Short Hairpin RNA Expression Research Articles

Expression of exogenous proteins and short hairpin RNAs in human primary thyrocytes

Recently, it has been shown that commercial human thyroid lines were in fact derived from colon, mammary carcinoma, or melanoma. Others have demonstrated the absence of a common pattern of gene expression between available thyroid cancer cell lines and tumors from patients. Thus, it is important to use several primary cells with a common pathological origin to achieve reproducible results, and it is necessary to find common methods for manipulation of protein expression in such various cultures. We have standardized a transfection method for efficient expression of exogenous proteins in human primary thyroid cultures. We compared lipid-based techniques with three electroporation systems (Electroporator PulseAgile [PA]-4000, Microporator MP-100, and Nucleofector II). Nucleofection was unquestionably the most efficient even for promoter regulation studies, and it was effective in cultures from different origins as normal thyroid, papillary carcinoma, or lymphoid node metastasis. We also standardized, through lentiviral infection, the short hairpin RNA downregulation of protein expression generating human thyrocytes with low levels of p27KIP1 as a model system.

Connective Tissue Growth Factor Knockdown Attenuated Matrix Protein Production and Vascular Endothelial Growth Factor Expression Induced by Transforming Growth Factor‐β1 in Cultured Human Peritoneal Mesothelial Cells

Connective tissue growth factor (CTGF), a downstream mediator of transforming growth factor-beta1 (TGF-beta1) inducing fibrosis, has recently been implicated in peritoneal fibrosis. Extracellular matrix (ECM) accumulation and angiogenesis are characteristic changes in peritoneal fibrosis. In this study we investigated the effect of CTGF knockdown via interference RNA (RNAi) on ECM production and vascular endothelial growth factor (VEGF) expression induced by TGF-beta1 in human peritoneal mesothelial cells (HPMCs). Four CTGF short hairpin RNA (shRNA) expression constructs were generated using the pRetroSuper vector, and infectious retroviral particles were prepared to infect HPMCs. Expression levels of CTGF, fibronectin(FN), collagen 1 (col 1), laminin, and VEGF mRNA and protein were measured by semi-quantitative reverse transcription polymerase chain reaction and western blot assay. CTGF expression was increased after stimulation with TGF-beta1, but inhibited using each of the four independent CTGF shRNA constructs (P < 0.01). Moreover, expression of ECM proteins (FN, col 1, and laminin) and VEGF were upregulated after incubation with TGF-beta1, but elevated levels of ECM and VEGF induced by TGF-beta1 were significantly inhibited by RNAi (P < 0.01), but not by the empty retroviral vector (P > 0.05). From these results, we concluded that retrovirus-mediated CTGF shRNA can effectively inhibit ECM production and VEGF expression induced by TGF-beta1 in HPMCs. This study suggests that downregulation of CTGF may represent a potential therapeutic approach for peritoneal fibrosis through decreasing ECM accumulation and angiogenesis.

ShRNA Expression Constructs Designed Directly from siRNA Oligonucleotide Sequences

Gene silencing by RNA interference (RNAi) is a widely used approach for target-specific knockdown of gene expression. Induction of RNAi in mammalian cells can be achieved by introduction of synthetic small interfering RNA (siRNA) resulting in transient knockdown, or alternatively by stable expression of short hairpin RNA (shRNA). Several algorithms for efficient siRNA design exist, but recent reports have suggested that these cannot be directly used to design efficient shRNAs. In this study, 25 siRNAs targeting independent sequences in five transcripts were used for the construction of shRNA cassettes. Both the siRNAs and shRNA constructs were transfected into HEK293T cells. Quantitative real-time PCR analysis revealed that 19 out of the 25 shRNA constructs reduced the average expression level to less than 30%. Our data support that sequences designed by siRNA algorithms efficiently reduce the expression of the target gene when converted into shRNA expression constructs.

Dickkopf‐1 is overexpressed in human pancreatic ductal adenocarcinoma cells and is involved in invasive growth

The protein products of the Dickkopf (DKK) genes are antagonists of Wnt glycoproteins, which participate in tumor development and progression by binding to frizzled receptors. In this study, the expression of DKK-1 was analyzed in a panel of 43 human cultured carcinoma cell lines. DKK-1 expression was consistently and significantly upregulated in pancreatic carcinoma cell lines. Low level of DKK-3 expression was also seen. In contrast, the expression of DKK-2 and -4 was not detectable in most pancreatic carcinoma cell lines. The overexpression of DKK-1 was confirmed in surgically resected human pancreatic cancer tissues, in which the mRNA level was evaluated in paired samples from cancerous and noncancerous pancreatic tissues. In ductal adenocarcinomas (23 cases), DKK-1 mRNA levels were significantly upregulated compared to corresponding noncancerous tissues in a statistically significant level. To test the biological role of DKK-1 in pancreatic carcinoma cells, we performed a knockdown of DKK-1 in SUIT-2 human pancreatic adenocarcinoma cell line and S2-CP8, its metastatic subline, using a retroviral short hairpin RNA expression vector. DKK-1 knockdown resulted in reduced migratory activity of SUIT-2 in vitro. The in vitro growth rate and Matrigel invasion were also suppressed by DKK-1 knockdown in S2-CP8 cells. Collectively, the evidence suggests that, despite of its presumed antagonistic role in Wnt signaling, DKK-1 may have a role in the aggressiveness of pancreatic carcinoma cells and could, therefore, serve as a novel biomarker of pancreatic cancer.

Aspartate racemase, generating neuronal D-aspartate, regulates adult neurogenesis

D-aspartic acid is abundant in the developing brain. We have identified and cloned mammalian aspartate racemase (DR), which converts L-aspartate to D-aspartate and colocalizes with D-aspartate in the brain and neuroendocrine tissues. Depletion of DR by retrovirus-mediated expression of short-hairpin RNA in newborn neurons of the adult hippocampus elicits profound defects in the dendritic development and survival of newborn neurons and survival. Because D-aspartate is a potential endogenous ligand for NMDA receptors, the loss of which elicits a phenotype resembling DR depletion, D-aspartate may function as a modulator of adult neurogenesis.

Effect of short-hairpin RNA expression vector-mediated osteopontin RNA interference on proliferation and invasion of prostate cancer PC-3 cells

Studies showed that osteopontin (OPN) regulates cell migration and invasion in a variety of cancers, which associates with the activities of matrix metalloproteinase (MMP)-2 and MMP-9. This study was to investigate the role of OPN in the proliferation and invasion of human prostate cancer PC-3 cells and the possible functions of IgammaB kinase (IKK) in nuclear factor kappa B (NF-kappaB)-mediated signaling pathways. OPN short-hairpin RNA (shRNA) recombinant plasmids were transfected into PC-3 cells and different concentrations of IKK inhibitors were used to inhibit the activities of IKKalpha and IKKbeta. The mRNA and protein expression levers of OPN, MMP-2, and MMP-9 were detected by real-time polymerase chain reaction (PCR) and Western blot. Cell cycle was detected by flow cytometry, cell proliferation by MTT assay, and cell invasion by Transwell chamber assay. Compared with untreated cells, the protein levers of OPN, MMP-2, and MMP-9 in OPN shRNA-transfected PC-3 cells were reduced by 55.22%, 51.71%, and 28.35%, respectively, and the ability of cell migration and invasion were decreased by 45.48% and 51.96%, respectively (P<0.05). Moreover, the inhibition of IKKbeta inhibited the expressions of MMP-2 and MMP-9. A shRNA expression vector-mediated OPN gene silencing can inhibit the malignant biological behaviors of PC-3 cells. IKKbeta may play a crucial role in the OPN-induced activation of MMP-2 and MMP-9 via NF-kappaB-mediated IkappaB/IKKbeta pathways.

Controlled expression of functional miR-122 with a ligand inducible expression system

BackgroundTo study the biological function of miRNAs, and to achieve sustained or conditional gene silencing with siRNAs, systems that allow controlled expression of these small RNAs are desirable. Methods for cell delivery of siRNAs include transient transfection of synthetic siRNAs and expression of siRNAs in the form of short hairpins using constitutive RNA polymerase III promoters. Systems employing constitutive RNA polymerase II promoters have been used to express miRNAs. However, for many experimental systems these methods do not offer sufficient control over expression.ResultsWe present an inducible mammalian expression system that allows for the conditional expression of short hairpin RNAs that are processed in vivo to generate miRNAs or siRNAs. Using modified nuclear receptors in a two hybrid format and a synthetic ligand, the Rheoswitch system allows rapid and reversible induction of mRNA expression. We evaluated the system's properties using miR-122 as a model miRNA. A short hairpin encoding miR-122 cloned into the expression vector was correctly processed to yield mature miRNA upon induction with ligand and the amount of miRNA produced was commensurate with the concentration of ligand. miR-122 produced in this way was capable of silencing both endogenous target genes and appropriately designed reporter genes. Stable cell lines were obtained, resulting in heritable, consistent and reversible expression of miR-122, a significant advantage over transient transfection. Based on these results, obtained with a microRNA we adapted the method to produce a desired siRNA by designing short hairpins that can be accurately and efficiently processed.ConclusionWe established an Inducible expression system with a miR-122 backbone that can be used for functional studies of miRNAs and their targets, in heterologous cells that do not normally express the miRNA. Additionally we demonstrate the feasibility of using the miR-122 backbone to express shRNA with a desired siRNA guide strand for inducible RNAi silencing.

RNA INTERFERENCE-MEDIATED SILENCING OF UBCH10 GENE INHIBITS COLORECTAL CANCER CELL GROWTH IN VITRO AND IN VIVO

1. UbcH10 is the cancer-related E2 ubiquitin-conjugating enzyme, and its overexpression has been demonstrated in a variety of malignancies. The aim of the present study is to silence UbcH10 gene by RNA interference (RNAi) and to observe its inhibitory effect on the colorectal cancer cell growth in vitro and in vivo. 2. We constructed the expression vector pGPU6/GFP/Neo/UbcH10-RNAi (pUbcH10-RNAi), which contained a UbcH10 short hairpin RNA expression cassette. Then the UbcH10 gene silencing cell lines LoVo/UbcH10-RNAi and HT-29/UbcH10-RNAi were established. Reverse transcription-polymerase chain reaction and western blot analysis were used to evaluate the expression of the UbcH10 gene. Cell Counting Kit-8 was used to assess properties of tumour cell growth in vitro. Flow cytometry was used to detect the effect of pUbcH10-RNAi on the cell cycle of colorectal cancer cells. Furthermore, the anti-tumour effects of pUbcH10-RNAi were evaluated in vivo in a nude mouse xenografts model. 3. Results demonstrated that UbcH10 gene expression was significantly decreased in pUbcH10-RNAi treated cells. Colorectal cancer cells growth was markedly suppressed in the pUbcH10-RNAi group compared with control conditions and colorectal cancer cells were arrested in the G2-M phase. In vivo, the downregulation of UbcH10 gene expression by pUbcH10-RNAi also inhibited tumour growth in a nude mice xenograft model. 4. Our study suggests that RNA interference-mediated silencing of UbcH10 gene has anti-tumour activity on colorectal cancer and might have therapeutic potential for the treatment of colorectal cancer.

Baculovirus-Mediated Bispecific Short-Hairpin Small-Interfering RNAs Have Remarkable Ability to Cope With Both Influenza Viruses A and B

Influenza viruses A and B cause widespread infections of the human respiratory tract; however, existing vaccines and drug therapy are of limited value for their treatment. Here, we show that bispecific short-hairpin small-interfering RNA constructs containing an 8-nucleotide intervening spacer, targeted against influenza virus A or influenza virus B, can inhibit the production of both types of virus in infected cell lines. This multiple vector showed remarkable ability to cope with both influenza viruses A and B. Furthermore, the Autographa californica multiple nuclear polyhedrosis virus can infect a range of mammalian cells, facilitating its use as a baculovirus vector for gene delivery into cells. In this study, baculovirus-mediated bispecific short-hairpin RNA expression markedly inhibited both influenza viruses A and B production.

AML Survival and In Vivo Progression Is Dependent On β-Catenin Signaling.

Abstract 2398Poster Board II-375B-catenin is the central effector molecule of the canonical wnt signaling pathway which governs cell fate and differentiation during embryogenesis as well as self-renewal of hematopoietic stem cells. Deregulation of the pathway has been observed in various malignancies including myeloid leukemias where over-expression of β-catenin is an independent adverse prognostic factor. In the present study we examined the functional outcome of stable β-catenin down-regulation through lentivirus-mediated expression of short hairpin RNA (shRNA). Reduction of the β-catenin levels in AML cell lines and patient samples diminished their in vitro proliferation ability without significantly affecting cell viability. In order to study the role of β-catenin in vivo, we transplanted leukemic cell lines with control or reduced levels of β-catenin in NOD/SCID animals and analyzed the engraftment levels in the bone marrow. We observed that while the immediate homing of the cells was not affected by the β-catenin levels, the bone marrow engraftment was directly dependent on its levels. Subsequent examination of bone marrow sections revealed that the reduced engraftment was partly due to the inability of the cells with lower β-catenin levels to dock to the endosteal niches, a finding that was confirmed in competitive repopulation assays with untransduced cells. When we examined the expression levels of adhesion molecules and integrins in engrafted cells in vivo, we observed a significant down-regulation of CD44 expression, a molecule that participates in the interaction of HSCs with the niche. Gene expression analysis of the components of the wnt signaling pathway showed that the pathway is subject to tight transcriptional regulation with minor expression deviations. We did, however, observe an up-regulation in components that participate in the non-canonical wnt signaling pathways such as the WNT5B ligand. Ongoing experiments in normal cord blood CD34+ cells will determine the in vivo role of β-catenin signaling in normal hematopoietic progenitors. In conclusion, our study showed that β-catenin comprises an integral part in the development and progression of AML in vivo, indicating that manipulation of the wnt pathway may hold a therapeutic potential in the management of AML. Disclosures:No relevant conflicts of interest to declare.

Effective generation of iPS cells from CD34 + cord blood cells by inhibition of p53

Cord blood banks provide fully human leukocyte antigen-typed cells, from which a set of standard induced pluripotent stem (iPS) cells for use in allogenic transplantation can be derived. Hence, the ability to generate iPS cells from cord blood cells has the potential to provide a suitable source for clinical transplantation. The aim of this work is to determine the reprogramming methods, culture conditions, and cell fractions that can be used to generate iPS cells from cord blood cells effectively. CD34(+), mononucleated, and derived adherent cells from cord blood were cultured in hematopoietic medium (X-vivo10 containing 50 ng/mL interleukin-6, 50 ng/mL soluble interleukin-6 receptor, 50 ng/mL stem cell factor, 10 ng/mL thrombopoietin, and 20 ng/mL Flit3/4 ligand) 3 days prior to viral infection. Cells were then infected with retroviral constructs driving the expression of OCT3/4, SOX2, Krüppel-like factor 4, c-MYC, and enhanced green fluorescent protein together with or without the p53 knockdown lentiviral construct Shp53 pLKO.1-puro. Infected cells were then cultured for an additional 4 days in hematopoietic culture medium before being transferred onto mouse embryonic fibroblast (MEF) or SNL76/7 feeder cells in human embryonic stem cell medium (Dulbecco's modified Eagle medium/F-12 containing 20% knockout serum replacement, 200 mM L-glutamine, 1% non-essential amino acids (NEAA), 0.1 mM 2-mercaptoethanol, and 4 ng/mL basic fibroblast growth factor). Subsequently, the number of embryonic stem cell-like colonies that emerged in the following 4 weeks was scored. Expression of a number of pluripotency makers were examined by immunochemistry and reverse transcriptase polymerase chain reaction. Finally, the differentiation potential of selected colonies was determined by teratoma formation in severe combined immunodeficient mice and in vitro culture. Repression of p53 expression by the addition of a lentiviral p53 short-hairpin RNA expression vector increased the frequency of formation of iPS-like colonies from 1 (on average) to around 100 per 2 x 10(4) cells when infected cells were grown on SNL feeder cells. iPS cells can be generated easily from CD34(+) cord blood cells through the addition of p53 inhibition to standard reprogramming conditions.

Procaspase 8 and Bax Are Up-regulated by Distinct Pathways in Streptococcal Pyrogenic Exotoxin B-induced Apoptosis

We have previously identified integrin alpha(v)beta(3) and Fas as receptors for the streptococcal pyrogenic exotoxin B (SPE B), and G308S, a mutant of SPE B that binds to Fas only. In the current study we found that after binding to alpha(v)beta(3), SPE B stimulated the tyrosine phosphorylation of JAK2 and STAT1. STAT1 tyrosine phosphorylation was inhibited by a JAK2 inhibitor, AG490, short interfering RNA (siRNA) silencing of JAK2, and anti-alpha(V)beta(3) antibody. AG490 also decreased the binding of tyrosine-phosphorylated STAT1 to the procaspase 8 promoter, decreasing procaspase 8 expression, suggesting that SPE B up-regulates procaspase 8 expression via the JAK2/STAT1 pathway. Alternatively, both SPE B and G308S increased STAT1 phosphorylation at serine 727, which was inhibited by anti-Fas antibody, a p38 inhibitor, SB203580, and siRNA silencing of p38. In addition, SPE B and G308S increased binding of serine-phosphorylated STAT1 to the Bax promoter and Bax expression, which was decreased by SB203580. SPE B and G308S-stimulated Bax expression was also inhibited by anti-Fas antibody. These findings suggest that Fas mediate SPE B-induced Bax expression through p38. Silencing of JAK2 or p38 by siRNA blocked procaspase 8 expression, whereas only p38 siRNA decreased Bax expression. Furthermore, JAK2 inhibition and p38 inhibition reduced SPE B-induced apoptosis, but only p38 inhibition blocked G308S-induced apoptosis.

Farnesoid X Receptor Alleviates Age-Related Proliferation Defects in Regenerating Mouse Livers by Activating Forkhead Box M1b Transcription

Elucidating the mechanism of liver regeneration could lead to life-saving therapy for a large number of patients, especially elderly patients, after segmental liver transplantation or resection of liver tumors. The forkhead box m1b (Foxm1b) transcription factor is required for normal liver regeneration. Here we report that Foxm1b is the first direct farnesoid X receptor (FXR) target gene known to be involved in cell cycle regulation and that aging regenerating livers have delayed activation of FXR, which results in defective induction of Foxm1b and thereby contributes to defective liver regeneration. An inverted repeat 0 (IR-0) FXR response element, acting as an enhancer in intron 3 of the Foxm1b gene, was identified by a combination of transcriptional reporter, electrophoretic mobility shift, and chromatin immunoprecipitation assays. Diminished FXR binding to the IR-0 element was found in aging regenerating livers. FXR activation by a novel ligand in aging livers induced Foxm1b expression and elevated hepatocyte DNA replication to about 70% of the levels found in young regenerating livers, which were specifically suppressed by hepatic expression of anti-Foxm1b short hairpin RNA. Our results have revealed Foxm1b as the first known direct FXR target gene involved in cell cycle regulation and have demonstrated that defective activation of FXR could be an intrinsic defect in aging regenerating livers. Activation of FXR alone is largely able to alleviate age-related liver regeneration defects. These findings highlight FXR as a potential target of drug design for promoting liver regeneration in older subjects.

Hepatitis B virus inhibition in mice by lentiviral vector mediated short hairpin RNA

BackgroundChronic hepatitis B virus (HBV) infection is an important cause of cirrhosis and hepatocellular carcinoma. The major challenges for current therapies are the low efficacy of current drugs and the occurrence of drug resistant HBV mutations. RNA interference (RNAi) of virus-specific genes offers the possibility of developing a new anti-HBV therapy. Recent reports have shown that lentiviral vectors based on HIV-1 are promising gene delivery vehicles due to their ability to integrate transgenes into non-dividing cells. Herein, a lentivirus-based RNAi system was developed to drive expression and delivery of HBV-specific short hairpin RNA (shRNA) in a mouse model for HBV replication.MethodsHepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in the sera of the mice were analyzed by quantitative sandwich enzyme linked immunosorbent assay (ELISA) technique, hepatitis B core antigen (HBcAg) and HBsAg in the livers of the mice were detected by immunohistochemical assay, HBV DNA and HBV mRNA were measured by fluorogenic quantitative polymerase chain reaction (FQ-PCR) and quantitative real-time PCR respectively.ResultsCo-injection of HBV plasmids together with the lentivirus targeting HBV shRNA induced an RNAi response. Secreted HBsAg was reduced by 89% in mouse serum, and HBeAg was also significantly inhibited, immunohistochemical detection of HBcAg or HBsAg in the liver tissues also revealed substantial reduction. Lentiviral mediated shRNA caused a significant suppression in the levels of viral mRNA and DNA synthesis compared to the control group.ConclusionLentivirus-based RNAi can be used to suppress HBV replication in vivo, it might become a potential therapeutic strategy for treating HBV and other viral infections.

Suppression of zebrafish VEGF gene by cytomegalovirus promoter-driven short hairpin constructs induces vascular development defects and down regulation NRP1 expression

The usage of RNA interference for gene knockdown in zebrafish through expression of the small interfering RNA mediators from DNA vectors has created a lot of excitement in the research community. In this work, the ability of human cytomegalovirus immediate early promoter (CMV promoter)-driven short hairpin RNA (shRNA) expression vector to induce shRNA against vascular endothelial growth factor (VEGF) gene in zebrafish was tested, and its effects on VEGF-mediated vasculogenesis and angiogenesis were evaluated. Altogether four vectors targeting various locations of VEGF gene were constructed, and pSI-V4 was proven to be the most effective one. Microinjection of pSI-V4 into the zebrafish embryos resulted in defective vascular formation and down regulation of VEGF expression. In situ hybridization analysis indicated that silencing VEGF gene expression by pSI-V4 resulted in down regulation of neuropilin-1 (NRP1), a potent VEGF receptor. Knockdown of VEGF expression by morpholino gave the same result. This provided evidence that the VEGF-mediated angiogenesis in zebrafish was in part dependent on NRP1 expression. The results contributed to a better understanding of molecular mechanisms of cardiovascular development and provided a potential promoter for making inducible knockdown in zebrafish.

VEGF gene silencing by cytomegalovirus promoter driven ShRNA expression vector results in vascular development defects in zebrafish

Zebrafish has been generally considered as an excellent model in case of drug screening, disease model establishment, and vertebrate embryonic development study. In this work, the ability of human cytomegalovirus immediate early promoter (CMV promoter)-driven short hairpin RNA (shRNA) expression vector to induce shRNA against VEGF gene in zebrafish was tested, and its effect on vascular development was assed, too. Using RT-qPCR, blood vessel staining, and in situ hybridization, we confirmed certain transcriptional activity and down regulation of gene expression by the vector. In situ hybridization analysis indicated selective inhibition of NRP1 expression in the VEGF gene loss of function model, which might imply in turn that VEGF could not only activate endothelial cells directly but also could contribute to stimulating angiogenesis in vivo by a mechanism that involved up-regulation of its cognate receptor expression in zebrafish. This contributed to a better understanding of molecular mechanisms of cardiovascular development. The system improved the success rate in making inducible knockdown and widened the possibilities for better therapeutic targets in zebrafish.

Inhibition of influenza virus by baculovirus-mediated shRNA

Influenza viruses A and B cause widespread infections of the human respiratory tract; however, existing vaccines and drug therapy are of limited value for their treatment. Here we show that bispecific short hairpin small-interfering RNA constructs containing an eight-nucleotide intervening spacer, targeted against influenza virus A or influenza virus B, can inhibit the production of both types of virus in infected cell lines. This multiple vector showed remarkable ability to cope with both influenza virus A or B. Furthermore, the Autographa californica multiple nuclear polyhedrosis virus can infect a range of mammalian cells, facilitating its use as a baculovirus vector for gene delivery into cells. In this study, baculovirus-mediated bispecific short-hairpin RNA expression markedly inhibited both influenza virus A and B production.

Blockade of Protein Phosphatase 2B Activity in the Amygdala Increases Anxiety- and Depression-Like Behaviors in Mice

Organ transplant patients receive chronic administration of the calcineurin inhibitor cyclosporin-A (CsA) and demonstrate increased incidence of mood disorders. Significant calcineurin expression can be observed with immunohistochemistry in the amygdala, a brain area important for behaviors related to mood disorders and anxiety. It is therefore important to determine whether chronic blockade of calcineurin might contribute to symptoms of anxiety and depression in these patients. Pharmacological CsA and viral-mediated gene transfer (adeno-associated viral expression of short hairpin RNA [AAV-shRNA]) approaches were used to inhibit calcineurin activity globally and selectively in the amygdala of the mouse brain to determine the role of calcineurin in behaviors related to depression and anxiety. Systemic inhibition of calcineurin activity with CsA or local downregulation of calcineurin levels in the amygdala with AAV-delivered shRNAs targeting calcineurin A increased behavioral measures of anxiety in both the elevated plus maze and light/dark tests with no changes in locomotor activity. In the forced swim and tail suspension models of depression-like behavior, calcineurin blockade in the amygdala increased immobility similarly to manipulations that lead to a depression-like phenotype. Taken together, these data demonstrate that decreasing calcineurin activity in the amygdala increases anxiety- and depression-like behaviors. These studies suggest that chronic administration of CsA to organ transplant patients could have significant effects on anxiety and mood and that this should be recognized as a clinical consequence of treatment to prevent transplant rejection.

RNAi-mediated inhibition of HIV-1 by targeting partially complementary viral sequences.

Potent antiviral RNAi can be induced by intracellular expression of short hairpin RNAs (shRNAs) and artificial microRNAs (miRNAs). Expression of shRNA and miRNA results in target mRNA degradation (perfect base pairing) or translational repression (partial base pairing). Although efficient inhibition can be obtained, error-prone viruses such as human immunodeficiency virus type 1 (HIV-1) can escape from RNAi-mediated inhibition by mutating the target sequence. Recently, artificial miRNAs have been shown to be potent RNAi inducers due to their efficient processing by the RNAi machinery. Furthermore, miRNAs may be more proficient in suppressing imperfect targets than shRNAs. In this study, we tested the knockdown efficiency of miRNAs and shRNAs against wild-type and RNAi-escape HIV-1 variants with one or two mutations in the target sequence. ShRNAs and miRNAs can significantly inhibit the production of HIV-1 variants with mutated target sequences in the open reading frame. More pronounced mutation-tolerance was measured for targets in the 3′ untranslated region (3′ UTR). Partially complementary sequences within the 3′ UTR of the HIV-1 RNA genome efficiently act as target sites for miRNAs and shRNAs. These data suggest that targeting imperfect target sites by antiviral miRNAs or shRNAs provides an alternative RNAi approach for inhibition of pathogenic viruses.

Lentiviral Delivery of RNAi Effectors Against HIV-1

RNA interference (RNAi) holds great promise as gene therapy approach against viral pathogens, including HIV-1. A specific anti-HIV-1 response can be induced via transfection of synthetic small interfering RNAs (siRNAs) or via intracellular transgene expression of short hairpin RNAs (shRNAs) or microRNAs (miRNAs). Both targeting of the viral mRNAs or the mRNAs for cellular co-factors that are required for viral replication have been shown successful in suppressing HIV-1 replication. However, like conventional mono-therapies, the use of a single anti-HIV-1 RNAi inducer results in the emergence of RNAi-escape mutants. To prevent viral escape, a combinatorial RNAi approach should be used in which multiple RNAi effectors against HIV-1 are simultaneously expressed. Although induced RNAi is able to trigger a robust and specific knockdown of virus replication, it is becoming apparent that RNAi therapeutics encounter difficulties concerning off-target effects, cellular toxicity and specific delivery to the right cells. This review covers the recent progress in combinatorial RNAi-based approaches against HIV-1 using lentiviral vectors as a delivery system. The potential for a clinical gene therapy application will be discussed.