Specific Gene Inhibition Research Articles

Ruxolitinib-loaded poly-ɛ-caprolactone (PCL) nanoparticles inhibit JAK2/STAT5 signaling in BT474 breast cancer cells by downregulating Bcl-2 and Mcl-1.

JAK/STAT signaling plays an important role in regulating cell proliferation. Reducing proliferation and inducing cell death with gene-specific inhibitors such as ruxolitinib, Receptor tyrosine kinases (RTK) inhibitor targeting JAK1/2, are therapeutic approaches. The use of nanoparticles can reduce the toxicity and side effects of drugs, as they act directly on cancer cells and can selectively increase drug accumulation in tumor cells. Poly-ɛ-caprolactone (PCL) is a polymer that is frequently used in drug development. In this study, Rux-PCL-NPs were synthesized to increase the effectiveness of ruxolitinib. In addition, this study aimed to determine the effect of Rux-PCL-NPs on JAK/STAT signaling and apoptotic cell death. Rux-PCL-NPs were synthesized by nanoprecipitation. The Rux-PCL-NPs had a spherical and mean particle size of 219 ± 88.66nm and a zeta potential of 0.471 ± 0.453 mV. In vitro cytotoxicity and antiproliferative effects were determined by MTT and soft agar colony formation assays, respectively. The effects of ruxolitinib, PCL-NPs, and Rux-PCL-NPs on apoptosis and the JAK/STAT pathway in cells were examined by western blot analysis. PCL-NPs did not have a toxic effect on the cells. The IC50 value of Rux-PCL-NPs was decreased 50-fold compared to that of ruxolitinib. Rux-PCL-NPs promoted cell death by downregulating JAK2 and STAT5, thereby inhibiting the JAK/STAT pathway. Our results revealed that Rux-PCL-NPs, which increased the efficacy of ruxolitinib, regulated apoptosis and the JAK2/STAT5 pathway.

SLC16a6, mTORC1, and Autophagy Regulate Ketone Body Excretion in the Intestinal Cells.

Ketone bodies serve several functions in the intestinal epithelium, such as stem cell maintenance, cell proliferation and differentiation, and cancer growth. Nevertheless, there is limited understanding of the mechanisms governing the regulation of intestinal ketone body concentration. In this study, we elucidated the factors responsible for ketone body production and excretion using shRNA-mediated or pharmacological inhibition of specific genes or functions in the intestinal cells. We revealed that a fasting-mimicked culture medium, which excluded glucose, pyruvate, and glutamine, augmented ketone body production and excretion in the Caco2 and HT29 colorectal cells. This effect was attenuated by glucose or glutamine supplementation. On the other hand, the inhibition of the mammalian target of rapamycin complex1 (mTORC1) recovered a fraction of the excreted ketone bodies. In addition, the pharmacological or shbeclin1-mediated inhibition of autophagy suppressed ketone body excretion. The knockdown of basigin, a transmembrane protein responsible for targeting monocarboxylate transporters (MCTs), such as MCT1 and MCT4, suppressed lactic acid and pyruvic acid excretion but increased ketone body excretion. Finally, we found that MCT7 (SLC16a6) knockdown suppressed ketone body excretion. Our findings indicate that the mTORC1-autophagy axis and MCT7 are potential targets to regulate ketone body excretion from the intestinal epithelium.

Control of aflatoxin biosynthesis by sulfur containing benzimidazole derivatives: In-silico interaction, biological activity, and gene regulation of Aspergillus flavus.

Aspergillus flavus producing aflatoxins is one of the potent contaminants of raw food commodities during pre-and post-harvest crops. Aflatoxins are the group of secondary metabolites a subset of natural polyketides. Our major focus is on the inhibition of the biosynthesis pathway of aflatoxin by targeting the enzymes involved. Benzimidazoles are known antimicrobial compounds. In this study the sulfur containing benzimidazole derivatives were tested for their antifungal and antiaflatoxigenic activity. The fungal growth and aflatoxin production was analysed in culture medium as well as in the rice. Inhibition of specific genes was studied in terms of mRNA expression and the interaction of test compound with polyketide synthases by in-silico molecular docking. Substitution at the 6th position of 2-(2-thienyl) benzimidazole (2-TBD) reduced the antifungal property of benzimidazole but effectively inhibited the aflatoxin synthesis in the culture medium as well as in the rice from the toxigenic strain of A. flavus. Among the derivatives tested, the methyl group containing 2-(2-thienyl)-6-methylbenzimidazole (6-MTBD) inhibited aflatoxin B1 most effectively followed by carboxylic group containing 2-(2-thienyl) benzimidazole-6-carboxylic acid (6-TBCA) with IC50 value of 12.36 and 18.25µg/mL respectively. Molecular docking study shows that 2-(2-thienyl) benzimidazole-6-carbonitrile (6-CTBD) and 6-MTBD occupy same pocket on TE domain of PksA with similar range of binding energy, however the experimental data show a different effect on the biosynthesis of AFB1. 6-MTBD effectively inhibited the AFB1 synthesis (97%) while 6-CTBD could not (39.5%). Data obtained from the expression study also supports the experimental observations. These compounds are non-toxic to mammalian cells. These benzimidazole derivatives inhibit toxic secondary metabolites without affecting the growth of the fungi hence can be used during fermentation to avoid mycotoxin contamination.

Antisense inhibition of RNA polymerase α subunit of Clostridioides difficile.

Clostridioides difficile, the causative agent of antibiotic-associated diarrhea and pseudomembranous colitis, has emerged as a major enteric pathogen in recent years. Antibiotic treatment perturbs the gut microbiome homeostasis, which facilitates the colonization and proliferation of the pathogen in the host intestine. Paradoxically, the clinical repertoire for C. difficile infection includes the antibiotics vancomycin and/or fidaxomicin. The current therapies do not address the perturbed gut microbiome, which supports the recurrence of infection after cessation of antibiotic therapy. Peptide nucleic acids (PNAs) are novel alternatives to traditional antimicrobial therapy capable of forming strong and stable complexes with RNA and DNA, thus permitting targeted inhibition of specific genes. Here, we report a novel PNA that can target the RNA polymerase α subunit (rpoA) in C. difficile. The designed anti-rpoA construct inhibited clinical isolates of C. difficile (minimum inhibitory concentration values ranged between 4 and 8 µM) and exhibited bactericidal activity. Furthermore, silencing of the rpoA gene suppressed the expression of genes that encode virulence factors [toxin A (tcdA), toxin B (tcdB)] in C. difficile, and the gene that encodes the transcription factor stage 0 sporulation protein (spoOA). Interestingly, the efficacy of the designed PNA conjugate remained unaffected even when tested at different pH levels and against a high inoculum of the pathogen. The rpoA-TAT conjugate was very specific against C. difficile and did not inhibit members of the beneficial gut microflora. Taken altogether, our study confirms that the rpoA gene can be a promising narrow-spectrum therapeutic target to curb C. difficile infection. IMPORTANCE The widespread use of antibiotics can destroy beneficial intestinal microflora, opening the door for spores of Clostridioides difficile to run rampant in the digestive system, causing life-threatening diarrhea. Alternative approaches to target this deadly pathogen are urgently needed. We utilized targeted therapeutics called peptide nucleic acids (PNAs) to inhibit gene expression in C. difficile. Inhibition of the RNA polymerase α subunit gene (rpoA) by PNA was found to be lethal for C. difficile and could also disarm its virulence factors. Additionally, antisense inhibition of the C. difficile rpoA gene did not impact healthy microflora. We also propose a novel approach to manipulate gene expression in C. difficile without the need for established genetic tools.

Gene Therapy: Will the Promise of Optimizing Lung Allografts Become Reality?

Lung transplantation is the definitive therapy for patients living with end-stage lung disease. Despite significant progress made in the field, graft survival remains the lowest of all solid organ transplants. Additionally, the lung has among the lowest of organ utilization rates—among eligible donors, only 22% of lungs from multi-organ donors were transplanted in 2019. Novel strategies are needed to rehabilitate marginal organs and improve graft survival. Gene therapy is one promising strategy in optimizing donor allografts. Over-expression or inhibition of specific genes can be achieved to target various pathways of graft injury, including ischemic-reperfusion injuries, humoral or cellular rejection, and chronic lung allograft dysfunction. Experiments in animal models have historically utilized adenovirus-based vectors and the majority of literature in lung transplantation has focused on overexpression of IL-10. Although several strategies were shown to prevent rejection and prolong graft survival in preclinical models, none have led to clinical translation. The past decade has seen a renaissance in the field of gene therapy and two AAV-based in vivo gene therapies are now FDA-approved for clinical use. Concurrently, normothermic ex vivo machine perfusion technology has emerged as an alternative to traditional static cold storage. This preservation method keeps organs physiologically active during storage and thus potentially offers a platform for gene therapy. This review will explore the advantages and disadvantages of various gene therapy modalities, review various candidate genes implicated in various stages of allograft injury and summarize the recent efforts in optimizing donor lungs using gene therapy.

Rapid identification of genome-edited mesenchymal stem cell colonies via Cas9.

Mesenchymal stem cells (MSCs) have been intensively investigated and widely applied in regenerative medicine and immune modulation. However, their efficacy declines during the aging or disease process. Thus, genome-edited MSCs with over-expression or inhibition ofspecific genes hold a great deal ofpromise in terms of theirtherapeutic application. Here we optimized the direct PCR approach for rapid identification of genome-edited MSCs with only ten cells required, which reduces the time and labor to expand the MSC colonies. Combined with our previouslyoptimized guide RNA structure and plasmid construction strategy for Cas9, we successfully identified MSCcolonies over-expressing IL-10 in the AAVS1 locus.

Delivery of BACE1 siRNA mediated by TARBP-BTP fusion protein reduces β-amyloid deposits in a transgenic mouse model of Alzheimer’s disease

Systemic delivery of nucleic acids to the central nervous system (CNS) is a major challenge for the development of RNA interference-based therapeutics due to lack of stability, target specificity, non-permeability to the blood-brain barrier (BBB), and lack of suitable carriers. Using a designed bi-functional fusion protein TARBP-BTP in a complex with siRNA, we earlier demonstrated knockdown of target genes in the brain of both AβPP-PS1 (Alzheimer's disease, AD) and wild-type C57BL/6 mice. In this report, we further substantiate the approach through an extended use in AβPP-PS1 mice, which upon treatment with seven doses of β-secretase AβPP cleaving Enzyme 1 (BACE1) TARBP-BTP:siRNA, led to target-specific effect in the mouse brain. Concomitant gene silencing of BACE1, and consequent reduction in plaque load in the cerebral cortex and hippocampus (greater than 60%) in mice treated with TARBP-BTP:siRNA complex, led to improvement in spatial learning and memory. The study validates the efficiency of TARBP-BTP fusion protein as an efficient mediator of RNAi, giving considerable scope for future intervention in neurodegenerative disorders through the use of short nucleic acids as gene specific inhibitors.

VEGFA Gene Silencing in CXCR4-Expressing Cells via siRNA Delivery by Means of Targeted Peptide Carrier.

Discovery of small interfering RNA as a tool for specific gene inhibition led to the development of new therapeutic strategy for the treatment of cancers. The efficacious delivery of therapeutic siRNAs into the cells is a crucial step in RNA interference (RNAi) application, but it remains challenging. Non-viral vectors can provide specific cellular uptake, stable siRNA complex formation, and intracellular siRNA release. Recently, we evaluated modular peptide carrier L1 bearing CXCR4 targeting ligand for its ability to condense siRNA and facilitate endosomal escape and VEGFA gene silencing in CXCR4-expressing endothelial and glioblastoma cells. The present chapter showcases the ability of L1 targeted peptide carrier to form complexes with siRNA and provide efficient VEGFA gene knockdown. We showed that siRNA delivery by means of L1 peptide carrier can result in significant decrease of VEGFA gene expression in A172 glioblastoma cells and in EA.hy 926 endothelial cells. Also, delivery of anti-VEGFA siRNA/peptide complexes led to significant inhibition of endothelial cell migration. Our results showed that L1 peptide carrier modified with CXCR4 ligand is a promising tool for targeted siRNA delivery into CXCR4-expressing cancer and endothelial cells.

Effects of methyl jasmonate and carotenogenic inhibitors on gene expression and carotenoid accumulation in coriander (Coriandrum sativum L.) foliage

Coriander (Coriandrum sativum L.), a commonly used annual herb that accumulates carotenoids upon methyl jasmonate (MeJA) treatment, provides an excellent model to investigate carotenogenesis and gene regulation. To explore key mechanisms involved in enhancing carotenoids, transcriptional expression profile of ten carotenogenic genes in the presence of MeJA and various gene specific inhibitors were investigated. Foliar application of MeJA (10 μM) increased expression levels of CsPDS (phytoene desaturase), CsZDS (ς-carotene desaturase), CsCHYE (carotene ε - hydroxylase) and CsLCYE (lycopene β-cyclase) genes, and their transcript levels were strongly associated with carotenoid content, where, three days after treatment, 3.9 & 6.1 fold increase was observed for β-carotene and lutein respectively. The regulatory effect of key genes, CsPDS, CsZDS, CsLCYE and LCYB were further confirmed by using gene-specific inhibitors fosmidomycin, norflurazon and amitrol. Norflurazon- the phytoene desaturation inhibitor leads to a decrease in β-carotene and lutein content correlated with CsPDS, CsZDS gene induction. Our results clearly demonstrate that MeJA induced-signalling network evokes carotenogenic genes, leading to the accumulation of carotenoids. This knowledge may help to develop precise strategies for remodelling carotenoid pathway so that desired levels of a particular carotenoid in leafy vegetables is achievable.

Imaging of conditional gene silencing in vivo using a bioluminescence-based method with thermo-inducible microRNAs

RNA interference (RNAi)-based gene therapy has great potential in cancer and infectious disease treatment to correct abnormal up-regulation of gene expression. We show a new original method uses synthetic microRNAs combined with a thermo-inducible promoter to reduce specific gene expression. The targeted gene is the luciferase firefly reporter gene overexpressed in a subcutaneous tumor which allows the RNAi monitoring by bioluminescence imaging (BLI). The inducible inhibition was first demonstrated in vitro using genetically modified cells lines and then in vivo using the corresponding xenograft model in mice. Achieving spatio-temporal control, we demonstrate the feasibility to induce, in vivo, a specific gene inhibition on demand. Future applications of this RNAi-based gene therapy, which can be restricted to pathological tissue, would offer wide-ranging potential for disease treatment.

Impact of DNA and RNA Methylation on Radiobiology and Cancer Progression.

Radiotherapy is a well-established regimen for nearly half the cancer patients worldwide. However, not all cancer patients respond to irradiation treatment, and radioresistance is highly associated with poor prognosis and risk of recurrence. Elucidation of the biological characteristics of radioresistance and development of effective prognostic markers to guide clinical decision making clearly remain an urgent medical requirement. In tumorigenic and radioresistant cancer cell populations, phenotypic switch is observed during the course of irradiation treatment, which is associated with both stable genetic and epigenetic changes. While the importance of epigenetic changes is widely accepted, the irradiation-triggered specific epigenetic alterations at the molecular level are incompletely defined. The present review provides a summary of current studies on the molecular functions of DNA and RNA m6A methylation, the key epigenetic mechanisms involved in regulating the expression of genetic information, in resistance to irradiation and cancer progression. We additionally discuss the effects of DNA methylation and RNA N6-methyladenosine (m6A) of specific genes in cancer progression, recurrence, and radioresistance. As epigenetic alterations could be reversed by drug treatment or inhibition of specific genes, they are also considered potential targets for anticancer therapy and/or radiotherapy sensitizers. The mechanisms of irradiation-induced alterations in DNA and RNA m6A methylation, and ways in which this understanding can be applied clinically, including utilization of methylation patterns as prognostic markers for cancer radiotherapy and their manipulation for anticancer therapy or use as radiotherapy sensitizers, have been further discussed.

CXCR4-targeted modular peptide carriers for efficient anti-VEGF siRNA delivery

The application of small interfering RNA (siRNA) for specific gene inhibition is a promising strategy in gene therapy treatments. The efficient cellular delivery of therapeutic siRNA is a critical step in RNA interference (RNAi) application. Highly efficient siRNA carriers should be developed for specific cellular uptake, stable RNA-complexes formation and intracellular RNA release. To study these features, we evaluated modular peptide carriers bearing CXCR4 targeting ligand for their ability to condense siRNA, facilitate endosomal escape and VEGFA gene silencing in CXCR4-expressing endothelial and glioblastoma cells. Peptide carriers were shown to condense and protect siRNA from RNAse degradation. Various N/P ratios were used for physicochemical characterization to optimize siRNA/peptide complexes for in vitro studies. On average, cytotoxicity of siRNA-polyplexes depended on cell type and was not higher than that of PEI/siRNA complexes. VEGFA gene knockdown was significantly improved with CXCR4-targeted carriers in contrast to nontargeted peptides. siRNA delivery by means of ligandconjugated carriers resulted in 2.5–3-fold decrease of VEGF expression in glioblastoma cells and in 1.5–2-fold decrease of VEGF expression in endothelial cells. Delivery of siRNA/peptide complexes resulted in 2–6- fold decrease in VEGF protein yield and in significant inhibition of endothelial cells migration. The study shows that implication of peptide carriers modified with CXCR4 ligand is a promising approach to develop targeted siRNA delivery system into CXCR4-expressing cancer and endothelial cells.

Effect of RNA Interference-Mediated Suppression of p75 on the Viability of Rat Notochordal Cells.

Study DesignIn vitro cell culture model.PurposeTo investigate the effects of RNA interference (RNAi) on p75 expression and viability of rat notochordal cells treated with serum deprivation.Overview of LiteratureRNAi enables the inhibition of specific genes by sequence-specific gene silencing using a double-stranded RNA.MethodsNotochordal cells were isolated, cultured, and placed in 10% (control) or 0% (apoptosis-promoting) fetal bovine serum (FBS) for 48 hours. The expression of p75, apoptosis, and cell proliferation were determined. To suppress p75 expression, a small interfering RNA (siRNA) was synthesized against p75 (p75 siRNA) and transfected into cells. The suppression of p75 mRNA expression was investigated using the reverse transcription-polymerase chain reaction. The degree of p75 suppression was semiquantitatively analyzed using densitometry. The effect of p75 siRNA on apoptosis and proliferation of cells was determined. Solutions of an unrelated siRNA and transfection agent alone served as controls.ResultsSerum deprivation significantly increased apoptosis by 40.3%, decreased proliferation of notochordal cells by 45.3% (both, p<0.001), and upregulated p75 expression. The p75 siRNA suppressed p75 expression in cells cultured in 0% FBS. The rate of suppression by p75 siRNA of p75 mRNA was 72.9% (p<0.001). Suppression of p75 expression by p75 siRNA inhibited apoptosis by 7% and increased proliferation by 14% in cells cultured in 0% FBS (both, p<0.05).ConclusionssiRNA-mediated suppression of p75 inhibited apoptosis and increased proliferation of notochordal cells under conditions of serum deprivation, suggesting that RNAi might serve as a novel therapeutic approach for disc degeneration caused by insufficient viability of disc cells through the suppression of the expression of harmful genes.

Comparative analysis of polymers for short interfering RNA delivery in vascular smooth muscle cells

The use of short interfering RNA (siRNA) to degrade messenger RNA in the cell cytoplasm and transiently attenuate intracellular proteins shows promise in the inhibition of vascular pathogenesis. However, a critical obstacle for therapeutic application is a safe and effective delivery system. Biodegradable polymers are promising alternative molecular carriers for genetic material. Here, we aim to perform a comparative analysis of poly(B-amino ester) (PBAE) and polyethylenimine (PEI) polymers in their efficacy for vascular smooth muscle cell transfection using siRNA against the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene as our test target. MethodsHuman aortic smooth muscle cells (HASMC) were transfected in vitro with polymers conjugated to GAPDH or negative control (NC) siRNAs. Increasing siRNA:polymer ratios were tested for optimal transfection efficiency. DharmaFECT2 chemical transfection complexes were used for comparative analysis. Live/dead dual stain was used to measure cell viability, and GAPDH gene silencing was measured by quantitative polymerase chain reaction normalized to 18S. ResultsThe highest rate of PEI-mediated silencing was achieved with a 9μL polymer:220 pmol/mL siRNA conjugate (16 ± 2% expression versus NC; n = 6). Comparable PBAE-mediated silencing could be achieved with a 1.95μL polymer:100 pmol/mL siRNA conjugate (10 ± 1% expression versus NC; n = 5). Transfection using PEIs resulted in silencing equivalent to other methods but with less efficiency and increased cell toxicity at 24h polymer exposure. Decreasing PEI exposure time to 4 h resulted in similar silencing efficacy (21 ± 9% expression versus NC, n = 6) with an improved toxicity profile. ConclusionsPolymeric bioconjugates transfected HASMCs in a manner similar to chemical complexes, with comparable cell toxicity and silencing efficiency. PEI bioconjugates demonstrated silencing equivalent to PBAE bioconjugates, although less efficient in terms of required polymer concentrations. Given the cost-to-benefit difference between the assayed polymers, and PEI's ability to transfect HASMCs within a short duration of exposure with an improved toxicity profile, this study shows that PEI bioconjugates are a potential transfection agent for vascular tissue. Future studies will expand on this method of gene therapy to validate delivery of gene-specific inhibitors aimed at attenuating smooth muscle cell proliferation, adhesion, and migration. These studies will lay the framework for our future experimental plans to expand on this method of gene therapy for in vivo transfection in animal models of vascular disease.

Identification of novel genes that regulate androgen receptor signaling and growth of androgen-deprived prostate cancer cells.

Prostate cancer progression to castration refractory disease is associated with anomalous transcriptional activity of the androgen receptor (AR) in an androgen-depleted milieu. To identify novel gene products whose downregulation transactivates AR in prostate cancer cells, we performed a screen of enzymatically-generated shRNA lenti-libraries selecting for transduced LNCaP cells with elevated expression of a fluorescent reporter gene under the control of an AR-responsive promoter. The shRNAs present in selected populations were analyzed using high-throughput sequencing to identify target genes. Highly enriched gene targets were then validated with siRNAs against selected genes, testing first for increased expression of luciferase from an AR-responsive promoter and then for altered expression of endogenous androgen-regulated genes in LNCaP cells. We identified 20 human genes whose silencing affected the expression of exogenous and endogenous androgen-responsive genes in prostate cancer cells grown in androgen-depleted medium. Knockdown of four of these genes upregulated the expression of endogenous AR targets and siRNAs targeting two of these genes (IGSF8 and RTN1) enabled androgen-independent proliferation of androgen-dependent cells. The effects of IGSF8 appear to be mediated through its interaction with a tetraspanin protein, CD9, previously implicated in prostate cancer progression. Remarkably, homozygous deletions of IGSF8 are found almost exclusively in prostate cancers but not in other cancer types. Our study shows that androgen independence can be achieved through the inhibition of specific genes and reveals a novel set of genes that regulate AR signaling in prostate cancers.

Small interfering RNA targeting nonstructural protein1 α (nsp1α) of porcine reproductive and respiratory syndrome virus (PRRSV) can reduce the replication of PRRSV in MARC-145 cells

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically devastating and pandemic diseases of swine, which is poorly controlled by current methods. The inhibition of specific genes by small interfering RNA (siRNA) has been proven to be a potential therapeutic strategy against viral infection. Previous studies have indicated that the nonstructural protein 1α (nsp1α) of PRRSV may take an important role in virulence of PRRSV. The present work was involved to explore the effect of siRNA targeting nsp1α on the replication of PRRSV in MARC-145 cells, and the results showed that over-expression of nsp1α enhanced the replication of PRRSV and that siRNAs specifically targeting nsp1α significantly inhibited the replication of PRRSV in MARC-145 cells. In conclusion, this work indicated that nsp1α may be a viral pathogenicity factor of PRRSV and that siRNAs specifically targeting nsp1α may be a new strategy to control PRRSV in the future.

Interference identifies immune modulators

A broad in vivo screen of the effects of specific gene inhibition on the antitumour activity of immune cells in mice bearing melanomas has revealed potential targets for cancer therapy. See Article p.52 Immunotherapy is an important area of cancer research, and recent work has shown that targeting inhibitory receptors on T cells can result in clinical benefit in patients with advanced cancers. A major problem in the field is the difficulty of finding potential targets. Here Kai Wucherpfennig and colleagues show that in vivo discovery of therapeutic targets is a practical proposition by using short hairpin RNA (shRNA) screening to identify genes that modify the action of tumour-infiltrating CD8 T cells in tumour-bearing mice. They identify the regulatory phosphatase Ppp2r2d as a target and show that knockdown of Ppp2r2d in T cells enabled their accumulation in tumours and significantly delayed tumour growth.

Gold-Nanobeacons as a theranostic system for the detection and inhibition of specific genes

This protocol describes the synthesis and detailed calibration of a gold nanoparticle-based nanobeacon (Au-nanobeacon) as an innovative theranostic approach for detection and inhibition of sequence-specific DNA and RNA for in vitro and ex vivo applications. Under hairpin configuration, proximity to gold nanoparticles leads to fluorescence quenching; hybridization to a complementary target restores fluorescence emission due to the gold nanobeacons’ conformational reorganization that causes the fluorophore and the AuNP to part from each other. This concept can easily be extended and adapted to assist the in vitro evaluation of silencing potential of a given sequence to be later used for ex vivo gene silencing and RNAi approaches, with the ability to monitor real-time gene delivery action. The time range for the entire protocol is ~8 days, including synthesis, functionalization and calibration of Au-nanobeacons, RNAi and gene silencing assays.

MicroRNA-199a-3p Regulates Endometrial Cancer Cell Proliferation by Targeting Mammalian Target of Rapamycin (mTOR)

Abnormal expression of miR-199a-3p, which has similar effects to oncogenes or tumor suppressor genes, can occur in various malignant tumors and is closely linked with tumor cell proliferation, invasion, and metastasis. However, its expression and effects in endometrial endometrioid adenocarcinoma (EEC) are still unclear. This study was designed to identify the impact of miR-199a-3p on the proliferation of EEC cells and its role in the carcinogenesis of EEC. The expression levels of miR-199a-3p in EEC and paired adjacent nontumor tissues were analyzed by real-time polymerase chain reaction. The effects of miR-199a-3p on proliferation, cell cycle and apoptosis in EEC cells were analyzed in Ishikawa cells transfected with miR-199a-3p mimics and inhibitors. The target genes of miR-199a-3p were predicted using bioinformatics methods. The extent of regulation of the predicted target genes by miR-199a-3p was determined using luciferase reporter assays, Western blotting, and quantitative polymerase chain reaction. The EEC cells were pretreated with target gene-specific inhibitors to further identify the relationship between the effects of miR-199a-3p and the predicted target genes. Compared with the adjacent tissues and normal endometrium, reduced expression of miR-199a-3p was found in human EEC specimens. Compared with the control group transfected with control microRNA mimics, the proliferative capacity of EEC cells transfected with miR-199a-3p mimics was inhibited, whereas cells transfected with miR-199a-3p inhibitors showed increased proliferation. The inhibitory effect was associated with increased cell populations at the G1-phase, and decreased cell populations at the S-phase. The results demonstrated that miR-199a-3p could inhibit the protein expression of mammalian target of rapamycin (mTOR) by targeted binding to the mTOR-3' untranslated region. Inhibition of EEC cell proliferation by miR-199a-3p was mediated by its targeted regulation of mTOR. MiR-199a-3p inhibits tumor cell proliferation through negative regulation of mTOR expression. Restoration of intracellular miR-199a-3p levels may serve as a potential option for EEC treatment.

Development of Quantitative Molecular Clinical End Points for siRNA Clinical Trials

RNA interference (RNAi) is an evolutionarily conserved mechanism that results in specific gene inhibition at the mRNA level. The discovery that short interfering RNAs (siRNAs) are selective, potent, and can largely avoid immune surveillance has resulted in keen interest to develop these inhibitors as therapeutics. A single nucleotide-specific siRNA (K6a_513a.12, also known as TD101) was recently evaluated in a phase 1b clinical trial for the rare skin disorder, pachyonychia congenita (PC). To develop a clinical trial molecular end point for this type of trial, methods were developed to: (1) isolate total RNA containing amplifiable mRNA from human skin and callus material; (2) quantitatively distinguish the single-nucleotide mutant mRNA from wild-type K6a mRNA in both patient-derived keratinocytes and patient callus; and (3) demonstrate that repeated siRNA treatment results in sustained inhibition of mutant K6a mRNA in patient-derived keratinocyte cultures. These methods allow noninvasive sampling and monitoring of gene expression from patient-collected shavings and may be useful in evaluating the effectiveness of RNAi-based therapeutics, including inhibitors that specifically target single-nucleotide mutations.