Targeting MAGI2-AS3-modulated Akt-dependent ATP-binding cassette transporters as a possible strategy to reverse temozolomide resistance in temozolomide-resistant glioblastoma cells.

Drug resistance is a major impediment to the successful treatment of glioma. This study aimed to elucidate the effects and mechanisms of the long noncoding RNA membrane-associated guanylate kinase inverted-2 antisense RNA 3 (MAGI2-AS3) on temozolomide (TMZ) resistance in glioma cells. MAGI2-AS3 expression in TMZ-resistant glioblastoma (GBM) cells was analyzed using the Gene Expression Omnibus data set GSE113510 and quantitative real-time PCR (qRT-PCR). Cell viability and TMZ half-maximal inhibitory concentration values were determined using the MTT assay. Apoptosis and cell cycle distribution were evaluated using flow cytometry. The expression of multidrug resistance 1 (MDR1), ATP-binding cassette superfamily G member 2 (ABCG2), protein kinase B (Akt), and phosphorylated Akt was detected using qRT-PCR and/or western blot analysis. MAGI2-AS3 was expressed at low levels in TMZ-resistant GBM cells relative to that in their parental cells. MAGI2-AS3 re-expression alleviated TMZ resistance in TMZ-resistant GBM cells. MAGI2-AS3 overexpression also accelerated TMZ-induced apoptosis and G2/M phase arrest. Mechanistically, MAGI2-AS3 overexpression reduced MDR1 and ABCG2 expression and inhibited the Akt pathway, whereas Akt overexpression abrogated the reduction in MDR1 and ABCG2 expression induced by MAGI2-AS3. Moreover, activation of the Akt pathway inhibited the effects of MAGI2-AS3 on TMZ resistance. MAGI2-AS3 inhibited tumor growth and enhanced the suppressive effect of TMZ on glioma tumorigenesis in vivo. In conclusion, MAGI2-AS3 reverses TMZ resistance in glioma cells by inactivating the Akt pathway.

Bone Marrow Mesenchymal Stem Cells-derived Exosomal Long Non-coding RNA KLF3 antisense RNA 1 Enhances Autophagy to Protect Against Cerebral Ischemia/Reperfusion Injury Via ETS Variant Transcription Factor 4/Silent Information Regulator 1 Axis.

Mesenchymal stem cells (MSCs)-derived exosomes are demonstrated to exert neuroprotective effects in stroke. We aimed to explore the role and mechanism of long non-coding RNA (lncRNA) KLF3 antisense RNA 1 (KLF3-AS1) in bone marrow mesenchymal stem cells-derived exosomes (BMSCs-Exos) in cerebral ischemia/reperfusion (I/R) injury. Exosomes were isolated from the culture medium of BMSCs. A mouse model of middle cerebral artery occlusion (MCAO) in vivo and a BV-2 cell model of oxygen and glucose deprivation/reoxygenation (OGD/RX) in vitro were established. Cell viability and apoptosis were detected using MTT assay, TUNEL staining and flow cytometry, respectively. Related proteins were determined with western blot and immunohistochemistry, while related RNAs were analyzed by RT-qPCR. Neurological deficit and cerebral infarct volume were evaluated by the modified neurological severity score (mNSS) and TTC staining, respectively. Our observations indicate that exosomes derived from BMSCs-preconditioned medium exerted neuroprotective effects, as indicated by the increased cell viability and the suppressed apoptosis in OGD/RX-suffered BV-2 cells. KLF3-AS1 expression was upregulated in BMSCs-Exos. Furthermore, KLF3-AS1 knockdown antagonized the protective effects of BMSCs-Exos. Mechanistically, BMSCs-Exos carrying KLF3-AS1 inhibited apoptosis via enhancing autophagy. KLF3-AS1 was found to recruit ETS variant transcription factor 4 (ETV4), which upregulated Sirt1 expression. Knockdown of KLF3-AS1 neutralized the protective effects of BMSCs-Exos on MCAO-induced brain injury, which was then reversed by the treatment with Sirt1 inhibitor EX527. We concluded that KLF3-AS1 derived from BMSCs-Exos promoted autophagy to alleviate I/R injury via ETV4/Sirt1 axis.

Anti-aging Effects of Alu Antisense RNA on Human Fibroblast Senescence Through the MEK-ERK Pathway Mediated by KIF15.

OBJECTIVE: To investigate whether human short interspersed nuclear element antisense RNA (Alu antisense RNA; Alu asRNA) could delay human fibroblast senescence and explore the underlying mechanisms. METHODS: We transfected Alu asRNA into senescent human fibroblasts and used cell counting kit-8 (CCK-8), reactive oxygen species (ROS), and senescence-associated beta-galactosidase (SA-ß-gal) staining methods to analyze the anti-aging effects of Alu asRNA on the fibroblasts. We also used an RNA-sequencing (RNA-seq) method to investigate the Alu asRNA-specific mechanisms of anti-aging. We examined the effects of KIF15 on the anti-aging role induced by Alu asRNA. We also investigated the mechanisms underlying a KIF15-induced proliferation of senescent human fibroblasts. RESULTS: The CCK-8, ROS and SA-ß-gal results showed that Alu asRNA could delay fibroblast aging. RNA-seq showed 183 differentially expressed genes (DEGs) in Alu asRNA transfected fibroblasts compared with fibroblasts transfected with the calcium phosphate transfection (CPT) reagent. The KEGG analysis showed that the cell cycle pathway was significantly enriched in the DEGs in fibroblasts transfected with Alu asRNA compared with fibroblasts transfected with the CPT reagent. Notably, Alu asRNA promoted the KIF15 expression and activated the MEK-ERK signaling pathway. CONCLUSION: Our results suggest that Alu asRNA could promote senescent fibroblast proliferation via activation of the KIF15-mediated MEK-ERK signaling pathway.

Loss of Detection of sgN Precedes Viral Abridged Replication in COVID-19-Affected Patients-A Target for SARS-CoV-2 Propagation.

The development of prophylactic agents against the SARS-CoV-2 virus is a public health priority in the search for new surrogate markers of active virus replication. Early detection markers are needed to follow disease progression and foresee patient negativization. Subgenomic RNA transcripts (with a focus on sgN) were evaluated in oro/nasopharyngeal swabs from COVID-19-affected patients with an analysis of 315 positive samples using qPCR technology. Cut-off Cq values for sgN (Cq < 33.15) and sgE (Cq < 34.06) showed correlations to high viral loads. The specific loss of sgN in home-isolated and hospitalized COVID-19-positive patients indicated negativization of patient condition, 3-7 days from the first swab, respectively. A new detection kit for sgN, gene E, gene ORF1ab, and gene RNAse P was developed recently. In addition, in vitro studies have shown that 2'-O-methyl antisense RNA (related to the sgN sequence) can impair SARS-CoV-2 N protein synthesis, viral replication, and syncytia formation in human cells (i.e., HEK-293T cells overexpressing ACE2) upon infection with VOC Alpha (B.1.1.7)-SARS-CoV-2 variant, defining the use that this procedure might have for future therapeutic actions against SARS-CoV-2.

Long noncoding RNA TSPOAP1 antisense RNA 1 negatively modulates type I IFN signaling to facilitate influenza A virus replication.

Although the expression of thousands of host long noncoding RNAs (lncRNAs) can be regulated by viral infection, the number of lncRNAs with experimentally verified function is limited. In this study, the expression of host lncRNA TSPOAP1-AS1 was significantly induced by influenza A virus (IAV) infection in a dose- and time-dependent manner. Polyinosine-polycytidylic acid (poly (I:C)), a synthetic analog of double-stranded RNA, also increased TSPOAP1-AS1 expression. RNA fractionation revealed that TSPOAP1-AS1 was a nucleocytoplasmic lncRNA, and an increased nuclear/cytoplasmic ratio was detected after IAV infection. The nuclear factor-κB signaling acting as a critical factor in the transcription of TSPOAP1-AS1 was determined through the use of pharmacological and genetic approaches. Functionally, overexpression of TSPOAP1-AS1 resulted in a significant increase in IAV replication. In contrast, the abolition of TSPOAP1-AS1 by RNA interference restricted viral replication. Furthermore, we demonstrated that TSPOAP1-AS1 negatively modulated the IAV-induced Ifnb1 transcription, interferon-sensitive response element (ISRE) activation, and downstream interferon-stimulated genes expression. Collectively, our data provides evidence for the host lncRNA utilized by viruses to support its replication.

Anti-aging and anti-oxidant activities of murine short interspersed nuclear element antisense RNA.

Short interspersed nuclear elements (SINEs) play a key role in regulating gene expression, and SINE RNAs are involved in age-related diseases. We investigated the anti-aging effects of a genetically engineered murine SINE B1 antisense RNA (B1as RNA) and explored its mechanism of action in naturally senescent BALB/c (≥14 months) and moderately senscent C57BL/6N (≥9 months) mice. After tail vein injection, B1as RNA was available in the blood of mice for approximately 30 min, persisted for approximately 2-4 h in most detected tissues and persisted approximately 48 h in lungs. We found that treatment with B1as RNA improved stamina and promoted hair re-growth in aged mice. Treatment with B1as RNA also partially rescued the increase in mitochondrial DNA copy number in liver and spleen tissues observed in aged and moderately senescent mice. Finally, treatment with B1as RNA increased the activities of superoxide dismutase and glutathione peroxidase in aged and moderately senescent mice, reduced these animals' malondialdehyde and reactive oxygen species levels, and modulated the expression of several aging-associated genes, including Sirtuin 1, p21, p16Ink4a, p15Ink4b and p19Arf, and anti-oxidant genes (Sesn1 and Sesn 2). These data suggest that B1as RNA inhibits the aging process by enhancing antioxidant activity, promoting the scavenging of free radicals, and modulating the expression of aging-associated genes. This is the first report describing the anti-aging activity of SINE antisense RNA, which may serve as an effective nucleic acid drug for the treatment of age-related diseases.

Targeting the Mitochondrial Genome Via a MITO-Porter : Evaluation of mtDNA and mtRNA Levels and Mitochondrial Function.

Genetic mutations and defects in mitochondrial DNA (mtDNA) are associated with certain types of mitochondrial dysfunctions, ultimately resulting in the emergence of a variety of human diseases. To achieve an effective mitochondrial gene therapy, it will be necessary to deliver therapeutic agents to the innermost mitochondrial space (the mitochondrial matrix), which contains the mtDNA pool. We recently developed a MITO-Porter, a liposome-based nanocarrier that delivers cargo to mitochondria via a membrane-fusion mechanism. In this chapter, we discuss the methodology used to deliver bioactive molecules to the mitochondrial matrix using a Dual Function (DF)-MITO-Porter, a liposome-based nanocarrier that delivers it cargo by means of a stepwise process, and an evaluation of mtDNA levels and mitochondrial activities in living cells. We also discuss mitochondrial gene silencing by the mitochondrial delivery of antisense RNA oligonucleotide (ASO) targeting mtDNA-encoded mRNA using the MITO-Porter system.

Tetrahedral DNA nanostructures functionalized by multivalent microRNA132 antisense oligonucleotides promote the differentiation of mouse embryonic stem cells into dopaminergic neurons.

MicroRNA132 (miR132) negatively regulates the differentiation of mouse embryonic stem cells (ESCs) into dopaminergic (DAergic) neurons; in contrast, antisense oligonucleotide against miR132 (miR132-ASO) effectively blocks the activity of endogenous miR132 and thereafter promotes the differentiation of DAergic neurons. However, it is difficult for miR132-ASO to enter cells without a suitable delivery system. Tetrahedral DNA nanostructures (TDNs), as a new type of DNA-based nanocarrier, have great potential in biomedical applications and even have been reported to promote stem cell differentiation. In this study, we developed functional multivalent DNA nanostructures by appending miR132-ASO motifs to three-dimensional TDNs (miR132-ASO-TDNs). Our data clearly revealed that miR132-ASO-TDNs exposure can promote the differentiation of ESCs into DAergic neurons as well as elevate DA release from differentiated DAergic neurons. MiR132-ASO-TDNs could serve as a novel biofunctional nanomaterial to improve the efficiency of DAergic neurons differentiation. Our findings may also provide a new approach for stem cell therapy against neurodegenerative diseases.

Effects of fibrinogen synthesis inhibition on vascular cognitive impairment during traumatic brain injury in mice.

Traumatic brain injury (TBI) is associated with increased blood content of fibrinogen (Fg), called hyperfibrinogenemia (HFg), which results in enhanced cerebrovascular permeability and leads to short-term memory (STM) reduction. Previously, we showed that extravasated Fg was deposited in the vasculo-astrocyte interface and was co-localized with cellular prion protein (PrPC) during mild-to-moderate TBI in mice. These effects were accompanied by neurodegeneration and STM reduction. However, there was no evidence presented that the described effects were the direct result of the HFg during TBI. We now present data indicating that inhibition of Fg synthesis can ameliorate TBI-induced cerebrovascular permeability and STM reduction. Cortical contusion injury (CCI) was induced in C57BL/6J mice. Then mice were treated with either Fg antisense oligonucleotide (Fg-ASO) or with control-ASO for two weeks. Cerebrovascular permeability to fluorescently labeled bovine serum albumin was assessed in cortical venules following evaluation of STM with memory assessement tests. Separately, brain samples were collected in order to define the expression of PrPC via Western blotting while deposition and co-localization of Fg and PrPC, as well as gene expression of inflammatory marker activating transcription factor 3 (ATF3), were characterized with real-time PCR. Results showed that inhibition of Fg synthesis with Fg-ASO reduced overexpression of AFT3, ameliorated enhanced cerebrovascular permeability, decreased expression of PrPC and Fg deposition, decreased formation of Fg-PrPC complexes in brain, and improved STM. These data provide direct evidence that a CCI-induced inflammation-mediated HFg could be a triggering mechanism involved in vascular cognitive impairment seen previously in our studies during mild-to-moderate TBI.

Mini-III RNase-based dual-color system for in vivo mRNA tracking.

Mini-III RNase (mR3), a member of RNase III endonuclease family, can bind to and cleave double-stranded RNAs (dsRNAs). Inactive mR3 protein without the α5ß-α6 loop loses the dsRNA cleavage activity, but retains dsRNA binding activity. Here, we establish an inactive mR3-based non-engineered mR3/dsRNA system for RNA tracking in zebrafish embryos. In vitro binding experiments show that inactive Staphylococcus epidermidis mR3 (dSmR3) protein possesses the highest binding affinity with dsRNAs among mR3s from other related species, and its binding property is retained in zebrafish embryos. Combined with a fluorescein-labeled antisense RNA probe recognizing the target mRNAs, dSmR3 tagged with a nuclear localization sequence and a fluorescent protein could allow visualization of the dynamics of endogenous target mRNAs. The dSmR3/antisense probe dual-color system provides a new approach for tracking non-engineered RNAs in real-time, which will help understand how endogenous RNAs dynamically move during embryonic development.

RNA Drugs and RNA Targets for Small Molecules: Principles, Progress, and Challenges.

RNA-based therapies, including RNA molecules as drugs and RNA-targeted small molecules, offer unique opportunities to expand the range of therapeutic targets. Various forms of RNAs may be used to selectively act on proteins, transcripts, and genes that cannot be targeted by conventional small molecules or proteins. Although development of RNA drugs faces unparalleled challenges, many strategies have been developed to improve RNA metabolic stability and intracellular delivery. A number of RNA drugs have been approved for medical use, including aptamers (e.g., pegaptanib) that mechanistically act on protein target and small interfering RNAs (e.g., patisiran and givosiran) and antisense oligonucleotides (e.g., inotersen and golodirsen) that directly interfere with RNA targets. Furthermore, guide RNAs are essential components of novel gene editing modalities, and mRNA therapeutics are under development for protein replacement therapy or vaccination, including those against unprecedented severe acute respiratory syndrome coronavirus pandemic. Moreover, functional RNAs or RNA motifs are highly structured to form binding pockets or clefts that are accessible by small molecules. Many natural, semisynthetic, or synthetic antibiotics (e.g., aminoglycosides, tetracyclines, macrolides, oxazolidinones, and phenicols) can directly bind to ribosomal RNAs to achieve the inhibition of bacterial infections. Therefore, there is growing interest in developing RNA-targeted small-molecule drugs amenable to oral administration, and some (e.g., risdiplam and branaplam) have entered clinical trials. Here, we review the pharmacology of novel RNA drugs and RNA-targeted small-molecule medications, with a focus on recent progresses and strategies. Challenges in the development of novel druggable RNA entities and identification of viable RNA targets and selective small-molecule binders are discussed. SIGNIFICANCE STATEMENT: With the understanding of RNA functions and critical roles in diseases, as well as the development of RNA-related technologies, there is growing interest in developing novel RNA-based therapeutics. This comprehensive review presents pharmacology of both RNA drugs and RNA-targeted small-molecule medications, focusing on novel mechanisms of action, the most recent progress, and existing challenges.

Antimicrobial antisense RNA delivery to F-pili producing multidrug-resistant bacteria via a genetically engineered bacteriophage.

Multidrug-resistant bacteria are a growing issue worldwide. This study developed a convenient and effective method to downregulate the expression of a specific gene to produce a novel antimicrobial tool using a small (140 nucleotide) RNA with a 24-nucleotide antisense (as) region from an arabinose-inducible expression phagemid vector in Escherichia coli. Knockdown effects of rpoS encoding RNA polymerase sigma factor were observed using this inducible artificial asRNA approach. asRNAs targeting several essential E. coli genes produced significant growth defects, especially when targeted to acpP and ribosomal protein coding genes rplN, rplL, and rpsM. Growth inhibited phenotypes were facilitated in hfq- conditions. Phage lysates were prepared from cells harboring phagemids as a lethal-agent delivery tool. Targeting the rpsM gene by phagemid-derived M13 phage infection of E. coli containing a carbapenem-producing F-plasmid and multidrug-resistant Klebsiella pneumoniae containing an F-plasmid resulted in the death of over 99.99% of infected bacteria. This study provides a possible strategy for treating bacterial infection and can be applied to any F-pilus producing bacterial species.

Antisense antibacterial compounds.

Extensive antibiotic use combined with poor historical drug stewardship practices have created a medical crisis in which once treatable bacterial infections are now increasingly unmanageable. To combat this, new antibiotics will need to be developed and safeguarded. An emerging class of antibiotics based upon nuclease-stable antisense technologies has proven valuable in preclinical testing against a variety of bacterial pathogens. This review describes the current state of development of antisense-based antibiotics, the mechanisms thus far employed by these compounds, and possible future avenues of research.

An RNA biology perspective on species-specific programmable RNA antibiotics.

Our body is colonized by a vast array of bacteria the sum of which forms our microbiota. The gut alone harbors >1,000 bacterial species. An understanding of their individual or synergistic contributions to human health and disease demands means to interfere with their functions on the species level. Most of the currently available antibiotics are broad-spectrum, thus too unspecific for a selective depletion of a single species of interest from the microbiota. Programmable RNA antibiotics in the form of short antisense oligonucleotides (ASOs) promise to achieve precision manipulation of bacterial communities. These ASOs are coupled to small peptides that carry them inside the bacteria to silence mRNAs of essential genes, for example, to target antibiotic-resistant pathogens as an alternative to standard antibiotics. There is already proof-of-principle with diverse bacteria, but many open questions remain with respect to true species specificity, potential off-targeting, choice of peptides for delivery, bacterial resistance mechanisms and the host response. While there is unlikely a one-fits-all solution for all microbiome species, I will discuss how recent progress in bacterial RNA biology may help to accelerate the development of programmable RNA antibiotics for microbiome editing and other applications.

Targeted mitochondrial delivery of antisense RNA-containing nanoparticles by a MITO-Porter for safe and efficient mitochondrial gene silencing.

Mitochondrial gene therapy will be needed to treat mitochondrial diseases. We previously demonstrated mitochondrial gene silencing by the mitochondrial delivery of antisense RNA oligonucleotide (ASO) targeting mtDNA-encoded mRNA using a MITO-Porter, a liposomal nano carrier system designed for mitochondrial delivery. Here, we report on the efficient packaging of ASO in the MITO-Porter via a nanoparticle packaging method, which showed a 10-fold higher packaging efficiency than the conventional method. The constructed carrier showed a decrease in the target mRNA levels and ATP production. These results indicate that such a MITO-Porter has potential for use in therapies designed to regulate mitochondrial function.

Nano-graphene oxide improved the antibacterial property of antisense yycG RNA on Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus (S. aureus) has the potential to opportunistically cause infectious diseases, including osteomyelitis, skin infections, pneumonia, and diarrhea. We previously reported that ASyycG RNA reduced the transcripts of virulent genes, and biofilm formation of S. aureus. Currently, graphene oxide (GO) nanosheets are used to efficiently deliver nucleic acids with favorable biocompatibility. METHODS: In the current study, a GO-based recombinant pDL278 ASyycG vector transformation strategy was developed. The particle size distributions and zeta-potential of the GO-PEI-based ASyycG were evaluated. The ASyycG plasmids were labeled with gene-encoding enhanced green fluorescent protein (ASyycG-eGFP). Quantitative real-time PCR assays were performed to investigate the expression of yycF/G/H and icaADB genes. Biofilm biomass and bacterial viability of S. aureus were evaluated by scanning electron microscopy and confocal laser scanning microscopy. We found that the expression of the yycG gene was inversely correlated with levels of the ASyycG transcripts and that the GO-PEI-ASyycG strain had the lowest expression of biofilm organization-associated genes. RESULTS: The results showed that the GO-based strategy significantly increased ASyycG transformation as a delivery system compared to the conventional competence-stimulating peptide strategy. Furthermore, GO-PEI-ASyycG suppressed bacterial biofilm aggregation and improved bactericidal effects on S. aureus after 24 h biofilm establishment. CONCLUSIONS: Our findings demonstrated that nano-GO with antisense yycG RNA is a more effective and relatively stable strategy for the management of S. aureus infections.

Two tales of Annexin A2 knock-down: One of compensatory effects by antisense RNA and another of a highly active hairpin ribozyme.

Besides altering its own expression during cell transformation, Annexin A2 is upregulated during the progression of many cancer types and also plays key roles during viral infection and multiplication. Consequently, there has been great interest in Annexin A2 as a potential drug target. The successful design of efficient in vivo delivery systems constitutes an obstacle in full exploitation of antisense and RNA-cleaving technologies for the knock-down of specific targets. Efficiency is dependent on the method of delivery and accessibility of the target. Here, hairpin ribozymes and an antisense RNA against rat annexin A2 mRNA were tested for their efficiencies in a T7-driven coupled transcription/translation system. The most efficient ribozyme and antisense RNA were subsequently inserted into a retroviral vector under the control of a tRNA promoter, in a cassette inserted between retroviral Long Terminal Repeats for stable insertion into host DNA. The Phoenix package system based on defective retroviruses was used for virus-mediated gene transfer into PC12 cells. Cells infected with the ribozyme-containing particles died shortly after infection. However, the same ribozyme showed a very high catalytic effect in vitro in cell lysates, explained by its loose hinge helix 2 region. This principle can be transferred to other ribozymes, such as those designed to cleave the guide RNA in the CRISPR/Cas9 technology, as well as to target specific viral RNAs. Interestingly, efficient down-regulation of the expression of Annexin A2 by the antisense RNA resulted in up-regulation of Annexin A7 as a compensatory effect after several cell passages. Indeed, compensatory effects have previously been observed during gene knock-out, but not during knock-down of protein expression. This highlights the problems in interpreting the phenotypic effects of knocking down the expression of a protein. In addition, these data are highly relevant when considering the effects of the CRISPR/Cas9 approach.

Silk Biomaterials-Mediated miRNA Functionalized Orthopedic Devices.

Silk-based bioresorbable medical devices, such as screws, plates, and rods, have been under investigation due to their promising properties for orthopedic repairs. Options to functionalize these new devices for enhanced control of bone regeneration would also exploit the compatible processing methods used to generate the devices. MicroRNAs are important regulators of bone maintenance and formation, and miRNA-based therapeutics have the potential to aid bone repair, utilizing a transient therapeutic approach with local bioactivity. We hypothesized that silk-based orthopedic devices could be used for the local delivery of miRNAs, using anti-sense miR-214 (AS-miR-214), to inhibit endogenous expression of osteoinductive antagonist and thereby supporting the upregulation of osteoinductive target molecules activating transcription factor 4 (ATF4) and Osterix (Osx). AS-miR-214 silk devices, prepared using surface coating, demonstrated continuous release of miRNA inhibitors up to 7 days in vitro. Additionally, human mesenchymal stem cells seeded on AS-miR-214 silk films expressed higher levels of osteogenic genes ATF4, Osx, Runx2, and Osteocalcin. Interestingly, these cells exhibited lower cell viability and DNA content over 21 days. Conversely, the cells demonstrated significantly higher levels of alkaline phosphatase expression and calcium deposition compared with cells seeded on silk films with nontargeting miRNA controls. The study demonstrated that the silk-based orthopedic devices, in conjunction with bioactive miRNA-based therapeutics, may serve as a novel system for localized bone tissue engineering, enhancing osteogenesis at the implant interface while avoiding detrimental systematic side effects.

Harnessing an Inhibitory Role of miR-16 in Osteogenesis by Human Mesenchymal Stem Cells for Advanced Scaffold-Based Bone Tissue Engineering.

MicroRNA (miRNA) therapeutics is increasingly being developed to either target bone-related diseases such as osteoporosis and osteoarthritis or as the basis for novel bone tissue engineering strategies. A number of miRNAs have been reported as potential osteo-therapeutics but no consensus has yet been established on the optimal target. miR-16 has been studied extensively in nonosteogenic functions and used as functionality reporter target in the development of nonviral miRNA delivery platforms. This study hypothesized that miR-16 may also play an inhibitory role in osteogenesis due to its ability to directly target Smad5 and AcvR2a. This study thus aimed to assess the potential of miR-16 inhibition to increase osteogenesis in human mesenchymal stem cells (hMSCs) using a previously established miRNA delivery platform composed of nanohydroxyapatite (nHA) particles as nonviral vectors in combination with collagen-nHA scaffolds designed specifically for bone repair. Initial results showed that antagomiR-16 delivery efficiently increased the relative levels of both putative targets and Runx2, the key transcription factor for osteogenesis, while also increasing osteocalcin levels. Furthermore, significant increases in mineral calcium deposition by hMSCs were found in both monolayer and most importantly in scaffold-based osteodifferentiation studies, ultimately demonstrating that miR-16 inhibition further enhances the therapeutic potential of a scaffold with known potential for bone repair applications and thus holds significant therapeutic potential as a novel bone tissue engineering strategy. Furthermore, we suggest that harnessing the additional functions known to miR-16 by incorporating either its enhancers or inhibitors to tissue-specific tailored scaffolds provides exciting opportunities for a diverse range of therapeutic indications.

Long non-coding RNA TP73-AS1 sponges miR-194 to promote colorectal cancer cell proliferation, migration and invasion via up-regulating TGFα.

OBJECTIVE: Colorectal cancer (CRC) is the 3rd most common cancer worldwide. Recently, long non-coding RNAs (lncRNAs) were found to be critical modulators in the CRC progression. The aim of this study is to investigate the potential roles of lncRNA P73 antisense RNA 1T (TP73-AS1) in CRC development and progression. METHODS: Quantitative real-time PCR (qRT-PCR) was performed to determine relevant gene expression levels; western blot was performed to determine protein expression levels; CCK-8, colony formation, wound healing and Transwell invasion assays were used to determined CRC cell proliferation, migration and invasion; in vivo tumor growth was assessed in xenograft mice model. RESULTS: TP73-AS1 was up-regulated in both CRC tissues and CRC cell lines. Overexpression of TP73-AS1 was associated with metastasis and advanced clinical stages in CRC patients. Overexpression of TP73-AS1 promoted CRC cell growth, proliferation, migration and invasion in vitro; and knockdown of TP73-AS1 significantly inhibited CRC cell growth, proliferation, migration and invasion in vitro as well as tumor growth in vivo. Bioinformatics analysis and luciferase reporter assay indicated that TP73-AS1 could bind directly with miR-194, and TP73-AS1 negatively regulated the expression of miR-194 in CRC cells. Further study indicated that miR-194 negatively regulated the downstream target of transforming growth factor alpha (TGFα) via targeting its 3' untranslated region, and TP73-AS1 positively regulated the expression of TGFα in CRC cells. Moreover, overexpression of miR-194 suppressed CRC cell proliferation and invasion, and attenuated the effects of TP73-AS1 overexpression on CRC cell proliferation and invasion. Silence of TGFα inhibited CRC cell proliferation and invasion, and also reversed the effects of TP73-AS1 overexpression on CRC cell proliferation and invasion. CONCLUSIONS: this study demonstrated that TP73-AS1 regulated CRC progression by acting as a competitive endogenous RNA to sponge miR-194 to modulate the expression of TGFα.