.86 ± 0.13,P = 0.001).No significant difference was found between CA1 and CA2 groups in cell cycle distribution and apoptosis.CONCLUSION:PIK3CA-targeted short hairpin RNAs can block the phosphoinositide 3-kinase-Akt signaling pathway and inhibit cell growth,increase apoptosis,and induce cell cycle arrest in the PIK3CA-mutant colon cancer SW948 cells.

Silencing phospholipid scramblase 1 expression by RNA interference in colorectal cancer and metastatic liver cancer

BACKGROUND:Phospholipid scramblase 1(PLSCR1) not only participates in the transbilayer movement of phospholipids,but also plays a role in the pathogenesis and progression of cancers.The present study aimed to evaluate the effect of silencing PLSCR1 expression by RNA interference in colorectal cancer(CRC) and metastatic liver cancer.METHODS:The expression of PLSCR1 in CRC and metastatic liver cancer samples was assessed by immunohistochemistry.The cultured cells with the highest expression were selected for subsequent experiments.We designed three siRNA oligonucleotide segments targeted at PLSCR1.Successful transfection was confirmed.The biological behavior of the cells in proliferation,adhesion,migration and invasion was determined.RESULTS:PLSCR1 protein expression increased significantly in the majority of CRC and metastatic liver cancer samples compared with normal samples.Lovo cells had the highest expression of PLSCR1.The siRNA-390 oligonucleotide segment had the best silencing effect.After transfection,Lovo cell proliferation was significantly inhibited compared with the controls in the MTT assay.Laminin and fibronectin adhesion assays showed Lovo cell adhesion was also significantly inhibited.In the migration assay,the number of migrating cells in the PLSCR1 siRNA-390 group was 50±12,significantly lower than the number in the siRNA-N group(115±28) and in the control group(118±31).In an invasion test,the number of invading cells in the PLSCR1 siRNA-390 group was 60±18,significantly lower than that in the siRNA-N group(97±26) and the control group(103±24).CONCLUSIONS:PLSCR1 is overexpressed in CRC and metastatic liver cancer.Silencing of PLSCR1 by siRNA inhibits the proliferation,adhesion,migration and invasion of Lovo cells,which suggests that PLSCR1 contributes to the tumorigenesis and tumor progression of CRC.PLSCR1 may be a potential gene therapy target for CRC and associated metastatic liver cancer.

Effects of RNA interference-induced tryptase down-regulation in P815 cells on IL-6 and TNF-α release of endothelial cells

Objective： To explore the effects of down-regulated tryptase expression in mast cells on the synthesis and release of interleukin-6 （IL-6） and tumor necrosis factor-alpha （TNF-α） of vascular endothelial cells. Methods： Tryptase-siRNA （small-interfering RNA） vector was constructed to inhibit tryptase expression in P815 cells. The medium of P815 cells treated by the tryptase-siRNA （RNAi-P815 group） or pure vector （P815 group） was collected and used to culture bEnd.3 cells. The messenger RNAs （mRNAs） of IL-6 and TNF-α in bEnd.3 cells and their protein levels in the medium were measured by reverse transcription polymerase chain reaction （RT-PCR） and enzyme-linked immunosorbent assay （ELISA）, respectively. Results： IL-6 and TNF-α mRNAs in bEnd.3 cells cultured in RNAi-P815-conditioned medium decreased significantly compared to those in P815-conditioned medium. Consistently, lL-6 and TNF-α protein levels in the medium of bEnd.3 of RNAi-P815 group were lower than those of P815 group. Conclusion： Reduced tryptase expression significantly inhibited the synthesis and release of IL-6 and TNF-α in vascular endothelial cells. RNA interference targeting tryptase expression may be a new anti-inflammatory strategy for vascular diseases.

PPM1D Silencing by Lentiviral-mediated RNA Interference Inhibits Proliferation and Invasion of Human Glioma Cells

To construct a lentiviral shRNA vector targeting human protein phosphatase 1D magnesium-dependent（PPM1D） gene and detect its effectiveness of gene silencing in human gliomas,specific siRNA targets with short hairpin frame were designed and synthesized.DNA oligo was cloned into the pFU-GW-iRNA lentiviral expression vector,and then PCR and sequencing analyses were conducted to verify the constructs.After the verified plasmids were transfected into 293T cells,the lentivirus was produced and the titer of virus was determined.Real-time quantitative PCR and Western blot were performed to detect the PPM1D expression level in the infected glioma cells.PCR and Western blot analyses revealed the optimal interfering target,and the virus with a titer of 6×10^8 TU/mL was successfully packaged.The PPM1D expression in human glioma cells was knocked down at both mRNA and protein levels by virus infection.The expression of PPM1D mRNA and protein was decreased by 76.3% and 87.0% respectively as compared with control group.The multiple functions of human glioma cells after PPM1D RNA interference were detected by flow cytometry and cell counting kit-8（CCK-8）.Efficient down-regulation of PPM1D resulted in significantly increased cell apoptosis and reduced cell proliferation and invasion potential in U87-MG cells.We have successfully constructed the lentiviral shRNA expression vector capable of stable PPM1D gene silencing at both mRNA and protein levels in glioma cells.And our data gave evidence that the reduced cell growth observed after PPM1D silencing in glioma cells was at least partly due to increased apoptotic cell death.