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.

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.

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.

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.

Influence of RNA interference on the mitochondrial subcellular localization of alpha-synuclein and on the formation of Lewy body-like inclusions in the cytoplasm of human embryonic kidney 293 cells induced by the overexpression of alphasynuclein

The specific and effective a-synuclein RNA interference （RNAi） plasmids, and the a-synuclein-pEGFP recombinant plasmids were co-transfected into human embryonic kidney 293 （HEK293） cells using the lipofectamine method. Using an inverted fluorescence microscope, a-synuclein proteins were observed to aggregate in the cytoplasm and nucleus. Wild-type a-synuclein proteins co-localized with mitochondria. Hematoxylin-eosin staining revealed round eosinophilic bodies （Lewy body-like inclusions） in the cytoplasm of some cells transfected with a-synuclein-pEGFP plasmid. However, the formation of Lewy body-like inclusions was not observed following transfection with the RNAi pSYN-1 plasmid. RNAi blocked Lewy body-like inclusions in the cytoplasm of HEK293 cells induced by wild-type a-synuclein overexpression, but RNAi did not affect the subcellular localization of wild-type a-synuclein in mitochondria.

RNA interference and its application in plants

RNA interference （RNAi）, a process that inhibits gene expression by the double-stranded RNA （dsRNA）, causes the degradation of target messenger RNA molecules. RNAi exists in almost all organisms. We review the recent history of RNAi studies, RNAi molecular mechanisms, characteristics and RNAi applications in higher plants. At the same time, the prospect of RNAi applications in functional genomics and genetic improvement of higher plants and possible future problems and possibilities are also discussed.

LXRα gene downregulation by lentiviral-based RNA interference enhances liver function after fatty liver transplantation in rats

Steatotic liver grafts, although accepted, increase the risk of poor posttransplantation liver function. However, the growing demand for adequate donor organs has led to the increased use of so-called marginal grafts. Liver X receptor alpha （LXRα） is important in fatty acid metabolism and inter- related with the specific ischemia-reperfusion injury in fatty liver transplantation. This study aimed to investigate whether LXRa RNA interference （RNAi） could improve the organ func- tion of liver transplant recipients. METHODS： Fifty Sprague-Dawley rats were fed with a high-fat diet and 56% alcohol. The livers of these animals had greater than 60% macrovesicular steatosis and were used as liver do- nors. The experimental donors were treated with 7×10^7 TU LXRα-RNAi-LV of a mixture injection and control donors with negative control-LV vector injection into the portal vein 72 hours before the operation. The effects of LXRa-RNAi-LV were assessed by serum aminotransferases, histology, immunostain- ing, and protein levels. The transcription of LXRα mRNA was assessed by reverse transcription-polymerase chain reaction. RESULTS： Compared with controls, LXRa RNAi inhibited the expression of LXRα at the mRNA （0.53±0.03 vs 0.94±0.02, P〈0.05） and protein levels （0.51±0.08 vs 1.09±0.12, P〈0.05）. LXRa RNAi also decreased the expressions of sterol regula- tory element-binding protein lc （SREBP-Ic） and CD36. LXRa RNAi consequently reduced fatty acid accumulation in hepa- tocytes. Compared with control animals, LXRα RNAi-treated group had lower serum alanine aminotransferase, aspartate aminotransferase, interleukin-1β, and tumor necrosis factor- alpha levels and milder pathologic damages. TUNEL analysisrevealed a significant reduction of apoptosis in the livers of rats treated with LXRa-RNAi-LV, and overall survival as determined by the Kaplan-Meier method was improved among rats treated with LXRα-RNAi-LV （P〈0.05）. CONCLUSION： LXRa-RNAi-LV treatment significantly down- regulated LXRa expression and improve steatotic liver graft function and recipient survival after a fatty liver transplanta- tion in rats.

Effects of lentiviral RNA interference-mediated downregulation of integrin-linked kinase on biological behaviors of human lens epithelial cells

AIM：To investigate the effects of lentivirus（LV）mediated integrin-linked kinase（ILK）RNA interference（RNAi）on biological behaviors of human lens epithelial cells（LECs）.·METHODS：Human cataract LECs and immortalized human LEC line,human lens epithelial（HLE）B-3 cells were transfected by lentiviral vector expressing ILKspecific short hairpin RNA（sh RNA）and then stimulated by transforming growth factor-β（TGF-β）,the silencing of ILK gene and protein was identified by reverse transcription-polymerase chain reaction（RT-PCR）and Western blot methods;biological behaviors including cell cycle and apoptosis,cell morphology,α-smooth muscle actin（SMA）stress fiber formation and cell migration were examined.·RESULTS：Remarkable decreases of ILK protein expression were detected in LECs carrying lentiviral ILK-sh RNA vector;flow cytometry revealed arresting of cell cycle progression through the G1/S transition and higher apoptosis rate in ILK-RNAi-LV transfected cells.Lessα-SMA stress fiber formation and migration was observed in ILK-RNAi-LV transfected LECs.·CONCLUSION：The present study demonstrated that ILK was an important regulator for LECs proliferation and migration.LV mediated ILK RNAi is an effective way todecrease ILK-regulated cell growth by arresting cell cycle progression and increasing cell apoptosis,as well as,to prevent cell migration by inhibiting TGF-βinducedα-SMA stress fiber formation.Thus,LV mediated ILK RNAi might be useful to prevent posterior capsular opacification.

Lentivirus-mediated short hairpin RNA interference of CENPK inhibits growth of colorectal cancer cells with overexpression of Cullin 4A

BACKGROUND Colorectal cancer(CRC)is one of the most common malignant tumors worldwide.The identification of novel diagnostic and prognostic biomarkers for CRC is a key research imperative.Immunohistochemical analysis has revealed high expression of centromere protein K(CENPK)in CRC.However,the role of CENPK in the progression of CRC is not well characterized.AIM To evaluate the effects of knockdown of CENPK and overexpression of Cullin 4A(CUL4A)in RKO and HCT116 cells.METHODS Human colon cancer samples were collected and tested using a human gene expression chip.We identified CENPK as a potential oncogene for CRC based on bioinformatics analysis.In vitro experiments verified the function of this gene.We investigated the expression of CENPK in RKO and HCT116 cells using quantitative polymerase chain reaction(qPCR),western blot,and flow cytometry.The effect of short hairpin RNA(shRNA)virus-infected RKO cells on tumor growth was evaluated in vivo using quantitative analysis of fluorescence imaging.To evaluate the effects of knockdown of CENPK and overexpression of CUL4A in RKO and HCT116 cells,we performed a series of in vitro experiments,using qPCR,western blot,MTT assay,and flow cytometry.RESULTS We demonstrated overexpression of CENPK in human colon cancer samples.CENPK was an independent risk factor in patients with CRC.The downstream genes FBX32,CUL4A,and Yesassociated protein isoform 1 were examined to evaluate the regulatory action of CENPK in RKO cells.Significantly delayed xenograft tumor emergence,slower growth rate,and lower final tumor weight and volume were observed in the CENPK short hairpin RNA virus infected group compared with the CENPK negative control group.The CENPK gene interference inhibited the proliferation of RKO cells in vitro and in vivo.The lentivirus-mediated shRNA interference of CENPK inhibited the proliferation of RKO and HCT116 colon cancer cells,with overexpression of the CUL4A.CONCLUSION We indicated a potential role of CENPK in promoting tumor proliferation,and it may be a novel diagnostic and prognostic biomarker for CRC.

Molecular characterization and functional analysis of USP-1 by RNA interference in the Asian gypsy moth Lymantria dispar

Ecdysteroids play an important role in regulating diverse physiological processes in arthropods,such as molting,metamorphosis,reproduction and diapause.Ecdysteroids mediate the response by binding to a heterodimeric complex of two nuclear receptors:the ecdysone receptor and the ultraspiracle(USP).To investigate the role of USP in development of the Asian gypsy moth(Lymantria dispar),a USP cDNA was obtained from the transcriptome of L.dispar and verified by PCR.In-depth profiling of transcript levels of L.dispar USP-1(LdUSP-1)at different developmental stages and over time in thirdinstar larvae and different tissues isolated during the thirdinstar stage of L.dispar was then carried out.Transcript levels of LdUSP-1 were relatively high before 72 h in the third-instar larvae after ecdysis and in the adult male.The function of LdUSP-1 in molting was analyzed by knockdown of LdUSP-1 in third instar larvae using RNA interference.Silencing of LdUSP-1 significantly downregulated the transcript level of E75,an ecdysone-inducible gene,and of Sad,a Halloween gene.In addition,the duration of the third-instar stage was slightly shortened and larval mortality increased after the LdUSP-1 knockdown.

RNA interference in Colorado potato beetle(Leptinotarsa decemlineata): A potential strategy for pest control

Colorado potato beetle(CPB),Leptinotarsa decemlineata,is a notorious destructive pest that mainly feeds on the leaves of potato and several other solanaceous plants.CPB is widely recognized for its adaptation to a remarkable variety of host plants and diverse climates,and its high resistance to insecticides and Bacillus thuringiensis toxins.RNA interference(RNAi)is a sequence-specific,endogenous gene silencing mechanism evoked by small RNA molecules that is used as a robust tool for virus and pest control.RNAi has been extensively tested for CPB management by employing various target genes and delivery methods.This article reviews the screening of RNAi target genes,efficient RNAi delivery systems,and factors affecting RNAi efficiency in CPB,which may help understand the mechanisms of RNAi and its application in CPB control strategy.

Influence of Silencing Soluble Epoxide Hydrolase with RNA Interference on Cardiomyocytes Apoptosis Induced By Doxorubicin

In order to investigate the influence of silencing soluble epoxide hydrolase（sEH） with double-stranded small interfering RNA（siRNA） on cardiomyocytes apoptosis induced by doxorubicin（DOX）,two plasmids containing siRNA sequences specific to sEH were constructed and transfected into the primary cultured cardiomyocytes by using FuGENE HD transfection agents.The mRNA and protein expression levels of sEH were detected by semiquantitative RT-PCR and Western blotting respectively,and the plasmids that silenced sEH most significantly were selected,and renamed EH-R.The plasmids carrying a nonspecific siRNA coding sequence（PCN） served as the negative control.Cardiomyocytes were divided into four groups：control group,DOX group,PCN＋DOX group,and EH-R＋DOX group.Apoptosis of cardiomyocytes was induced by DOX at a concentration of 1 μmol/L.Apoptosis rate of cardiomyocytes was determined by flow cytometery.The protein expression levels of Bcl-2 and Bax were detected by Western blotting.The results showed that the expression of sEH was down-regulated by EH-R plasmid.The expression levels of sEH mRNA and protein in the EH-R＋DOX group were significantly decreased as compared with other groups（P0.01）.As compared with the control group,the apoptosis rate of cardiomyocytes in three DOX-treated groups was obviously increased,the expression levels of Bax increased,and those of Bcl-2 decreased（P0.01）.However,the expression levels of Bax were decreased,those of Bcl-2 increased and the apoptosis rate of cardiomyocytes obvi-ously decreased in EH-R＋DOX group when compared with those in the DOX group and the PCN＋DOX group（P0.01 for each）.It was concluded that the recombinant plasmids could be successfully constructed,and transfected into the primary cultured cardiomyocytes.They could ameliorate the DOX-induced cardiomyocytes apoptosis by selectively inhibiting the expression of sEH with RNAi and increasing the expression of Bcl-2.

Effect of RNA Interference Hsp72 Gene Expression on Development of Mouse Preimplantation Embryos

The method of RNAi was used to inhibit the expression of induced heat shock protein 70 （Hsp72） in the 4-cell stage mouse embryos and the embryo development competence was analyzed to identify the functions of Hsp72 on embryonic heat resistance. The results indicated that the inhibition rates of siRNA1 for Hsp72 mRNA and Hsp72 protein were 87.1 and 78.5%, respectively. The blastocysts development rates were 41, 86, and 84% for the siRNA1 group, the LipofectamineTM 2 000 exposed group, and the 37℃ group, respectively, and the hatched blastocysts development rates for the above three groups were 35, 72, and 68%, respectively. The data suggest that the siRNAI has a significant inhibiting effect on Hsp72 gene, and Hsp72 gene silence reduces the blastocysts development rate and hatched blastocysts rate after heat shock during the development of mouse preimplantation embryos.

RNA interference blocking the apoptosis in HEK293 cells induced by overexpression of alpha-synuclein

BACKGROUND： Overexpression of α-synuclein can induce cell apoptosis. RNA interference （RNAi） may block specific gene function and cause gene silencing. OBJECTIVE： To construct a specific and effective RNAi plasmid for the α-synuclein gene and investigate if RNAi can block apoptosis in HEK293 cells, induced by overexpression of wild-type α-synuclein. DESIGN, TIME AND SETTING： A contrast experiment based on genetically engineered cytobiology was performed at the State Key Lab of Medical Genetics of China, Xiangya Medical College of Central South University, between October 2004 and October 2008. MATERIALS： HEK293 cells and pBSHH1 plasmid were provided by the State Key Lab of Medical Genetics of China; OligDNA sequence by Sagon Bioengineering Company, Shanghai; Lipofectamine 2000 by Invitrogen, USA; α-synuclein monoclonal antibody, Hoechst 33258, and MTT by Sigma, USA; Horseradish peroxidase-coupled goat anti-rat IgG by KPL, USA; FACSan flow cytometry by BD, USA. METHODS： Four target sites were used to construct hairpin RNA pBSHH1 vectors - pSYNi-1, pSYNi-2, pSYNi-3 and pSYNi-4 - which were cloned in the pBSHH1 plasmid. HEK293 cells were transfected using Lipofectamine 2000. In addition, a non-transfect group and a negative plasmid transfect group were established. The cultured HEK293 cells were processed as follows： transfection of blank plasmid （blank control group）, transfection of α-synuclein-pEGFP and RNAi negative vector （negative control group）, and transfection of α-synuclein-pEGFP and pSYNi-1 （transfection group）. Cells in all groups were transfected with Lipofectamine 2000 for 48 hours. MAIN OUTCOME MEASURES： Expression of α-synuclein mRNA and protein were detected by RT-PCR and Western blot. Cell morphology was observed under an inverted fluorescence microscope; cell viability was measured using MTT method; and cell apoptosis was determined with Annexin V-PE flow cytometry. RESULTS： α-synuclein mRNA and protein expressions were significantly decreased in the pSYNi-1 group when compared with the non-transfect and negative plasmid transfect groups （P 〈 0.05）. The expressions were partially decreased in the pSYNi-2 group, but there was no significant difference in the pSYNi-3 and pSYNi-4 groups. Hoechst staining indicated that cell nuclei were enlarged in the negative control group, coloring was not uniform, and chromatin was accumulated and appeared spot-like. The nucleus coloring was uniform in the transfection group compared to negative control group. Cell viability in the negative control group was significantly lower than blank control group with cell apoptosis being significantly increased （P 〈 0.05）. In comparison with negative control group, cell viability was significantly increased in the transfection group and cell apoptosis was significantly decreased （P 〈 0.05）. CONCLUSION： pSYNi-1 can inhibit α-synuclein gene expression and block apoptosis of HEK293 cells induced by overexpression of wild-type α-synuclein.

Effects of RNA Interference Combined with Ultrasonic Irradiation and SonoV ue Microbubbles on Expression of STAT3 Gene in Keratinocytes of Psoriatic Lesions

The most effective sequence of small interfering RNA（si RNA） silencing STAT3 of psoriatic keratinocytes（KCs） was screened out,and the effects of the most effective si RNA combined with ultrasonic irradiation and Sono Vue microbubbles on the expression of STAT3 of KCs and the dose-and time-response were investigated.Three chemically-synthetic si RNAs targeting STAT3 carried by Lipofectamine 3000 were transfected into KCs,and the effects on STAT3 expression were detected,then the most effective si RNA was selected for the subsequent experiments.The negative controls of siR NA（si RNA-NC） labeled with Cy3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and Sono Vue microbubbles were transfected into KCs,then the optimal parameters of ultrasonic irradiation were determined.The most effective si RNA carried by Lipofectamine 3000 combined with ultrasonic irradiation at the optimal parameters and Sono Vue microbubbles was transfected into KCs,and the dose-and time-response of RNA interference was determined.The effect of RNA interference by the most effective si RNA at the optimal time and dose carried by Lipofectamine 3000 combined with ultrasonic irradiation and Sono Vue microbubbles（LUS group） was compared with that only carried by Lipofectamine 3000（L group）.The results showed that si RNA-3 achieved the highest silencing efficacy.0.5 W/cm2 and 30 s were selected as the parameters of ultrasonic irradiation.The si RNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and Sono Vue microbubbles could effectively knock down the STAT3 expression at m RNA and protein levels in dose-and time-dependent manners determined at 100 nmol/L with maximum downregulation on m RNA at 48 h,and on protein at 72 h after transfection.The LUS group achieved the highest silencing efficacy.It was concluded that si RNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoV ue microbubbles could effectively knock down the STAT3 expression in psoriatic KCs,and the optimized transfection condition and the sequence of si RNA-3 could serve for further research on gene therapy of psoriasis.

Adenovirus-mediated short hairpin RNA interference against p75 neurotrophin receptor in pheochromocytoma cells

Previous studies have confirmed that motor neuron apoptosis in the anterior horn of the lumbosacral spinal cord is positively correlated with p75 neurotrophin receptor （p75NTR） expression in rat models of cauda equina syndrome. This study used adenovirus to carry a short hairpin RNA （shRNA） for p75NTR gene silencing, to reduce p75NTR expression in the damaged phase and to decrease motor neuron apoptosis. Three p75 siRNA template oligonucleotide segments （shRNA） were designed, and cloned into the 1.0 CMV shuttle vector. HEK293 cells were cotransfected with shuttle vector （carrying shRNA） and an adenovirus vector framework expressing enhanced green fluorescent protein. Thus, this study successfully obtained adenovirus carrying p75shRNA. The obtained viruses were named Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3. The recombinant adenoviruses were separately used to infect cultured pheochromocytoma cells （PC12）. Forty-eight hours later, p75NTR mRNA and total protein were analyzed from the PC12 cells. Compared with the negative controls, RNA interference rates were separately 98.49 ± 0.68%, 95.08 ± 1.79% and 96.60 ± 1.14% at the mRNA level, and 72.89 ± 2.17%, 58.83 ± 1.15% and 59.88 ± 0.44% at the protein level in the Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3 groups, respectively. Thus, recombinant adenovirus shRNA-mediated gene silencing successfully suppressed p75NTR expression.

RNA interference targeting ω-secalin genes differentially affects the processing quality in a wheat T1BL·1RS translocation line

Wheat-rye T1BL·1RS translocation lines are widely used,especially in China,but their processing quality is generally poor.An interfering expression vector targeting theω-secalin genes was constructed with the 1Bx7 seed-specific promoter.Biolistic-mediated genetic transformation of the wheat cultivar KN199 carrying the T1BL·1RS translocation generated 10 transgenic lines.Two representative transgenic lines,8-2 and 13-7,were selected for analysis.Compared with the control,the two transformants showed an up to 4.5-fold decrease in totalω-secalins and various levels of decrease inω-gliadins,γ-gliadins,and low-molecular-weight glutenins.A decrease in high molecular weight(HMW)glutenin 1Bx7 was detected only in 8-2,owing possibly to promoter methylation.Increased levels ofα-gliadins were observed in both transformants,but increased levels of HMW glutenins were observed only in 13-7.Line 13-7 showed increases in gluten index,Zeleny sedimentation value,stabilization time,and maximum resistance.Its bread volume was 849.6 mL,an 11.9%increase over that of the control.Line 8-2 showed decreases in these parameters,but its total cake-making quality score was 88,an 17.3%increase over that of the control.The study demonstrates that the same RNAi construct may produce different effects on wheat processing quality and highlights the influence of the vector promoter in RNA interference.

INFLUENCE OF p53 SMALL DOUBLE STRANDED RNA INTERFERENCE ON HEPATOMA CELL LINE SK-HEP-1

Objective: The effects on cell-cycle and p53 expression in hepatoma cell line SK-Hep-1 were explored by transfecting exogenous p53 small double stranded RNA (dsRNA) into the SK-Hep-1 cells. Methods: p53 dsRNA and EGFP dsRNA were synthesized. SK-Hep-1 (wtp53) cell line was transfected with 200 ng and 400 ng p53 dsRNA or EGFP and EGFP+EGFP dsRNA (as positive control) or 9% NaCl (as blank control) by liposome transfection technique. Flow cytometry was adopted to measure the effects of p53 dsRNA on cell cycle. Expression of p53 protein was detected by Western-Blotting at 48 h after transfecting p53 dsRNA. Results: The number of G0-G1 phase SK-Hep-1 cells, which were transfected with 200 ng p53 dsRNA, was decreased by 52.53% comparing with the control, and decreased by 50.29% (P<0.05) comparing with the positive control cells transfected with same dosage of EGFP+EGFP dsRNA. The number of S phase cells, which were transfected with 200 ng p53 dsRNA, was increased by 146.8% comparing with the control, and increased by 128.62% (P<0.05) comparing with the positive control cells transfected with same dosage of EGFP+EGFP dsRNA. The number of G2-M phase cells, which were transfected with 200 ng p53 dsRNA, was increased by 30.56% (P<0.05) comparing with the control, and increased by 21.63% (P>0.05) comparing with the positive control cells transfected with same dosage of EGFP+EGFP dsRNA. After 48 h, p53 protein expression was not detected in the SK-Hep-1 cells transfected with p53 dsRNA. Conclusion: p53 dsRNA can obviously improve the proliferation of SK-Hep-1 cells, and suppress p53 protein expression of SK-Hep-1 cells, the former may be related to of the latter.

Expression Silence of DNA Repair Gene hMGMT Induced by RNA Interference

Objective： MGMT protein expression has been associated with tumor resistance to alkylating agents. The objective of this paper is to construct the RNA interference vector which can specifically induce the expression silence of human DNA repair gene hMGMT. Methods： The hMGMT specific siRNA expression cassette was made by two steps PCR, linked with pUCI 9 to get pU6-MGMTi, co-transfected with pEGFP-CI into 16HBE and screened by G418. The MGMT mRNA and protein levels were detected by RT-PCR and Western Blot respectively. Results： hMGMT specific RNA interfere vector pU6-MGMTi was constructed successfully. In transfected 16HBE cells MGMT mRNA level could hardly be detected and the protein level was only 10% of control. Conclusion： MGMT specific RNAi expression cassette can effectively inhibit MGMT expression. MGMT silence cell line was built by co-transfection technology, which offered condition for studying the gene function of MGMT.

Effect of RNA interference on WD101 gene of Schistosoma japonicum

Objective: To study the effect of RNA interference(RNAi) on WD101 gene and its effect on the expression of WD101 mRNA and protein in Schistosoma japonicum.Methods: Doublestranded RNA(dsRNA) WD101 gene and control gene(lacZ) were generated by in vitrotranscription and transfected into mechanically transformed schistosomula.The total RNA and protein were isolated simultaneously using TRIzol reagent.The expression levels of mRNA and the protein were determined by quantitative real-time PCR(qPCR) and Western blotting, respectively.After injected dsRNA-electroporated schistosomula into BALB/c mouse six weeks, the male and female reproductive organs were observed and measured under the confocal laser scanning microscope.Results: After 1, 3 and 5 d of RNAi, WD101 mRNA level was decreased by 15%, 39%, and 58% in experiment group compared to that in control group; meanwhile, WD101 protein level was decreased by 11%, 28%, and 43% in experiment group compared to that in control group.There were significantly more sperms in testicular lobes in experiment group than that in control group, while there were no significant differences in terms of ovary and vitelline glands between two groups.Conclusions: The ds WD101-RNAi can effectively induce suppression of WD101 gene expression at both mRNA and protein levels.WD101 gene might be a reproduction-related gene in Schistosoma japonicum.