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.

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.

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.

Construction and Identification of a Vector Expressing RNA Interference Aimed at the Human CyclinD1 Gene and its Expression in Vitro

OBJECTIVE To construct a eukaryotic expression vector for RNA interference of the human cyclinD1 gene, and to detect its interference effect in human ovarian cancer cells （HO-8910）. METHODS Four target gene segments were synthesized and cloned into the pSUPER vector respectively to construct four recombinant eukaryotic expression vectors, pSUPER-C1-4. The four recombinant vectors were identified by enzyme digestion analysis and DNA sequencing. Then HO-8910 cells were transfected with the pSUPER-C1-4 vectors and subjected to G418 selection. In G418-resistant cells, the interference effect was detected by RT-PCR. RESULTS Enzyme digestion analysis and DNA sequencing showed that the target segments were cloned into the pSUPER vector. The four recombinant vectors inhibited transcription of the cyclinD1 gene. The pSUPER-C2 vector had a better interference effect. CONCLUSION The sequence-specific siRNA effectively interfered with expression of the cyclinD1 gene that was selected. The transcription and expression of the cyclinD1 gene were inhibited effectively by the constructed RNAi eukaryotic expression vectors in the ovarian cancer cells. These results indicate that it is possible to search for a new tumor gene therapy method,

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.

Cloning of CRABS CLAW Gene from Brassica napus and Construction of Its RNA Interference Expression Vector

CRABS ClAW （CRC） is a member of the YABBYA transcription factor gene family that plays an important role in floral organ development of plants. This study aimed to further investigate the regulatory function of CRC transcription factor in the development of floral organs of rape （Brassica napus L. ）. A 580 bp fragment of CRC gene was cloned by RT-PCR from total RNA of buds of rape cultivar Ningyou No. 10 to construct an inverted repeated expression cassette of CRC gene using intermediate vector pHturieane. Firstly, CRC gene fragment was positively inserted into the 5＇ end of a spliceable intron and negatively inserted into the 3＇ end of the intron. Subsequently, CaMV35S promoter sequence and inverted repeated expression cassette of CRC gene were transferred into pUC18 multiple clone site of binary expression vector pCAMBIAI1390. The constructed interference expression vector was named pA6-CRCi, which was further confirmed by restriction enzyme digestion and sequencing.

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.

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 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.

Host cellular micro RNA involvement in the control of hepatitis B virus gene expression and replication

A large number of studies have demonstrated that the synergistic collaboration of a number of micro RNAs(mi RNAs), their growth factors and their downstream agents is required for the initiation and completion of pathogenesis in the liver. mi RNAs are thought to exert a profound effect on almost every aspect of liver biology and pathology. Accumulating evidence indicates that several mi RNAs are involved in the hepatitis B virus(HBV) life cycle and infectivity, in addition to HBVassociated liver diseases including fibrosis, cirrhosis and hepatocellular carcinoma(HCC). In turn, HBV can modulate the expression of several cellular mi RNAs, thus promoting a favorable environment for its replication and survival. In this review, we focused on the involvement of host cellular mi RNAs that are directly and indirectly associated with HBV RNA or HBV associated transcription factors. Exploring different facets of the interactions among mi RNA, HBV and HCV infections, and the carcinogenesis and progress of HCC, could facilitate the development of novel and effective treatment approaches for liver disease.

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.

Inhibition of HOXB7 Gene Expression in Melanoma Cells by Small Interfering RNA

Objective： HOXB7 gene is a kind of transcription regulator over-expressed in malignant melanoma （MM） cell lines. It can specifically up-regulate the expression of angiogenic factors and tumor growth factors such as bFGF, GROa, VEGF and induce angiogenesis in melanoma, resulting in the proliferation and metastasis of tumor cells. We designed and synthesized HOXB7 specific siRNA to study its interfering effect on the expressions of HOXB7 and bFGF genes in melanoma A375 cell line and the biologic characteristics of A375 cells. Methods： Three synthesized siRNA with different sequences were separately transfected into A375 cells by lipofecter 2000. The expression of HOXB7 and bFGF mRNA in transfected cells was detected by RT-PCR 24 and 48 hours after transduction. The expression of bFGF protein in the transfected cells were detected by flowcytometry 48 hours after transfection. MTT assay was used to analyze the cell proliferation rate of siRNA transfected cells. Based on the in vitro experiment results, one effective siRNA sequence was selected for the construction of in vivo siRNA expression vector. Then, a malignant melanoma animal model was established. The siRNA expression plasmid was injected into the tumor foci and its influence on the growth and angiogenesis of tumor was observed. Results： The mRNA expressions of both HOXB7 and bFGF genes in the A375 cells reduced significantly 24 and 48 hour after transfection of siRNA. Expression level of the protein of angiogenic factor bFGF induced by HOXB7 gene in siRNA transfected cells was significantly lower than that in control cells 48 hours after transduction. Cell proliferation was also suppressed in siRNA transfected cells. Two of the three siRNA strands showed prominent interference effect. The in vivo study indicated that the tumor size and the microvessel density in the tumor both reduced after injection of HOXB7siRNA plasmid. Conclusion： Down-regulation of HOXB7 gene expression can effectively reduce the expression of angiogenic factor bFGF and the proliferation of MM cells. Besides, the growth and angiogenesis of MM tumor were also inhibited.

Challenges Analyzing RNA-Seq Gene Expression Data

The analysis of messenger Ribonucleic acid obtained through sequencing techniques (RNA-se- quencing) data is very challenging. Once technical difficulties have been sorted, an important choice has to be made during pre-processing: Two different paths can be chosen: Transform RNA- sequencing count data to a continuous variable or continue to work with count data. For each data type, analysis tools have been developed and seem appropriate at first sight, but a deeper analysis of data distribution and structure, are a discussion worth. In this review, open questions regarding RNA-sequencing data nature are discussed and highlighted, indicating important future research topics in statistics that should be addressed for a better analysis of already available and new appearing gene expression data. Moreover, a comparative analysis of RNAseq count and transformed data is presented. This comparison indicates that transforming RNA-seq count data seems appropriate, at least for differential expression detection.

Effects of Suppressing OsCRY1a Gene Expression on Rice Agronomic Traits

Using primers designed according to the published sequence of rice OsCRY1a gene, we obtained part of the gene fragment by PCR and constructed an RNA interference expression vector with it. To down-regulate the expression level of the gene or lead to the loss-of-function of the gene, the vector was then introduced into rice via Agrobacterium-mediated transformation. Based on the performance of the transgenic plants, the functions of the gene were analyzed and deduced. The results indicated that suppressing the expression of the gene retarded flowering for 16 d in rice with the plant height and grain length significantly increasing whereas other important agronomic traits observed remained unchanged apparently.

Experimental study on siRNA expressing vector-based RNA interference targeting c-Myc in human hepatocellular carcinoma cell line BEL-7402

Objective: To explore the inhibition of the expression of c-Myc in human hepatocellular carcinoma via vector-based RNA interference (RNAi). Methods: Two RNA interference DNA templates targeting c-Myc oncogene were designed via online software and synthesized. By ligation, the fragments were inserted into pSilencer 1.0-U6 to construct the recombinant siRNA expressing plasmids. The identified recombinants were introduced into BEL-7402 cells with Lipofactamine. The inhibition of c-Myc expression, together with the expression of CDK4, hTERT and Gadd45β in c-Myc down-regulated BEL-7402 cells, were analyzed by semi-quantitative RT-PCR. Results: Two recombinant plasmids pSic-myc-1 and pSic-myc-2, which direct the yields of siRNAs targeting c-Myc in cells, were constructed. Among which, pSic-myc-2 was shown to trigger a RNAi-mediated inhibition of expression of c-Myc in BEL-7402 by up to 90%. In c-Myc knockdown BEL-7402 cells, the expression of CDK4 and hTERT were down-regulated with a ratio of 85% and 57%, respectively, while the expression of Gadd45β was up-regulated by up to 110%. Conclusion: The expression of c-Myc in BEL-7402 could be suppressed by vector-based RNA interference successfully. The knockdown of c-Myc in turn resulted in the changes of expression of genes related to cell proliferation and apoptosis. Thus, our study provided a preliminary data in searching of a c-Myc-targeted RNAi therapy of human hepatocellular carcinoma.

Adhesion molecule and proinflammatory cytokine gene expression in hepatic sinusoidal endothelial cells following cecal ligation and puncture

INTRODUCTIONMultiple organ dysfunction syndrome (MODS) isthought to be a frequent consequence of sepsis[1-3].Despite substantial advances in our knowledge and understanding of the basic pathophysiologic mechanisms[4-7], in critically ill patients infections and sepsis are still associated with a high mortality[8,9].

Inhibiting effect of antisense oligonucleotides phosphorthioate on gene expression of TIMP-1 in rat liver fibrosis

AIM: To observe the inhibition of antisense oligonucleotides (asON) phosphorthioate to the tissue inhibitors metalloproteinase-1 (TIMP-1) gene and protein expression in the liver tissue of immunologically induced hepatic fibrosis rats. The possibility of reversing hepatic fibrosis through gene therapy was observed. METHODS: Human serum albumin (HSA) was used to attack rats, as hepatic fibrosis model, in which asONs were used to block the gene and protein expressing TIMP-1. According to the analysis of modulator, structure protein, coding series of TIMP-1 genome, we designed four different asONs. These asONs were injected into the hepatic fibrosis models through coccygeal vein. The results was observed by RT-PCR for measuring TIMP-1 mRNA expression, immunohistochemistry and in situ hybridization for collagen I, II, special staining of collagen fiber, and electron microscopic examination. RESULTS: Hepatic fibrosis could last within 363 days in our modified model. The expressing level of TIMP-1 was high during hepatic fibrosis process. It has been proved by the immunohistochemical and the electron microscopic examination that the asON phosphorthioate of TIMP-1 could exactly express in vivo. The effect of colchicine was demonstrated to inhibit the expressing level of mRNA and the content of collagen I, III in the liver of experimental hepatic fibrosis rats. However, the electron microscopy research and the pathologic grading of hepatic fibrosis showed that there was no significant difference between the treatment group and the model group (P】 0.05). CONCLUSION: The experimental rat model of hepatic fibrosis is one of the preferable models to estimate the curative effect of anti-hepatic fibrosis drugs. The asON phosphorthioate of TIMP-1 could block the gene and protein expression of TIMP-1 in the liver of experimental hepatic fibrosis rats at the mRNA level. It is possible to reverse hepatic fibrosis, and it is expected to study a new drug of antihepatic fibrosis on the genetic level. Colchicine has very limited therapeutic effect on hepatic fibrosis, furthermore, its toxicity and side effects are obvious.

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.

The Influence of Aerial Exposure on Sea Anemones Aulactinia veratra Mucin Genes Expression Using the RNA Sequencing

Mucin genes are the main component of mucus. The sea anemone species, Aulactinia veratra (Phylum Cnidaria) contains different types of mucin genes. In the intertidal zone, A. veratra is found to be exposed to air during the low tide and produces large quantities of mucus as an external covering. The relation between low tide and mucus secretion is still unclear, and what is the role of mucin during arial exposure is not yet investigated. This study hypothesised that the mucin genes in A. veratra would have significantly high expression in response to aerial exposure. Therefore, the aim of current study was to examine and analyses the response of A. veratra mucins in response to an experiment involving three hours of aerial exposure. To achieve this, aim the RNA-sequencing and bioinformatics analyses were used to examine the expression profile of A. veratra mucin genes in response to aerial exposure. The generated results have shown that, Mucin4-like and mucin5B-like were up-regulated in response to the three hours of aerial exposure in A. veratra. This finding shows a significant role of mucin5B-like and mucin4-like genes in response to air stress at low tide. The data generated from this study could be used in conjunction with future mucin gene studies of sea anemones and other cnidarians to compare A. veratra mucin gene expression results across time, and to extend our understanding of mucin stress response in this phylum.