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.

CopE and TLR6 RNAi-mediated tomato resistance to western flower thrips

The western flower thrips(WFT;Frankliniella occidentalis)is a mesophyll cell feeder that damages many crops.Management of WFT is complex due to factors such as high fecundity,short reproduction time,ability to feed on a broad range of host plants,and broad pesticide resistance.These challenges have driven research into developing alternative pest control approaches for WFT.This study analyzed the feasibility of a biological control-based strategy to manage WFT using RNA interference(RNAi)-mediated silencing of WFT endogenous genes.For the delivery of RNAi,we developed transgenic tomato lines expressing double-stranded RNA(dsRNA)of coatomer protein subunit epsilon(CopE)and Toll-like receptor 6(TLR6)from WFT.These genes are involved in critical biological processes of WFT,and their dsRNA can be lethal to these insects when ingested orally.Adult WFT that fed on the transgenic dsRNAexpressing tomato flower stalk showed increased mortality compared with insects that fed on wild-type samples.In addition,WFT that fed on TLR6 and CopE transgenic tomato RNAi lines showed reduced levels of endogenous CopE and TLR6 transcripts,suggesting that their mortality was likely due to RNAi-mediated silencing of these genes.Thus,our findings demonstrate that transgenic tomato plants expressing dsRNA of TLR6 and CopE can be lethal to F.occidentalis,suggesting that these genes may be deployed to control insecticide-resistant WFT.

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.

Silencing MTA1 by RNAi Reverses Adhesion, Migration and Invasiveness of Cervical Cancer Cells (SiHa) via Altered Expression of p53, and E-cadherin/β-catenin Complex

It has been reported that metastasis-associated gene 1 （Mta1） is overexpressed in many malignant tumors with high metastatic potential. In addition, some studies indicated that MTA1 participated in invasion, metastasis, and survival of cancer cells by regulating cell migration, adhesion and proliferation. But the role of MTA1 is unclear in vitro in the development of cervical cancer cells. This study investigated whether and how MTA1 mediated cell proliferation, migration, invasion and adhesion in cervical cancer. MTA1 expression level was detected by Western blot in two cervical cancer cell lines of different invasion potentials. The effects of MTA1 expression on SiHa cell apoptosis, cycle, proliferation, migration, invasion and adhesion were tested by flow cytometry, MTT, wound-healing assay, Transwell assay and adhesion assay, respectively. The expression levels of p53, E-cadherin, and β-catenin activity were evaluated in untreated and treated cells. The results showed that MTA1 protein expression was significantly higher in SiHa than in HeLa, which was correlated well with the potential of migration and invasion in both cell lines. Furthermore, the cell invasion, migration and adhesion capabilities were decreased after inhibition of MTA1 expression mediated by Mta1-siRNA transfection in SiHa. However, no significant differences were found in cell apoptosis, cycle, and proliferation. In addition, E-cadherin and p53 protein levels were significantly up-regulated, while β-catenin was significantly down-regulated in SiHa transfected with the siRNA. These results demonstrated that MTA1 played an important role in the migration and invasion of cervical cancer cells. It was speculated that the decreased migration and invasion capability by inhibiting the MTA1 expression in the SiHa cell line may be mediated through the altered expression of p53, and E-cadherin/β-catenin complex. MTA1 could serve as a potential therapeutic target in cervical cancer.

Downregulation of survivin by RNAi inhibits growth of human gastric carcinoma cells

AIM: To investigate the inhibitory effect of a specific small survivin interfering RNA (siRNA) on cell proliferation and the expression of survivin in human gastric carcinoma cell line SGC-7901. METHODS: To knockdown survivin expression, a small interfering RNA targeting against survivin was synthesized and transfected into SGC-7901 cells with lipofectamineTM2000. The downregulation of survivin expression at both mRNA and protein levels were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Cell proliferation inhibition rates were determined by methyl thiazolyl tetrazolium (MTT) assay. The effect of survivin siRNA on cell cycle distribution and cell apoptosis was determined by flow cytometry (FCM). RESULTS: RNA interference could efficiently suppress the survivin expression in SGC-7901 cells. At 48 h after transfection, the expression inhibition rate was 44.52% at mRNA level detected by RT-PCR and 40.17% at protein level by Western blot analysis. Downregulation of survivin resulted in significant inhibition of tumor cell growth in vitro. The cell proliferation inhibition rates at 24, 48 and 72 h after survivin siRNA and non-siliencing siRNA transfection, were 34.06%, 47.61% and 40.36%, respectively. The apoptosis rate was 3.56% and the number of cells was increased in G0/G1 phase from 38.2% to 88.6%, and decreased in S and G2/M phase at 48 h after transfection. CONCLUSION: Downregulation of survivin results in significant inhibition of tumor growth in vitro. The inhibition of survivin expression can induce apoptosis of SGC-7901 cells. The use of survivin siRNA deserves further investigation as a novel approach to cancer therapy.

慢病毒介导基于microRNA系统的HBs RNAi技术抑制HBV复制

目的:构建HBs基因RNAi慢病毒载体,观察其对HBV复制和抗原表达的作用。方法:针对已经筛选确定的HBs基因RNAi有效靶序列,合成靶序列的Oligo DNA,退火形成双链DNA,与经MluⅠ和ClaⅠ酶切后的pGCLM-GFP载体连接产生慢病毒载体,PCR筛选阳性克隆,测序鉴定。用pGCLM-GFP、pHelper1.0和pHelper2.0质粒共转染包装细胞293T,包装产生慢病毒,以293T细胞GFP蛋白的表达水平测定病毒滴度。培养HepG2.2.15细胞系,用慢病毒(MOI=1和MOI=10)对肝癌细胞进行感染,感染后细胞培养上清进行ELISA、Western印迹、HBV DNA定量分析。结果:PCR和测序结果证实,成功构建HBsshRNA的慢病毒载体。包装慢病毒后浓缩病毒悬液的滴度为5×108～2×109TU/ml。慢病毒HBsRNAi后,对HBV复制和抗原表达的抑制作用显著。感染4d后,抑制效应开始出现,一直持续到第9天,抑制效应达到高峰(P<0.05)。相对于阴性对照,HBs shRNA作用后细胞上清中HBsAg分泌量下降70%以上(P<0.05),而Western印迹和real-time PCR结果进一步证实了上述结果,在蛋白水平和mRNA水平都得到了进一步验证。经HBV DNA定量,发现慢病毒RNAi后DNA水平也显著下降(P<0.05)。结论:成功构建HBs基因RNAi慢病毒载体,以HBs基因为靶点的慢病毒介导的基于micro RNA系统的RNAi技术能有效抑制HBV的复制和抗原表达。

Knockdown of survivin gene expression by RNAi induces apoptosis in human hepatocellular carcinoma cell line SMMC-7721

AIM: To investigate the survivin gene expression in human hepatocellular carcinoma cell line SMMC-7721 and the effects of survivin gene RNA interference (RNAi) on cell apoptosis and biological behaviors of SMMC-7721 cells. METHODS: Eukaryotic expression vector of survivin gene RNAi and recombinant plasmid pSuppressorNeo-survivin (pSuNeo-SW), were constructed by ligating into the vector, pSupperssorNeo (pSuNeo) digested with restriction enzymes Xba I and Sail and the designed double-chain RNAi primers. A cell model of SMMC-7721 after treatment with RNAi was prepared by transfecting SMMC-7721 cells with the lipofectin transfection method. Strept-avidin-biotin-complex (SABC) immunohistochemical staining and RT-PCR were used to detect survivin gene expressions in SMMC-7721 cells. Flow cytometry was used for the cell cycle analysis. Transmission electron microscopy was performed to determine whether RNAi induced cell apoptosis, and the method of measuring the cell growth curve was utilized to study the growth of SMMC-7721 cells before and after treatment with RNAi. RESULTS: The eukaryotic expression vector of survivin gene RNAi and pSuNeo-SW, were constructed successfully. The expression level of survivin gene in SMMC-7721 cells was observed. After the treatment of RNAi, the expression of survivin gene in SMMC-7721 cells was almost absent, apoptosis index was increased by 15.6%, and the number of cells was decreased in G2/M phase and the cell growth was inhibited. CONCLUSION: RNAi can exert a knockdown of survivin gene expression in SMMC-7721 cells, and induce apoptosis and inhibit the growth of carcinoma cells.

Liver-specific Reduction of Mfn2 Protein by RNAi Results in Impaired Glycometabolism and Lipid Homeostasis in BALB/c Mice

Mitofusin-2 （Mfn2） gene expression is positively correlated with insulin sensitivity in patients with type 2 diabetes. However, it is unclear if Mfn2 is involved in carbohydrate metabolism and lipid homeostasis. In order to investigate the specific functions of Mfn2 in glycometabolism and lipid homeostasis in BALB/c mice, a RNA interference technique-mediated hydrodynamic injection was developed, in which short hairpin RNAs （shRNAs） were used to inhibit the Mfn2 expression in vivo. Seventy-two mice were randomly divided into two groups： the Mfn2 reduction group （Mfn2/shRNA） and the negative control group （NC）. Intraperitoneal glucose tolerance tests and intraperitoneal insulin tolerance tests were used to evaluate glycometabolism and insulin sensitivity. D-（3-3H） glucose or 3H2O was injected into the tail vein or intraperitoneally to facilitate the calculation of the rate of hepatic glucose production and fatty acid synthesis in vivo. The results showed that, in Mfn2/shRNA mice, the liver Mfn2 protein was significantly decreased, and fasting blood glucose concentrations were increased by approximately 48%, when compared with the NC mice. In parallel with the changes in fasting glucose levels, hepatic glucose production was significantly elevated in Mfn2/shRNA mice. When insulin was administrated, these mice exhibited impaired insulin tolerance. It was also found that the reduction of Mfn2 markedly decreased the rate of fatty acid synthesis in the liver, and the Mfn2/shRNA mice exhibited hypertriglyceridema. Taken together, our results indicate that Mfn2 plays an important role in maintaining glucose and lipid homeostasis, and in the development of insulin resistance in vivo.

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.

Inhibition of KIT RNAi mediated with adenovirus in gastrointestinal stromal tumor xenograft

AIM: To investigate a therapeutic method for gastrointestinal stromal tumor (GIST) based on KIT RNA interference (RNAi) with AdMax adenovirus. METHODS: KIT short hairpin RNA (shRNA), whose lateral sides were decorated with restriction endonuclease sequences, was designed. T 4 DNA ligase catalyzed the joint of the KIT shRNA and the green fluorescent protein-containing PDC316-EGFP-U6 to form PDC316EGFP-U6-KIT. Homologous recombination of AdEGFPU6-KIT was performed with the AdMax system. Heterotopically transplanted GISTs were established in nude mice. AdEGFP-U6-KIT was intratumorally injected. The volume, inhibition ratio of tumor and CD117 expression of GIST graft tumor in nude mice were compared between test and control groups. RESULTS: The length of KIT shRNA was determined to be about 50bp by agarose electrophoresis. Gene se-quencing detected the designed KIT RNAi sequence in PDC316-EGFP-U6-KIT. After transfection with AdEGFPU6-KIT, 293 cells displayed green fluorescence. The physical and infective titers of AdEGFP-U6-KIT were 5 × 10 11 viral particles/mL and 5.67 × 10 7 plaque forming units/mL, respectively. The mean volume of the grafted tumor was significantly smaller in test mice than in control mice (75.3 ± 22.9 mm 3 vs 988.6 ± 30.5 mm 3 , t = -18.132, P < 0.05). The inhibition ratio of the tumors was 59.6% in the test group. CD117 positive expression was evident in two cases (20%) in the test group and 10 cases (100%) in the control group (χ 2 = 10.2083, P < 0.005). CONCLUSION: AdEGFP-U6-KIT is successfully constructed, and KIT RNAi mediated with Admax vector system can effectively inhibit the expression of the KIT gene and the growth of GIST in nude mice.

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.

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.

农杆菌介导的花生Δ^(12)脂肪酸脱氢酶基因AhFAD2 RNAi抑制表达遗传转化研究

以成熟花生种子萌动4d的上胚轴为受体材料,采用农杆菌介导的转化方法,将倒位重复Δ12脂肪酸脱氢酶基因AhFAD2片段分别与CaMV35S启动子和种子特异性表达的大豆凝集素启动子相连导入花生。经PCR检测证实含有AhFAD2倒位重复基因片段的RNAi抑制表达框架已成功转入花生。

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.

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

Construction of shRNA Targeted to the Rat Angiotensin Ⅱ Type 1 Receptors and Its RNAi in Cytoplasma

The expression vector of shRNA targeted to the rat angiotensin Ⅱ receptor gene was constructed and the efficacy of siRNAs to modulate the expression of target gene in the in vitro cultured mammalian cells was investigated for antihypertensive therapy in spontaneous hypertensive rat （SHR） at post-transcriptional level. The sense and antisense RNA oligonucleotides strands targeting angiotensin Ⅱ receptor mRNA were synthesized individually according to the sequence of the rat angiotensin Ⅱ receptor. For preparation of duplexes, sense- and antisense-stranded oligonucleotides were mixed and annealed, and the annealed duplexes were cloned into the pGenesil-1 vector. The rat glioma cells were transfected with constructed pGenesil-1-shRNA plasmid and scrambled plasmid. The cultured cells were collected at different phases. RT-PCR and Western blot were performed. The AT1 mRNA and protein levels behaved ultimately same. Compared to control after 48 h, AT1 mRNA levels were decreased to 35.5%±3.0 %, and the levels reached their lowest point after 72 h （20.7% ±4 % of control）. At 24 and 48 h, AT1 protein was reduced to 46.9%±4.2% and 36.98%±3.7% respectively compared to control and a maximum reduction was observed after 72 h of incubation （28.1%± 4% compared to controls）. Plasmid-based shRNA expression systems targeted against the rat angiotensin Ⅱ receptor gene were generated successfully. The shRNAs with a 22-nt stem and a short loop were cleaved into small interfering dsRNA （siRNA） by the Dicer. The in vitro transcribed siRNA enables the effective silencing of gene expression to the target mRNA and leads to effective inhibition of translation of proteins and will be lay the foundation of application of gene silencing technology to hypertensive rats.

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.

Suppression of RNA Interference Pathway in vitro by Grass Carp Reovirus

The means of survival of genomic dsRNA of reoviruses from dsRNA-triggered and Dicer-initiated RNAi pathway remains to be defined. The present study aimed to investigate the effect of Grass carp reovirus (GCRV) replication on the RNAi pathway of grass carp kidney cells (CIK). The dsRNA-triggered RNAi pathway was demonstrated unimpaired in CIK cells through RNAi assay. GCRV-specific siRNA was generated in CIK cells transfected with purified GCRV genomic dsRNA in Northern blot analysis; while in GCRV-infected CIK cells, no GCRV-specific siRNA could be detected. Infection and transfection experiments further indicated that replication of GCRV correlated with the increased transcription level of the Dicer gene and functional inhibition of in vitro synthesized egfp-siRNA in silencing the EGFP reporter gene. These data demonstrated that although only the genomic dsRNA of GCRV was sensitive to the cellular RNAi pathway, unidentified RNAi suppressor protein(s) might contribute to the survival of the viral genome and efficient viral replication.

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.

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.