Zhuomin Wu

Regulation of lncRNA expression

Long non-coding RNAs (lncRNAs) are series of transcripts with important biological functions. Various diseases have been associated with aberrant expression of lncRNAs and the related dysregulation of mRNAs. In this review, we highlight the mechanisms of dynamic lncRNA expression. The chromatin state contributes to the low and specific expression of lncRNAs. The transcription of non-coding RNA genes is regulated by many core transcription factors applied to protein-coding genes. However, specific DNA sequences may allow their unsynchronized transcription with their location-associated mRNAs. Additionally, there are multiple mechanisms involved in the post-transcriptional regulation of lncRNAs. Among these, microRNAs might have indispensible regulatory effects on lncRNAs, based on recent discoveries.

Tumor-targeted in vivo gene silencing via systemic delivery of cRGD-conjugated siRNA.

RNAi technology is taking strong position among the key therapeutic modalities, with dozens of siRNA-based programs entering and successfully progressing through clinical stages of drug development. To further explore potentials of RNAi technology as therapeutics, we engineered and tested VEGFR2 siRNA molecules specifically targeted to tumors through covalently conjugated cyclo(Arg-Gly-Asp-d-Phe-Lys[PEG-MAL]) (cRGD) peptide, known to bind αvβ3 integrin receptors. cRGD-siRNAs were demonstrated to specifically enter and silence targeted genes in cultured αvβ3 positive human cells (HUVEC). Microinjection of zebrafish blastocysts with VEGFR2 cRGD-siRNA resulted in specific inhibition of blood vessel growth. In tumor-bearing mice, intravenously injected cRGD-siRNA molecules generated no innate immune response and bio-distributed to tumor tissues. Continuous systemic delivery of two different VEGFR2 cRGD-siRNAs resulted in down-regulation of corresponding mRNA (55 and 45%) and protein (65 and 45%) in tumors, as well as in overall reduction of tumor volume (90 and 70%). These findings demonstrate strong potential of cRGD-siRNA molecules as anti-tumor therapy.

Erratum to: LncRNA-N1LR Enhances Neuroprotection Against Ischemic Stroke Probably by Inhibiting p53 Phosphorylation

In recent years, long noncoding RNAs (lncRNAs) have been shown to have critical roles in a broad range of cell biological processes. However, the activities of lncRNAs during ischemic stroke remain largely unknown. In this study, we carried out a genome-wide lncRNA microarray analysis in rat brains with ischemia/reperfusion (I/R) injury. The results revealed the differential expression of a subset of lncRNAs. Through the construction of lncRNA-mRNA co-expression networks, we identified lncRNA-N1LR as a novel I/R-induced lncRNA. The functions of lncRNA-N1LR were assessed by silencing and overexpressing this lncRNA in vitro and in vivo. We found that lncRNA-N1LR enhanced cell cycle progression and cell proliferation, and inhibited apoptosis in N2a cells subjected to in vitro ischemia (oxygen-glucose deprivation/reoxygenation, OGD/R). Furthermore, we showed that lncRNA-N1LR reduced neuronal apoptosis and neural cell loss in I/R-induced mouse brains. Mechanistically, we discovered that lncRNA-N1LR promoted neuroprotection probably through the inhibition of p53 phosphorylation on serine 15 in a manner that was independent of its location-associated gene Nck1. In summary, our results indicated that lncRNA-N1LR promoted neuroprotection against ischemic stroke probably by inactivating p53. Thus, we propose that lncRNA-N1LR may serve as a potential target for therapeutic intervention following ischemic brain injury.

Self-assembled nanoparticles based on the c(RGDfk) peptide for the delivery of siRNA targeting the VEGFR2 gene for tumor therapy.

The clinical application of small interfering RNA (siRNA) has been restricted by their poor intracellular uptake, low serum stability, and inability to target specific cells. During the last several decades, a great deal of effort has been devoted to exploring materials for siRNA delivery. In this study, biodegradable, tumor-targeted, self-assembled peptide nanoparticles consisting of cyclo(Arg–Gly–Asp–d–Phe–Lys)-8–amino–3,6–dioxaoctanoic acid–β–maleimidopropionic acid (hereafter referred to as RPM) were found to be an effective siRNA carrier both in vitro and in vivo. The nanoparticles were characterized based on transmission electron microscopy, circular dichroism spectra, and dynamic light scattering. In vitro analyses showed that the RPM/VEGFR2-siRNA exhibited negligible cytotoxicity and induced effective gene silencing. Delivery of the RPM/VEGFR2 (zebrafish)-siRNA into zebrafish embryos resulted in inhibition of neovascularization. Administration of RPM/VEGFR2 (mouse)-siRNA to tumor-bearing nude mice led to a significant inhibition of tumor growth, a marked reduction of vessels, and a down-regulation of VEGFR2 (messenger RNA and protein) in tumor tissue. Furthermore, the levels of IFN-α, IFN-γ, IL-12, and IL-6 in mouse serum, assayed via enzyme-linked immunosorbent assay, did not indicate any immunogenicity of the RPM/VEGFR2 (mouse)-siRNA in vivo. In conclusion, RPM may provide a safe and effective delivery vector for the clinical application of siRNAs in tumor therapy.

A tumor-targeting cRGD-EGFR siRNA conjugate and its anti-tumor effect on glioblastoma in vitro and in vivo

Abstract The epidermal growth factor receptor (EGFR) is an important anti-tumor target. The development of novel molecular-targeted anti-tumor drugs that can target the interior of tumor cells and specifically silence EGFR expression is valuable and promising. In this work, a promising anti-tumor conjugate comprising methoxy-modified EGFR siRNA and cyclic arginine-glycine-aspartic acid (cRGD) peptides, which selectively bind to αvβ3 integrins, was synthesized and examined. To prepare cRGD-EGFR siRNA (cRGD-siEGFR), cRGD was covalently conjugated to the 5′-end of an siRNA sense strand using a thiol-maleimide linker. The cellular uptake and cytotoxicity of cRGD-siEGFR in vitro were tested using an αvβ3-positive U87MG cell line. In vivo bio-distribution, anti-tumor activity, immunogenicity and toxicity were investigated in a nude mouse tumor model through repeated i.v. administration of cRGD-siEGFR (7 times over a 48 h interval). Analyses of in vitro data showed that cRGD-siEGFR silenced EGFR expression effectively, with high tumor targeting ability. Administration of cRGD-siEGFR to tumor-bearing nude mice led to significant inhibition of tumor growth, obvious reduction of EGFR expression and down-regulation of EGFR mRNA and protein in tumor tissue. Furthermore, serum biochemistry and pathological section evaluation did not indicate any serious toxicity of cRGD-siEGFR in vivo. cRGD-siEGFR is likely a promising candidate with high targeting ability, substantial anti-tumor effects and low toxicity in vitro and in vivo.

α‑lipoic acid protects against cerebral ischemia/reperfusion‑induced injury in rats

It is well established that the brain is sensitive to ischemia/reperfusion (I/R)‑induced injury. α‑lipoic acid (LA), a free radical scavenger and antioxidant, has a neuroprotective effect against cerebral I/R‑induced injury, however, the underlying mechanisms remain to be elucidated. Therefore, the present study was undertaken to evaluate whether LA was able to protect against cerebral I/R‑induced injury and to examine the potential mechanisms. The neuroprotective effects of LA were investigated in a rat model of transient focal ischemia induced by middle cerebral artery occlusion (MCAO) followed by reperfusion. Adult male Sprague‑Dawley rats were randomly assigned into the sham, cerebral I/R injury model and model plus LA groups. Cerebral I/R injury was induced by 90 min MCAO followed by reperfusion for 24 h. Cerebral infarct size was detected by 2,3,5‑triphenyltetrazolium chloride staining. Neurological deficit score (NDS), brain water content and oxidative parameters, including malondialdehyde (MDA), nitric oxide (NO), total antioxidant capacity (T‑AOC) and superoxide dismutase (SOD) were measured. The expression of cleaved caspase‑3, brain‑derived neurotrophic factor (BDNF), phosphatidylinositol‑4,5‑bisphosphate 3‑kinase (PI3K), p‑Akt and phosphorylated extracellular signal‑regulated kinase 1/2 (p‑ERK1/2) were also analyzed using western blotting. The present study demonstrated that pretreatment with LA significantly decreased the infarction size, brain water content and improved NDS. LA reversed the levels of oxidative parameters, including MDA, NO, T‑AOC and SOD to their normal state in rat brains following cerebral I/R. Furthermore, the expression of cleaved caspase‑3 markedly decreased and the expression of BDNF, PI3K, p‑Akt and p‑ERK1/2 significantly increased following administration of LA. On the basis of these findings, it was concluded that LA protected the brain from cerebral I/R damage by attenuation of oxidative stress and caspase‑dependent apoptosis. Furthermore, LA exerts its neuroprotective effects potentially through activation of the BDNF‑PI3K/Akt‑ERK1/2 pathway.

Something between the amazing functions and various morphologies of self-assembling peptides materials in the medical field

In recent years, there are a growing number of researches in the field of self-assembling peptides. Due to their diversity structures and the promising applications, self-assembling peptides have already become the focus of studies in the fields of materials and biological science. Some amazing functions of these peptides in the medical field caught our attention, such as tissue repair and regeneration, therapeutic delivery, haemostasis, antimicrobial and so on. There are different morphologies of self-assembling peptides in different functions. This review provides an overview of the relationship between some amazing functions and various morphologies of self-assembling peptides principally. Furthermore, the mechanisms of peptide self-assembly are also discussed.

α-lipoic acid protects against hypoxia/reoxygenation-induced injury in human umbilical vein endothelial cells through suppression of apoptosis and autophagy

α-lipoic acid (ALA) is known as a powerful antioxidant, which has been reported to have protective effects against various cardiovascular diseases. The present study aimed to determine whether ALA pre- or post-treatment induced protective effects against hypoxia/reoxygenation-induced injury via inhibition of apoptosis and autophagy in human umbilical vein endothelial cells (HUVECs). In order to simulate the conditions of hypoxia/reoxygenation, HUVECs were subjected to 4 h of oxygen-glucose deprivation (OGD) followed by 12 h of reoxygenation. For the pre-treatment, ALA was added to the buffer 12 h prior to OGD, whereas for the post-treatment, ALA was added at the initiation of reoxygenation. The results demonstrated that ALA pre- or post-treatment significantly reduced lactate dehydrogenase (LDH) release induced through hypoxia/reoxygenation in HUVECs in a dose-dependent manner; of note, 1 mM ALA pre- or post-treatment exhibited the most potent protective effects. In addition, ALA significantly reduced hypoxia/reoxygenation-induced loss of mitochondrial membrane potential, apoptosis and the expression of cleaved caspase-3 in HUVECs. In the presence of the specific autophagy inhibitor 3-methyladenine, hypoxia/reoxygenation-induced apoptosis was significantly reduced. Furthermore, the formation of autophagosomes, cytosolic microtubule-associated protein 1A/1B-light chain 3 ratio and beclin1 levels significantly increased following hypoxia/reoxygenation injury; however, all of these effects were ameliorated following pre- or post-treatment with ALA. The results of the present study suggested that ALA may provide beneficial protection against hypoxia/reoxygenation-induced injury via attenuation of apoptosis and autophagy in HUVECs.

Protective effect of Gelofusine against cRGD-siRNA-induced nephrotoxicity in mice

Abstract Based on successful targeting to the αvβ3 integrin of cyclic arginine–glycine–aspartic acid (cRGD), cRGD-conjugated small interfering RNA (siRNA) exhibits tumor targeting and has become a new treatment strategy for solid tumors. However, the nephrotoxicity caused by its renal retention limits its clinical application. Here, we evaluated the protective effect of Gelofusine against cRGD-conjugated siRNA-induced nephrotoxicity in mice. Male Kunming mice (six per group) were either co-injected with Gelofusine and cRGD-siRNA or injected with cRGD-siRNA alone. After administration of these treatments five times, creatinine and blood urea nitrogen (BUN) levels were determined. Hematoxylin–eosin staining (HE staining) and transferase dUTP nick end labeling (TUNEL) analysis were used to compare the difference in renal damage between the groups. Additionally, fluorescence imaging was used to observe the distribution of cRGD-siRNA in vivo. The group co-injected with Gelofusine and cRGD-siRNA displayed lower creatinine and BUN levels than the cRGD-siRNA-alone group and showed less renal damage upon HE staining and TUNEL analysis. Gelofusine decreased the retention time and accelerated the elimination of cRGD-siRNA from the organs, as observed in the fluorescence images. These data indicate that Gelofusine significantly increased the excretion of cRGD-conjugated siRNA and reduced the associated renal damage.

Letter regarding Chang et al.: The safety and efficacy of minimally invasive discectomy: a meta-analysis of prospective randomised controlled trials

To the Editor, We read with great interest the article by Chang et al. [1] entitled “The safety and efficacy of minimally invasive discectomy: a meta-analysis of prospective randomised controlled trials” published in International Orthopaedics. In this meta-analysis, Chang et al. compared the safety and efficacy of minimally invasive discectomy with standard discectomy. Liu et al. [2] provided some questions regarding this article. However, after thoroughly reading the article and analysing the included studies, we found other problems that should be of great concern. First, Chang et al. stated that only randomised controlled trials comparingminimally invasive discectomywith standard discectomy for lumbar disc herniation were included. However, this meta-analysis incorrectly included a nonrandomised controlled trial. Michael et al. [3] reported their study was a prospective controlled trial rather than a randomised controlled trial. Second, repetitive data existed in this meta-analysis. Arts et al. [4–6] performed a randomised controlled trial and reported their study three times. The differences among the three articles were the periods of follow-up and variables. However, Chang et al. included them as three randomised controlled trials and repeatedly pooled them in Figure 4 and Figure 6. Obviously, this was not reasonable. Third, the number of patients was not in accordance with the original study in one study. Michael et al. [3] reported that 20 patients were actually included in their study, ten patients in the minimally invasive lumbar microdiscectomy group and ten patients in the traditional open discectomy group. However, Chang et al. inputted the number of patients as 30 respectively in the two groups. Fourth, methodological quality of all 16 included studies was assessed as either low risk or unclear risk, which was incredible. In fact, the assessment of methodological quality was inappropriate in this meta-analysis. For example, Huang et al. [7] did not report the allocation concealment and blinding of participants and personnel in their study. However, Chang et al. incorrectly assessed the selection bias and performance bias as low risk. In conclusion, the results of the meta-analysis by Chang et al. should be interpreted with caution. They should carefully and scientifically perform the meta-analysis and solve all these questions in their article.