Plasmid-based Short Hairpin RNA Research Articles

Carbon-based Nanomaterials for delivery of small RNA molecules: a focus on potential cancer treatment applications.

Nucleic acid-mediated therapy holds immense potential in the treatment of recalcitrant human diseases such as cancer. This is underscored by advances in understanding the mechanisms of gene regulation. In particular, the endogenous protective mechanism of gene silencing known as RNA interference (RNAi) has been extensively exploited. We review here the developments from 2011 to 2021, in the use of nanographene oxide, carbon nanotubes, fullerenes, carbon nanohorns, carbon nanodots and nanodiamonds for the delivery of therapeutic small RNA molecules. Appropriately designed effector molecules such as small interfering RNA (siRNA), can, in theory, silence the expression of any disease-causing gene. Alternatively, siRNA can be generated in vivo through the introduction of plasmid-based short hairpin RNA (shRNA) expression vectors. Other small RNAs such as micro RNA (miRNA) also function in post-transcriptional gene regulation and are aberrantly expressed under disease conditions. The miRNA-based therapy involves either restoration of miRNA function through the introduction of miRNA mimics; or the inhibition of miRNA function by delivering anti-miRNA oligomers. However, the large size, hydrophilicity, negative charge and nuclease-sensitivity of nucleic acids necessitate an appropriate carrier for their introduction as medicine into cells. While numerous organic and inorganic materials have been investigated for this purpose, the perfect carrier agent remains elusive. In recent years, carbon-based nanomaterials have received widespread attention in biotechnology due to their tunable surface characteristics, mechanical, electrical, optical and chemical properties.

Targeting Pulmonary Artery Infusion of Nuclear-Targeted Plasmid-Based Short Hairpin RNA (ShRNA) to Hypoxia Inducible Factor-1α3 (pshHIF-1α3) Nano-Microspheres for Treatment of Implanted Lung Cancer in Rats.

The pshHIF-1α3 stealth nanospheres have been studied if they have the function of arterial targeted drug delivery to provide a new arterial targeted drug delivery method for interventional therapy of lung cancer. The study is also aimed at exploring therapeutic effect of the checked drug delivery on lung cancer. The tested groups were designed as follows: Group I: blank control group (pulmonary artery perfusion of 0.5 mL 0.9% saline); group II: tail vein injection of pshHIF-1α3 nano-microsphere; group III: pshHIF-1α3 nano-microsphere pulmonary artery perfusion group. In vitro experiment assessed the effects of pulmonary artery perfusion of pshHIF-1α3 nanospheres on proliferation, apoptosis and colony forming ability of lung cancer A549 cells, which were all evaluated by using MTT method, flow cytometry and colony formation experiments, respectively. In vivo experiment tumor xenotransplantation was used to observe the effect of pulmonary artery perfusion of pshHIF-1α3 nanospheres on treatment of lung cancer. Both the In vivo pulmonary artery perfusion experiment and In vitro experiments in A549 cells confirmed that the pulmonary artery perfusion of pshHIF-1α3 nano-microspheres can inhibit the proliferation of lung cancer tissues and cells, promoting apoptosis and inhibiting migration, leading to enhanced therapeutic effect of lung cancer. One of characteristics of nanomaterials is their large surface area, high dispersion, specific adhesion, tumor-specific affinity and adhesion, thereby prolonging their circulation time in the body. Through aggregation of nanodrug delivery system in tumor cells, the local concentration of the drug is increased, thereby improving selectivity of chemotherapeutic drugs. The results from this study therefore suggest that pulmonary artery perfusion of pshHIF-1α3 may be used in arterial targeted drug delivery for treatment of lung cancer, providing a new and efficient targeted drug delivery arterial route for interventional therapy of lung cancer.

Experimental study on nuclear-targeted plasmid-based short hairpin RNA (ShRNA) to hypoxia inducible factor-1α (PshHIF-1α) PshHIF-1α nano-drug carrier system for breast cancer treatment in rats

This study was aimed at exploring the regulatory mechanism of auclear-targeted pshHIF-1α nano-drug carrier system (NPNCS) in the treatment of breast cancer in rats. MDA-MB-231 cell was cultured and DJ-1 and PTEN mRNA level was detected by qRT-PCR along with analysis of cell viability by CCK-8 and apoptosis by means of flow cytometry. The rats were assigned into control group, nuclear-targeted pshHIF-1α nanopharmaceutical system treatment group, and nuclear-targeted pshHIF-1α nano-pharmaceutical system+PTEN inhibitor SF1670 group, followed by analysis of cell proliferation by EdU staining, cell apoptosis by flow cytometry, and the levels of DJ-1, PTEN, and p-AKT. Compared with MDA-MB-231 cells treated with NPNCS, cells without intervention showed decreased PTEN and increased DJ-1 level. NPNCS induced cell apoptosis. Administration of nuclear-targeted pshHIF-1α nano-drug delivery system down-regulated DJ-1 and up-regulated PTEN. Combined treatment with SF1670 promoted p-AKT level and decreased the inhibitory effect of NPNCS on p-AKT level. In conclusion, the nuclear-targeted pshHIF-1α nano-drug delivery system down-regulated DJ-1 and exerted a tumor suppressor and pro-apoptotic effect, which wasrelated to p-AKT phosphorylation and up-regulation of PTEN.

Rosmarinic acid attenuates β-amyloid-induced oxidative stress via Akt/GSK-3β/Fyn-mediated Nrf2 activation in PC12 cells.

Oxidative stress is an important pathogenic factor in Alzheimer's disease (AD). Recently, nuclear factor E2-related factor 2 (Nrf2) has emerged as a master regulator for the endogenous antioxidant response, and thus represents an attractive therapeutic target against AD. The aim of this study is to test the hypothesis that rosmarinic acid (RosA) attenuates amyloid-β (Aβ)-evoked oxidative stress through activating Nrf2-inducible cellular antioxidant defense system. Here, we reported that RosA attenuated Aβ-induced cellular reactive oxygen species (ROS) generation and lipid hydroperoxides (LPO). Interestingly, knockdown of Nrf2 by plasmid-based short hairpin RNA (shRNA) abrogated, at least in part, RosA-mediated neuroprotection in Aβ-challenged PC12 cells. Mechanistically, RosA enhanced the nuclear translocation of Nrf2 and binding to antioxidant response element (ARE) core element but did not induced Nrf2 transcription. Simultaneously, RosA induced a set of Nrf2 downstream target genes encoding phase-II antioxidant enzymes. Furthermore, RosA enhanced protein kinase B (Akt) phosphorylation, glycogen synthase kinase-3β (GSK-3β) phosphorylation at Ser9, and Fyn phosphorylation. Noteworthy, pharmacological inhibition or gene knockdown studies demonstrated that Akt locate upstream of GSK-3β and regulate Nrf2 through Fyn in the context of PC12 cells pre-incubated with RosA following exposed to Aβ. Conversely, the antioxidant effects of RosA could be blocked by Akt inhibitors LY294002, GSK-3β inhibitor LiCl, Nrf2 shRNA, or Fyn shRNA in Aβ-challenged PC12 cells. Consequently, the antioxidant effects of RosA are mediated predominantly by Akt/GSK-3β/Fyn pathway through increased activity of Nrf2. These results suggest, although do not prove, that RosA can be a promising candidate for neuroprotective treatment of AD.

Plasmid-Based Short Hairpin RNA Against Serum Response Factor Attenuated Monocrotaline-Induced Pulmonary Vascular Remodeling in Rats

Plasmid-Based Short Hairpin RNA Against Serum Response Factor Attenuated Monocrotaline-Induced Pulmonary Vascular Remodeling in Rats

Plasmid-Based Short Hairpin RNA Against Connective Tissue Growth Factor Attenuated Monocrotaline-Induced Pulmonary Vascular Remodeling in Rats

Plasmid-Based Short Hairpin RNA Against Connective Tissue Growth Factor Attenuated Monocrotaline-Induced Pulmonary Vascular Remodeling in Rats

Plasmid-based short hairpin RNA against connective tissue growth factor attenuated monocrotaline-induced pulmonary vascular remodeling in rats.

Pulmonary hypertension is a life-threatening medical condition, and a growing body of evidence shows that the expression of connective tissue growth factor (CTGF) is significantly associated with its pathogenesis, making it an attractive therapeutic target. Our earlier work revealed that plasmid-based CTGF-specific short hairpin RNA (shRNA) could attenuate pulmonary artery smooth muscle cell (PASMC) proliferation and pulmonary vascular remodeling in rats exposed to cigarette smoke. In this study, we explored the therapeutic role of this shRNA plasmid in the treatment of monocrotaline-induced pulmonary vascular remodeling in rats, and demonstrated that the upregulation of CTGF in PASMCs following a single injection of monocrotaline could be attenuated by administration of the shRNA. Accordingly, this shRNA was found to repress monocrotaline-induced pulmonary vascular remodeling, as evidenced by its ability to reduce the percentage of muscularized vessels and the wall thickness of pulmonary vessels. We concluded that plasmid-based shRNA against CTGF attenuated pulmonary vascular remodeling in monocrotaline-treated rats. CTGF might be a potential target for the treatment of pulmonary vascular remodeling and pulmonary hypertension.

ShRNA Constructs Targeting IGFBP-3 Alleviate Age Related Erectile Dysfunction in the Rat

We investigated whether injecting shRNA constructs targeting IGFBP-3 in the penis of old rats would improve erectile function. The most validated IGFBP-3 shRNA plasmid vector (pGPU6/GFP/Neo-shIGFBP-3) was prepared and injected in penile corpus cavernosum tissue. A total of 30 old (age 24 months) male Sprague Dawley® rats were randomly divided into 3 groups, including 10 each that received phosphate buffered saline only (100 μl), pGPU6/GFP/Neo-shNC (100 μg) and the most validated plasmid constructs pGPU6/GFP/Neo-shIGFBP-3 (100 μg). At 4 weeks the erectile response was measured as intracavernous pressure. The percent ofsmooth muscle in corpus cavernosum tissue was evaluated. Nitric oxide synthase activity and the cGMP concentration in penile tissue were also analyzed. IGFBP-3 was estimated in penile tissue by Western blot, real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry. pGPU6/GFP/Neo-shIGFBP-3 corrected the impaired erectile response in aged rats compared with the response in those injected with phosphate buffered saline and pGPU6/GFP/Neo-shNC (each p <0.01). The percent of cavernous smooth muscle was increased in the pGPU6/GFP/Neo-shIGFBP-3 group. Nitric oxide synthase activity and the cGMP concentration were also significantly increased in rats treated with pGPU6/GFP/Neo-shIGFBP-3. IGFBP-3 shRNA effectively reduced IGFBP-3 mRNA and protein expression in penile corpus cavernosum tissue. Decreasing IGFBP-3 expression by plasmid expressed shRNA improved erectile function in aged rats. The therapy may modulate smooth muscle integrity and increase the cGMP concentration. This may be a new direction for treating erectile dysfunction in clinical practice.

Endosomal acidification by Na+/H+ exchanger NHE5 regulates TrkA cell-surface targeting and NGF-induced PI3K signaling.

To facilitate polarized vesicular trafficking and signal transduction, neuronal endosomes have evolved sophisticated mechanisms for pH homeostasis. NHE5 is a member of the Na(+)/H(+) exchanger family and is abundantly expressed in neurons and associates with recycling endosomes. Here we show that NHE5 potently acidifies recycling endosomes in PC12 cells. NHE5 depletion by plasmid-based short hairpin RNA significantly reduces cell surface abundance of TrkA, an effect similar to that observed after treatment with the V-ATPase inhibitor bafilomycin. A series of cell-surface biotinylation experiments suggests that anterograde trafficking of TrkA from recycling endosomes to plasma membrane is the likeliest target affected by NHE5 depletion. NHE5 knockdown reduces phosphorylation of Akt and Erk1/2 and impairs neurite outgrowth in response to nerve growth factor (NGF) treatment. Of interest, although both phosphoinositide 3-kinase-Akt and Erk signaling are activated by NGF-TrkA, NGF-induced Akt-phosphorylation appears to be more sensitively affected by perturbed endosomal pH. Furthermore, NHE5 depletion in rat cortical neurons in primary culture also inhibits neurite formation. These results collectively suggest that endosomal pH modulates trafficking of Trk-family receptor tyrosine kinases, neurotrophin signaling, and possibly neuronal differentiation.

Suppression of cyclin D1 by plasmid-based short hairpin RNA ameliorated experimental pulmonary vascular remodeling

Our previous study has demonstrated that a plasmid-based short hairpin RNA (shRNA) against cyclin D1 could attenuate the pulmonary artery smooth muscle cell (PASMC) proliferation and pulmonary vascular remodeling in smoking rats. In this report, we examined the efficiency of this shRNA plasmid in monocrotaline-induced pulmonary vascular remodeling. A single injection of monocrotaline induced pulmonary vascular remodeling and cyclin D1 over-expression in pulmonary vascular smooth muscle. The shRNA successfully suppressed the up-regulation of cyclin D1 in pulmonary vessels of monocrotaline-treated rats. Moreover, this shRNA decreased the percentage of muscularized vessels and the wall thickness of pulmonary vessels. So, we concluded that plasmid-based shRNA against cyclin D1 ameliorated pulmonary vascular remodeling in monocrotaline-treated rats. Cyclin D1 might be a potential target for the therapy of pulmonary vascular remodeling and pulmonary hypertension.

Construction of a Novel Single Double-Conditional shRNA Expression Vector

Silencing of gene expression by RNA interference (RNAi) has become a widely used tool for assessing gene function in a fast and easy manner. An important advance in the RNAi field was the discovery that plasmid-based short hairpin RNA transcription can substitute for synthetic small interference RNAs both in vitro and in vivo. The constitutive knockdown of gene expression by RNAi can limit the scope of experiments, especially if the inhibition of genes leads to cell lethality, which prevents in vivo functional analysis. We have generated double-conditional short hairpin RNA from a single vector that can regulate its own transcription by a combination of tetracycline-inducible and Cre-loxP systems. This vector will be useful for the control of short hairpin RNA expression in a spatially-regulated manner by Cre recombinase as well as a temporally-regulated manner by tetracycline induction. These control features provide for the specific silencing of genes to facilitate functional genetic analysis in mammals in vivo.

Efficient inhibition of intraperitoneal ovarian cancer growth in nude mice by liposomal delivery of short hairpin RNA against STAT3

Signal transducer and activator of transcription 3 (STAT3) plays an important role in the tumor formation, prognosis and chemoresistance of ovarian cancer. Our goal was to investigate the effect of silencing STAT3 on ovarian cancer cell apoptosis, proliferation, angiogenesis and expression of key targets in vitro and in vivo. The ovarian cancer cell lines A2780CP and A2780s were used. STAT3 was knocked down by the plasmid-based short hairpin RNA (shRNA) expression system. In vitro, a colony formation assay and Hoechst staining were used to examine cell proliferation and apoptosis. The expression level of STAT3 and apoptosis-related proteins were determined by Western blot. The A2780CP intraperitoneal model was used to evaluate the effect of shSTAT3 on tumor growth in mice. Proliferation, apoptosis, and angiogenesis in tumor tissues were measured by proliferating cell nuclear antigen, TUNEL and CD31 immunostaining, respectively. Treatment with shSTAT3 resulted in apoptosis and inhibition of cell proliferation in vitro. Western blot analysis demonstrated that shSTAT3 induced the expression of cleaved caspase-3 and reduced the expression of survivin, Bcl-2 and vascular endothelial growth factor. In vivo, the tumor weight was reduced to 13.46% of 5% glucose by shSTAT3/lipoplexes (P < 0.01), accompanied with apoptosis induction (P < 0.01), proliferation inhibition (P < 0.01) and angiogenesis inhibition (P < 0.01). We find that treatment with shSTAT3 inhibits tumor growth in vitro and in vivo by inducing apoptosis and inhibiting cell proliferation. This work should provide the scientific foundation for future investigation of shSTAT3 as a strategy for ovarian cancer gene therapy and the combination of gene therapy with chemotherapy.

Knockdown of connective tissue growth factor by plasmid-based short hairpin RNA prevented pulmonary vascular remodeling in cigarette smoke-exposed rats

Cigarette smoking may contribute to pulmonary hypertension in chronic obstructive pulmonary disease by resulting in pulmonary vascular remodeling that involves pulmonary artery smooth muscle cell proliferation. Connective tissue growth factor (CTGF) is a cysteine-rich peptide implicated in several biological processes such as cell proliferation, survival, and migration. This study investigated the potential role of CTGF in pulmonary vascular remodeling. We constructed a plasmid-based short hairpin RNA (shRNA) to knock down the expression of CTGF in primary cultured rat pulmonary artery smooth muscle cells (rPASMCs) and in rat lung vessels. Rat PASMCs were challenged with cigarette smoke extract (CSE). Rats were exposed to cigarette smoke for 3 months in the absence or in the presence of plasmid-based short hairpin RNA against CTGF which was administrated by tail vein injection. CTGFshRNA significantly prevented CTGF and cyclin D1 expression, arrested cell cycle at G0/G1 phase and suppressed cell proliferation in rPASMCs exposed to CSE. CTGFshRNA administration ameliorated pulmonary vascular remodeling, inhibited cigarette smoke-induced CTGF elevation and reversed the cyclin D1 increase in pulmonary vessels in rats. Collectively, our data demonstrated that plasmid-based shRNA against CTGF attenuated pulmonary vascular remodeling in cigarette smoke-exposed rats.

Novel Prolyl Hydroxylase Domain-2 Short Hairpin RNA Interference Therapy For Diabetic Wound Healing

Introduction: Chronic diabetic wounds, which fail to progress through normal stages of healing, represent a significant health burden. An impaired response to tissue hypoxia is a major factor responsible for poor healing in diabetic wounds. Cellular adaptive responses to hypoxia are mediated by hypoxia-inducible factor (HIF)-1α, and diabetic wounds demonstrate low levels of HIF-1α expression. HIF-1α is naturally degraded by prolyl hydroxylase domain-2 (PHD-2) protein. This study evaluates whether plasmid-based short hairpin RNA (shRNA) interference therapy targeting PHD-2 can be used for treatment of diabetic wounds while monitoring the process noninvasively by molecular imaging.

Plasmid-based short hairpin RNA against cyclin D1 attenuated pulmonary vascular remodeling in smoking rats

Accumulating evidence indicated that smoking might directly induce pulmonary vascular remodeling at the initial stage of chronic obstructive pulmonary disease (COPD). However, the molecular mechanism underlying this process remains poorly understood. To investigate the role of cyclin D1 in pulmonary vascular remodeling, we constructed a plasmid-based short hairpin RNA (shRNA) to knock down the expression of cyclin D1 in smoking rats. Specific shRNA against cyclin D1 significantly prevented the cyclin D1 expression and the cell proliferation in rat pulmonary artery smooth muscle cells (rPASMCs). Furthermore, the plasmid-based shRNA successfully decreased the cyclin D1 protein in intra-pulmonary arteries of smoking rats and subsequently decreased the wall thickness of pulmonary vessels and the percentage of muscularized vessels. We conclude that the plasmid-based shRNA against cyclin D1 gene attenuated pulmonary vascular remodeling in smoking rats. Cyclin D1 might play a critical role in cigarette smoke-induced pulmonary vascular remodeling via regulating rPASMCs proliferation.

A novel short hairpin RNA (shRNA) expression system promotes Sox9-dependent gene silencing

Cartilage development and function are dependent on a temporally integrated program of gene expression. With the advent of RNA interference (RNAi), artificial control of these complex programs becomes a possibility, limited only by the ability to regulate and express the RNAi. Using existing methods for production of RNAi’s, we have constructed a plasmid-based short hairpin RNA (shRNA) expression system under control of the human pol III H1 promoter and supplemented this promoter with DNA binding sites for the cartilage-specific transcription factor Sox9. The resulting shRNA expression system displays robust, Sox9-dependent gene silencing. Dependence on Sox9 expression was confirmed by electrophoretic mobility shift assays. The ability of the system to regulate heterologously expressed Sox9 was demonstrated by Western blot, as a function of both Sox9 to shRNA ratio, as well as time from transfection. This novel expression system supports auto-regulatory gene silencing, providing a tissue-specific feedback mechanism for temporal control of gene expression. Its applications for both basic mechanistic studies and therapeutic purposes should facilitate the design and implementation of innovative tissue engineering strategies.