Injection Of siRNA Research Articles

POU2F1/DNMT3a Pathway Participates in Neuropathic Pain by Hypermethylation-Mediated LRFN4 Downregulation Following Oxaliplatin Treatment.

Evidence demonstrates that DNA methylation is associated with the occurrence and development of various neurological diseases. However, the potential target genes undergoing DNA methylation, as well as their involvement in the chemotherapy drug oxaliplatin-induced neuropathic pain, are still unclear. Here, Lrfn4, which showed hypermethylation in the promoter regions, was screened from the SRA methylation database (PRJNA587622) following oxaliplatin treatment. MeDIP and qPCR assays identified that oxaliplatin treatment increased the methylation in Lrfn4 promoter region and decreased the expression of LRFN4 in the spinal dorsal horn. The assays with gain and loss of LRFN4 function demonstrated that LRFN4 downregulation in spinal dorsal horn contributed to the oxaliplatin-induced mechanical allodynia and cold hyperalgesia. Moreover, oxaliplatin treatment increased the DNA methyltransferases DNMT3a expression and the interaction between DNMT3a and Lrfn4 promoter, while inhibition of DNMT3a prevented the downregulation of LRFN4a induced by oxaliplatin. We also observed that the transcriptional factor POU2F1 can bind to the predicted sites in DNMT3a promoter region, oxaliplatin treatment upregulated the expression of transcriptional factor POU2F1 in dorsal horn neurons. Intrathecal injection of POU2F1 siRNA prevented the DNMT3a upregulation and the LRFN4 downregulation induced by oxaliplatin. Additionally, intrathecal injection of DNMT3a siRNA or POU2F1 siRNA alleviated the mechanical allodynia induced by oxaliplatin. These findings suggested that transcription factor POU2F1 upregulated the expression of DNMT3a, which subsequently decreased LRFN4 expression through hypermethylation modification in spinal dorsal horn, thereby mediating neuropathic pain following oxaliplatin treatment.

Hmgb1 Silencing in the Amygdala Inhibits Pain-Related Behaviors in a Rat Model of Neuropathic Pain.

Chronic pain presents a therapeutic challenge due to the highly complex interplay of sensory, emotional-affective and cognitive factors. The mechanisms of the transition from acute to chronic pain are not well understood. We hypothesized that neuroimmune mechanisms in the amygdala, a brain region involved in the emotional-affective component of pain and pain modulation, play an important role through high motility group box 1 (Hmgb1), a pro-inflammatory molecule that has been linked to neuroimmune signaling in spinal nociception. Transcriptomic analysis revealed an upregulation of Hmgb1 mRNA in the right but not left central nucleus of the amygdala (CeA) at the chronic stage of a spinal nerve ligation (SNL) rat model of neuropathic pain. Hmgb1 silencing with a stereotaxic injection of siRNA for Hmgb1 into the right CeA of adult male and female rats 1 week after (post-treatment), but not 2 weeks before (pre-treatment) SNL induction decreased mechanical hypersensitivity and emotional-affective responses, but not anxiety-like behaviors, measured 4 weeks after SNL. Immunohistochemical data suggest that neurons are a major source of Hmgb1 in the CeA. Therefore, Hmgb1 in the amygdala may contribute to the transition from acute to chronic neuropathic pain, and the inhibition of Hmgb1 at a subacute time point can mitigate neuropathic pain.

Effect of locoregional injection of RNA oligonucleotide targeting carbohydrate sulfotransferase 15 on both tumoral and lymph node MDSCs and T cell infiltration in pancreatic tumor in mice.

e16310 Background: Carbohydrate sulfotransferase 15 (CHST15) is a proteoglycan biosynthetic enzyme and is responsible for tumor matrix remodeling. Intratumoral injection of CHST15 siRNA was shown to increase tumor-infiltrating T cells in patients with pancreatic cancer in the phase I/IIa study. However, the mechanism underlying the enhanced T cell accumulation is not fully explored. Here we investigated the effect of locally injected CHST15 siRNA on modulating tumor immune microenvironment in mice. Methods: For in vivo pharmacokinetics after a single local injection of CHST15 siRNA to normal C57BL/6J mice, RNA oligonucleotides were isolated from tissues and detected by HPLC. Mouse pancreatic cancer KPC cells were implanted subcutaneously into the left hind footpad of syngeneic C57BL/6J mice and the tumor growth was monitored. From week 1, intratumoral injections with 1 mg/mL of CHST15 siRNA or vehicle were performed twice a week for 2 weeks and mice were sacrificed at week 3. In separate experiments, human BxPC-3 cells were implanted subcutaneously in T cell-deficient nude mice, and the effect of intratumoral injections of CHST15 siRNA or vehicle for 2 weeks was also investigated. Immunohistochemical staining was performed for the tumor and tumor-draining lymph node (TDLN). Results: CHST15 siRNA was detectable in not only the local sites but also the draining LNs of normal mice 1 and 4 hours after locoregional injection. In the KPC-implanted syngeneic mouse model, CHST15 siRNA showed significant tumor growth inhibition compared to the vehicle. In tumor, CHST15 was expressed by tumor cells, and to a lesser extent, Ly6C/G+ MDSCs. CHST15 siRNA altered tissue remodeling by repressing dense fibrotic architecture and NETosis. CHST15 siRNA significantly diminished Ly6C/G+ MDSCs while increasing CD4+ and CD8+ T cells. In TDLN, CHST15 was expressed by Ly6C/G+ MDSCs. Locally injected CHST15 siRNA markedly diminished Ly6C/G+ MDSCs, while increasing Ki-67+ proliferating CD4+ and CD8+ T cells. In the BxPC-3-implanted nude mouse model, locally injected CHST15 siRNA repressed excessive fibrosis and NETosis in tumor, diminishing both tumoral and TDLN MDSCs, but tumor growth inhibition was marginal. These results suggest that locally injected CHST15 siRNA could enter into TDLN, directly diminishing CHST15+ MDSCs, and expanded T cells. Tumor growth inhibition was largely T-cell medicated. Conclusions: The present study demonstrated that CHST15 siRNA locally and remotely diminished MDSCs, leading to the enhancement of anti-tumor T cell activity. A novel role of locally-delivered RNA oligonucleotide is suggested.

Reduction of SIRT1-Mediated Epigenetic Upregulation of Nav1.7 Contributes to Oxaliplatin-Induced Neuropathic Pain

BACKGROUND: Clinically, neuropathic pain is a severe side effect of oxaliplatin chemotherapy, which usually leads to dose reduction or cessation of treatment. Due to the unawareness of detailed mechanisms of oxaliplatin-induced neuropathic pain, it is difficult to develop an effective therapy and limits its clinical use. OBJECTIVES: The aim of the present study was to identify the role of sirtuin 1 (SIRT1) reduction in epigenetic regulation of the expression of voltage-gated sodium channels 1.7 (Nav1.7) in the dorsal root ganglion (DRG) during oxaliplatin-induced neuropathic pain. STUDY DESIGN: Controlled animal study. SETTING: University laboratory. METHODS: The von Frey test was performed to evaluate pain behavior in rats. Real-time quantitative polymerase chain reaction, western blotting, electrophysiological recording, chromatin immunoprecipitation, and small interfering RNA (siRNA) were used to illustrate the mechanisms. RESULTS: In the present study, we found that both the activity and expression of SIRT1 were significantly decreased in rat DRG following oxaliplatin treatment. The activator of SIRT1, resveratrol, not only increased the activity and expression of SIRT1, but also attenuated the mechanical allodynia following oxaliplatin treatment. In addition, local knockdown of SIRT1 by intrathecal injection of SIRT1 siRNA caused mechanical allodynia in naive rats. Besides, oxaliplatin treatment enhanced the action potential firing frequency of DRG neurons and the expression of Nav1.7 in DRG and activation of SIRT1 by resveratrol reversed this effect. Furthermore, blocking Nav1.7 by ProTx II (a selective Nav1.7 channel blocker) reversed oxaliplatin-induced mechanical allodynia. In addition, histone H3 hyperacetylation at the Nav1.7 promoter in DRG of rats following oxaliplatin treatment was significantly suppressed by activation of SIRT1 with resveratrol. Moreover, both the expression of Nav1.7 and histone H3 acetylation at the Nav1.7 promoter were upregulated in the DRG by local knockdown of SIRT1 with SIRT1 siRNA in naive rats. LIMITATIONS: More underlying mechanism(s) of SIRT1 reduction after oxaliplatin treatment needs to be explored in future research. CONCLUSIONS: These findings suggest that reduction of SIRT1-mediated epigenetic upregulation of Nav1.7 in the DRG contributes to the development of oxaliplatin-induced neuropathic pain in rats. The intrathecal drug delivery treatment of activating SIRT1 might be a novel therapeutic option for oxaliplatin-induced neuropathic pain. KEY WORDS: SIRT1, neuropathic pain, dorsal root ganglion, NAV1.7 voltage-gated sodium channel, oxaliplatin

Pum3 is dispensable for mouse oocyte maturation and embryo development in vitro.

Pumilio3 (Pum3), an evolutionarily distant homologue of the classical RNA-binding protein PUF (PUMILIO and FBF) family member, is also involved in the process of RNA metabolism through post-transcriptional regulation. However, the functions of Pum3 in mouse oocyte maturation and preimplantation embryonic development have not been elucidated. By comparing RNA levels in different tissues, we found that Pum3 was widely expressed in multiple tissues, but moderately predominant in the ovary. Histochemical staining suggested that the PUM3 protein exhibits positive signals in oocytes, granulosa cells and theca cells of different follicle stages. Oocyte immunofluorescence results showed a slightly higher level of PUM3 protein in metaphase II compared with the germinal vesicle (GV) stage. After knockdown of Pum3 in GV oocytes using siRNA injection (siPUM3), no obvious defect was observed in the processes of GV breakdown and polar body extrusion during in vitro maturation (IVM) for the siPum3 oocytes. Compared with the control group, the siPUM3 group displayed no significant abnormality in the cleavage and blastocyst formation rate of these fertilized oocytes. Therefore, we can conclude that depletion of Pum3 does not affect mouse oocyte maturation and early embryonic development in vitro.

Vascular cell-adhesion molecule 1 (VCAM-1) regulates JunB-mediated IL-8/CXCL1 expression and pathological neovascularization

Vascular adhesion molecules play an important role in various immunological disorders, particularly in cancers. However, little is known regarding the role of these adhesion molecules in proliferative retinopathies. We observed that IL-33 regulates VCAM-1 expression in human retinal endothelial cells and that genetic deletion of IL-33 reduces hypoxia-induced VCAM-1 expression and retinal neovascularization in C57BL/6 mice. We found that VCAM-1 via JunB regulates IL-8 promoter activity and expression in human retinal endothelial cells. In addition, our study outlines the regulatory role of VCAM-1-JunB-IL-8 signaling on retinal endothelial cell sprouting and angiogenesis. Our RNA sequencing results show an induced expression of CXCL1 (a murine functional homolog of IL-8) in the hypoxic retina, and intravitreal injection of VCAM-1 siRNA not only decreases hypoxia-induced VCAM-1-JunB-CXCL1 signaling but also reduces OIR-induced sprouting and retinal neovascularization. These findings suggest that VCAM-1-JunB-IL-8 signaling plays a crucial role in retinal neovascularization, and its antagonism might provide an advanced treatment option for proliferative retinopathies.

Knockdown of mechanosensitive adaptor Hic-5 ameliorates post-traumatic osteoarthritis in rats through repression of MMP-13

Osteoarthritis (OA) is the most common joint disease associated with articular cartilage destruction. Matrix metalloproteinase-13 (MMP-13) has an essential role in OA pathogenesis by degradation of collagen II, a major component of articular cartilage. Hydrogen peroxide-inducible clone-5 (Hic-5; TGFB1I1), a transforming growth factor-β-inducible mechanosensor, has previously been reported to promote OA pathogenesis by upregulating MMP-13 expression in mouse osteoarthritic lesions. In our current study, immunohistochemical analysis showed that Hic-5 protein expression was increased in human OA cartilage compared with normal cartilage. Functional experiments demonstrated that Hic-5 and MMP-13 expression was increased by mechanical stress, and mechanical stress-induced MMP-13 expression was suppressed by Hic-5 siRNA in human chondrocytes. Moreover, intracellular localization of Hic-5 shifted to the nucleus from focal adhesions in human chondrocytes subjected to mechanical stress, and nuclear Hic-5 increased MMP-13 gene expression. In vivo, intra-articular injection of Hic-5 siRNA decreased the Osteoarthritis Research Society International score and MMP-13 protein expression in articular cartilage of OA rats. Our findings suggest that Hic-5 regulates transcription of MMP-13 in human chondrocytes, and Hic-5 may be a novel therapeutic target for OA because OA progression was suppressed by intra-articular injection of Hic-5 siRNA in rats.

NADPH oxidase 4 contributes to oxidative stress in a mouse model of myocardial infarction

Oxidative stress closely related to the progression and severity of myocardial infarction (MI). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) is one of the major enzymes that generate reactive oxygen species (ROS) in cardiovascular system. Here, we aim to elucidate the pathological role of NOX4 in MI. MI mouse model was created by the coronary artery ligation. NOX4 was specifically knocked down in heart through intramyocardial injection of siRNA. NOX4 expression and oxidative stress indicators were determined at different time points using qRT-PCR, Western blot, and ELISA, and then analyzed by Pearson’s correlation. Cardiac function was evaluated by using echocardiographic technique. NOX4 was upregulated in myocardial tissues of MI mice, which positively correlated with the elevation of oxidative stress indicators. Knockdown of NOX4 in heart significantly reduced the production of ROS and the level of oxidative stress in left ventricle tissues, which was accompanied by significant improvement of cardiac function in MI mice. Selective knockdown of NOX4 in heart attenuates MI-induced oxidative stress and improves cardiac function, suggesting inhibition of NOX4/ROS axis in heart using siRNA is a potential therapeutic treatment for MI-induced cardiac dysfunction.

Inhibition of AIF-1 alleviates laser-induced macular neovascularization by inhibiting endothelial cell proliferation via restrained p44/42 MAPK signaling pathway

Age-related macular degeneration (AMD) is a leading blinding disease worldwide, and macular neovascularization (MNV) is a common complication encountered in the advanced stages of AMD. While the underlying causes of MNV remain elusive, aberrant multiplication of choroidal endothelial cells (CECs) and increased vascular endothelial growth factor (VEGF) are thought to play significant roles in the occurrence and development of MNV. Allograft inflammatory factor-1(AIF-1) is a crucial regulatory factor of vascular tubular structure formation and growth, involving the proliferation and migration of vascular endothelial cells and various tumor cells. This study aimed to understand how AIF-1 effects the proliferation of CECs and the subsequent progression of MNV. To study this, a mouse MNV model was established through laser injury, and the AIF-1 expression levels were then measured using western blot and immunohistochemistry. AIF-1 siRNA was intravitreally injected to silence AIF-1 gene expression. Western blot and choroidal flat mount were performed to measure the progression of MNV and proliferation of the CECs. These results showed that the protein expression of AIF-1 was significantly elevated in the laser-induced mouse MNV model, and the expression trend was consistent with VEGF. The protein level of AIF-1 was significantly decreased after the intravitreal injection of AIF-1 siRNA, the damage range of laser lesions was significantly reduced, and the proliferation of endothelial cells was inhibited. Knockdown of the AIF-1 gene significantly inhibited the expression of mitogen-activated protein kinase p44/42 in MNV lesions. In summary, this research demonstrates that AIF-1 promoted MNV progression by promoting the proliferation of CECs and that silencing AIF-1 significantly ameliorates MNV progression in mouse models, which may act through the p44/42 MAPK signaling pathway. AIF-1 could be a new potential molecular target for MNV.

Phosphate NIMA-Related Kinase 2-Dependent Epigenetic Pathways in Dorsal Root Ganglion Neurons Mediates Paclitaxel-Induced Neuropathic Pain.

The microtubule-stabilizing drug paclitaxel (PTX) is an important chemotherapeutic agent for cancer treatment and causes peripheral neuropathy as a common side effect that substantially impacts the functional status and quality of life of patients. The mechanistic role for NIMA-related kinase 2 (NEK2) in the progression of PTX-induced neuropathic pain has not been established. Adult male Sprague-Dawley rats intraperitoneally received PTX to induce neuropathic pain. The protein expression levels in the dorsal root ganglion (DRG) of animals were measured by biochemical analyses. Nociceptive behaviors were evaluated by von Frey tests and hot plate tests. PTX increased phosphorylation of the important microtubule dynamics regulator NEK2 in DRG neurons and induced profound neuropathic allodynia. PTX-activated phosphorylated NEK2 (pNEK2) increased jumonji domain-containing 3 (JMJD3) protein, a histone demethylase protein, to specifically catalyze the demethylation of the repressive histone mark H3 lysine 27 trimethylation (H3K27me3) at the Trpv1 gene, thereby enhancing transient receptor potential vanilloid subtype-1 (TRPV1) expression in DRG neurons. Moreover, the pNEK2-dependent PTX response program is regulated by enhancing p90 ribosomal S6 kinase 2 (RSK2) phosphorylation. Conversely, intrathecal injections of kaempferol (a selective RSK2 activation antagonist), NCL 00017509 (a selective NEK2 inhibitor), NEK2-targeted siRNA, GSK-J4 (a selective JMJD3 inhibitor), or capsazepine (an antagonist of TRPV1 receptor) into PTX-treated rats reversed neuropathic allodynia and restored silencing of the Trpv1 gene, suggesting the hierarchy and interaction among phosphorylated RSK2 (pRSK2), pNEK2, JMJD3, H3K27me3, and TRPV1 in the DRG neurons in PTX-induced neuropathic pain. pRSK2/JMJD3/H3K27me3/TRPV1 signaling in the DRG neurons plays as a key regulator for PTX therapeutic approaches.

Multivalent mannose-conjugated siRNA causes robust gene silencing in pancreatic macrophages in vivo

Nucleic acid therapeutics have been utilized for gene regulation, and their recent advancement has led to approval of novel drugs for liver-related disorders. However, systemic extrahepatic delivery remains challenging. Here, we report newly designed mannose-conjugated oligonucleotides for delivering oligonucleotides to macrophages by leveraging the mannose receptor, C-type 1 (MRC1, CD206), which is abundantly expressed in macrophages. We investigated the relationship between cellular uptake and multivalency (mono to tetra) of mannose ligands or linker length and selected a trivalent-mannose ligand. Trivalent-mannose (Man3)-conjugated siRNA induced concentration-dependent gene silencing in both human CD206-overexpressing cells and human macrophages in vitro. After subcutaneous injection into mice, we observed a high distribution of Man3-conjugated oligonucleotides in the liver and pancreata as well as cellular uptake into Kupffer cells and pancreatic macrophages. A single subcutaneous injection of Man3-conjugated siRNA (10 mg/kg) targeting β2-microglobulin (B2M) silenced B2m mRNA expression by ∼50% and decreased its protein levels in mouse pancreatic macrophages compared to those in saline-treated mice. Of note, multiple subcutaneous injections decreased B2m gene expression and B2M protein levels by ∼80% and ∼85%, respectively. These results show that mannose-conjugation with oligonucleotides is expected to help deliver oligonucleotides to macrophages and regulate gene expression in vivo, particularly in the pancreas.

Dnmt1/Tet2-mediated changes in Cmip methylation regulate the development of nonalcoholic fatty liver disease by controlling the Gbp2-Pparγ-CD36 axis

Dynamic alteration of DNA methylation leads to various human diseases, including nonalcoholic fatty liver disease (NAFLD). Although C-Maf-inducing protein (Cmip) has been reported to be associated with NAFLD, its exact underlying mechanism remains unclear. Here, we aimed to elucidate this mechanism in NAFLD in vitro and in vivo. We first identified alterations in the methylation status of the Cmip intron 1 region in mouse liver tissues with high-fat high-sucrose diet-induced NAFLD. Knockdown of DNA methyltransferase (Dnmt) 1 significantly increased Cmip expression. Chromatin immunoprecipitation assays of AML12 cells treated with oleic and palmitic acid (OPA) revealed that Dnmt1 was dissociated and that methylation of H3K27me3 was significantly decreased in the Cmip intron 1 region. Conversely, the knockdown of Tet methylcytosine dioxygenase 2 (Tet2) decreased Cmip expression. Following OPA treatment, the CCCTC-binding factor (Ctcf) was recruited, and H3K4me3 was significantly hypermethylated. Intravenous Cmip siRNA injection ameliorated NAFLD pathogenic features in ob/ob mice. Additionally, Pparγ and Cd36 expression levels were dramatically decreased in the livers of ob/ob mice administered siCmip, and RNA sequencing revealed that Gbp2 was involved. Gbp2 knockdown also induced a decrease in Pparγ and Cd36 expression, resulting in the abrogation of fatty acid uptake into cells. Our data demonstrate that Cmip and Gbp2 expression levels are enhanced in human liver tissues bearing NAFLD features. We also show that Dnmt1–Trt2/Ctcf-mediated reversible modulation of Cmip methylation regulates the Gbp2–Pparγ–Cd36 signaling pathway, indicating the potential of Cmip as a novel therapeutic target for NAFLD.

SCN5A promotes the growth and lung metastasis of triple-negative breast cancer through EF2-kinase signaling

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer often associated with early metastasis, poor prognosis, and shortened survival. The main causes of its high mortality level are attributed to significant genetic heterogenity, rapid development of chemoresistance, and a limited number of targeted therapeutics. Therefore, as described herein, we investigated the role of the SCN5A gene encoding the voltage-gated sodium ion channel NaV1.5 in vitro and in vivo in TNBC models. Cell proliferation, colony formation, invasion, western blot and in vivo analyzes were performed. We found that SCN5A gene/NaV1.5 and its neonatal spliced isoform nNaV1.5 were markedly upregulated in TNBC cells and that they promoted cell proliferation, colony formation, motility/migration, and invasion. Therapeutic targeting of NaV1.5/nNaV1.5 via systemic injection of specific siRNAs incorporated in single-lipid nanoparticles suppressed tumor growth and lung metastasis in orthotopic TNBC models. We further demonstrated that targeting of NaV1.5/nNaV1.5 expression associated with eukaryotic elongation factor-2 kinase (EF2K) expression. This approach led to the inhibition EF2K's oncogenic signaling and its clinically significant downstream targets, including integrin β1/Src/focal adhesion kinase, c-myc, cyclin D1, phosphoinositide 3-kinase/Akt, p-ERK, p-IF2α and p-PI3K. Targeting of NaV1.5/nNaV1.5 also induced robust apoptosis in TNBCs in vivo. Inhibition of both isoforms of SCN5A in TNBC cells enhanced the effect of first-line chemotherapeutics such as paclitaxel. In conclusion, our results provide the first evidence that expression of SCN5A has an important role in the growth of primary and lung-metastatic TNBCs and that targeting of NaV1.5/nNaV1.5 by systemically administered therapeutics is a potential molecularly targeted strategy to control TNBC progression and may enhance the efficacy of currently used chemotherapeutics.

Inhibition of METTL5 improves preimplantation development of mouse somatic cell nuclear transfer embryos.

Several factors affect the reprogramming efficiency of nuclear transfer embryos. This study shows that inhibiting 18S rRNA m6A methyltransferase METTL5 during nuclear transfer can improve the developmental rate of nuclear transfer embryos. N6-methyladenosine (m6A) is one of the most important epigenetic modifications in eukaryotic RNAs, which regulates development and diseases. It is identified by several proteins. Methyltransferase-like 5 (METTL5), an enzyme that methylates 18S rRNA m6A, controls the translation of proteins and regulates pluripotency in embryonic stem cells. However, the functions of METTL5 in embryonic development have not been explored. Here, we found that Mettl5 was upregulated in somatic cell nuclear transfer (SCNT) embryos compared with normal fertilized embryos. Therefore, we hypothesized that METTL5 knockdown during the early stage of SCNT would improve the developmental rate of SCNT embryos. Notably, injection of Mettl5 siRNA (si-Mettl5) into enucleated oocytes during nuclear transfer increased the rate of development and the number of cells in blastocysts. Moreover, inhibition of METTL5 reduced the activity of phosphorylated ribosomal protein S6, decreased the levels of the repressive histone modification H3K27me3 and increased the expression of activating histone modifications H3K27ac and H3K4me3 and mRNA levels of some 2-cell-specific genes. These results expand our understanding of the role of METTL5 in early embryonic development and provide a novel idea for improving the efficiency of nuclear transfer cloning.

Expression patterns and biological function of BCL2L10 during mouse preimplantation development

BCL2-like 10 (BCL2L10) is abundantly expressed in mammalian oocytes and plays a crucial role in the completion of oocyte meiosis. However, the expression patterns of BCL2L10 and its biological functions during preimplantation development have not been well characterized. Here, we investigated the spatiotemporal expressions of Bcl2l10 during mouse preimplantation development using RT-qPCR and immunofluorescence and its biological function using siRNA and morpholino injection into pronuclear embryos. Results from RT-qPCR showed that Bcl2l10 was highly expressed in the metaphase Ⅱ-stage oocytes and pronuclear-stage embryos, but expression markedly decreased from the two-cell stage onwards and was no longer detected at the four-cell stage and beyond. Immunofluorescence staining showed that BCL2L10 was detectable throughout preimplantation development and localized in the cytoplasm and nuclei. Knocking down Bcl2l10 resulted in a reduced blastocyst formation rate (P < 0.01) and decreased expression of OCT4, NANOG, and SOX17 (P < 0.05). We concluded that the role of BCL2L10 is strongly associated with developmental competence of preimplantation mouse embryos.

Plasmacytoma Variant Translocation 1 (PVT1) Gene as a Potential Novel Target for the Treatment of Diabetic Nephropathy.

Introduction: Diabetic nephropathy (DN), a severe microvascular complication in patients with diabetes, is clinically characterized by progressive decline in glomerular filtration rate (GFR). DN is the most common cause of end-stage renal disease (ESRD), and has a consistently high mortality rate. Despite the fact that the prevalence of DN is increasing worldwide, the molecular mechanism underlying the pathogenesis of DN is not fully understood. Previous studies indicated PVT1 as a key determinant of ESRD as well as a mediator of extracellular matrix (ECM) accumulation in vitro. More investigations into the role of PVT1 in DN development are needed. Objectives: To study the effect of PVT1 silencing on progression of DN in diabetic male C57BL/6 mice at early, intermediate and relatively advanced ages. Methods: Diabetic mice were treated with either scramble-siRNA (DM + siRNA (scramble)) or PVT1-siRNA (DM + siRNA (PVT1)), whereas the control mice were normal mice without siRNA injection (Control). Blood, urine and kidney were collected at the age of 9 (young), 16 (middle-aged) or 24 (old) weeks old. Kidney function, histology and molecular gene expression were evaluated. Results: Our findings showed that silencing of PVT1 reduced kidney hypertrophy, proteinuria (UAE, UACR, UPE, UPCR), serum creatinine, serum TGF-β1, serum insulin decline, glomerular and mesangial areas, and increased creatinine clearance in diabetic mice to levels closer to the age-matched controls. Also, silencing of PVT1 markedly suppressed the upregulation of PAI-1, TGF-β1, FN1, COL4A1, and downregulation of BMP7. Conclusion: Silencing of PVT1 ameliorates DN in terms of kidney function and histology in diabetic mice. The renoprotection is attributed to the reduction in ECM accumulation, TGF-β1 elevation and insulin decline. PVT1 is suggested to play an important role in ECM accumulation which makes it a possible target for the treatment of DN.

A Comparison of RNA Interference via Injection and Feeding in Honey Bees.

Simple SummaryRNA interference is an important way to analyze gene function. It is also widely used in honey bees. It was thought that RNA interference with honey bee brain genes could only be achieved by injection. However, this method of injection is very complicated and can easily cause damage to bees. Recent studies have shown that specially treated siRNA can knockdown brain genes in animals by feeding them. Therefore, we used different approaches to deliver modified and unmodified siRNAs to investigate what methods can successfully interfere with honey bee brain genes. The results of this study are helpful to improve the application of RNA interference techniques in honey bees and other insects.RNA interference (RNAi) has been used successfully to reduce target gene expression and induce specific phenotypes in several species. It has proved useful as a tool to investigate gene function and has the potential to manage pest populations and reduce disease pathogens. However, it is not known whether different administration methods are equally effective at interfering with genes in bees. Therefore, we compared the effects of feeding and injection of small interfering RNA (siRNA) on the messenger RNA (mRNA) levels of alpha-aminoadipic semialdehyde dehydrogenase (ALDH7A1), 4-coumarate-CoA ligase (4CL), and heat shock protein 70 (HSP70). Both feeding and injection of siRNA successfully knocked down the gene but feeding required more siRNA than the injection. Our results suggest that both feeding and injection of siRNA effectively interfere with brain genes in bees. The appropriateness of each method would depend on the situation.

Priming of Solanum Tuberosum in vitro plants with PVY-specific interfering RNAs activates anti-viral resistance

Helper component-proteinase (HC-Pro) is a multifunctional suppressor protein synthesized by potato virus Y (PVY), which is able to neutralize the protective mechanisms of RNA interference (RNA-i) in potato plants, thereby causing systemic infection of the host plant and significant damage to the tuber material. This article demonstrates how one of the main functions of HC-Pro, the capture and retention of short interfering RNAs, 21-23 nt in size (siRNA) on the surface of protein subunits, can be used to create virus-resistant potato plants in vitro. The proposed method is based on the selective dissociation of the HC-Pro/siRNA complex from PVY-infected plants and siRNA-priming of healthy plants. Purified preparations of PVY-specific siRNAs were obtained using column gel filtration followed by immunoprecipitation and phenol-chloroform extraction. Injections of siRNA into the leaf plate of virus-free potato microplants, cultivars bred at Amanzholov University, led to a decrease in the accumulation of viral particles in the cytoplasm of plants inoculated with the wild PVY strain and the formation of resistance to PVY for the entire growing season.

Inhibition of NLRP1-Dependent Pyroptosis Prevents Glycogen Synthase Kinase-3β Overactivation-Induced Hyperphosphorylated Tau in Rats.

Our previous study indicated that inhibition of NLRP1-dependent pyroptosis could decrease intracerebroventricular (ICV) injection of a protein kinase A (PKA) agonist- or streptozotocin (STZ)-induced hyperphosphorylated tau. In this study, we used a glycogen synthase kinase-3β (GSK-3β) overactivation rat model to reconfirm our previous results. ICV injection of wortmannin (WT, a PI3K inhibitor) and GF-109203X (GFX, a PKC inhibitor) was used to induce overactivation of GSK-3β in rats. We injected NLRP1 siRNA together with WT/GFX to evaluate the effect of the inhibition of NLRP1-dependent neuronal pyroptosis on hyperphosphorylated tau. Our results indicated that ICV injection of NLRP1 siRNA prevented ICV-WT/GFX-induced neuronal death, further improving the spatial memory of the rats in the Morris water maze test. ICV injection of NLRP1 siRNA downregulated the expression of ASC, caspase-1, and GSDMD and the contents of IL-1β and IL-18 in rat brains. ICV injection of NLRP1 siRNA also decreased hyperphosphorylated tau and the activity of GSK-3β. Thus, these results support our previous study that NLRP1-dependent pyroptosis could enhance hyperphosphorylation of tau protein.

Reduction of Laser-Induced Choroidal Neovascularization in Mice With Erythropoietin RNA Interference

PurposeThe purpose of this study was to evaluate the pathological involvement of erythropoietin (EPO) in experimental choroidal neovascularization (CNV) and its association with neovascular age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV) in the Chinese population.MethodsTreatment effect of recombinant EPO protein were assessed by human umbilical vein endothelial cell (HUVEC) proliferation, migration, and tube formation, and ex vivo choroid-sprouting ability. The effect of intravitreal injection of Epo siRNA against neovascularization was evaluated in the laser-induced CNV mouse model. In addition, the association of EPO variants with neovascular AMD and PCV was determined.ResultsExogenous supplementation of EPO significantly enhanced the migration and tube formation of HUVECs and promoted ex vivo choroid sprouting in mouse retinal pigment epithelium (RPE)-choroid-sclera complex culture. In the experimental CNV mouse model, Epo expression was found to be significantly upregulated by 3.5-folds in RPE-choroid-sclera complex at day 10 after laser induction as compared to the baseline. Immunofluorescence analysis showed that Epo was mainly expressed around the vascular endothelial cells in the RPE-choroid-sclera complex. Intravitreal injection of siRNA targeting Epo reduced 40% Epo expression and 40% CNV lesion areas as compared to the scramble control. However, EPO variants were not associated with neovascular AMD nor PCV in the Chinese population.ConclusionsThis study revealed the promotion of human endothelial cell tube formation in vitro and choroid sprouting ex vivo by EPO, and the reduction of laser-induced CNV in vivo by Epo RNA interference.Translational RelevanceTargeting EPO could be a potential additional treatment for CNV-related diseases.