Comparative Long Non-Coding Transcriptome Analysis of Three Contrasting Barley Varieties in Response to Aluminum Stress.

Aluminum toxicity is a major abiotic stress on acidic soils, leading to restricted root growth and reduced plant yield. Long non-coding RNAs are crucial signaling molecules regulating the expression of downstream genes, particularly under abiotic stress conditions. However, the extent to which lncRNAs participate in the response to aluminum (Al) stress in barley remains largely unknown. Here, we conducted RNA sequencing of root samples under aluminum stress and compared the lncRNA transcriptomes of two Tibetan wild barley genotypes, XZ16 (Al-tolerant) and XZ61 (Al-sensitive), as well as the aluminum-tolerant cultivar Dayton. In total, 268 lncRNAs were identified as aluminum-responsive genes on the basis of their differential expression profiles under aluminum treatment. Through target gene prediction analysis, we identified 938 candidate lncRNA-messenger RNA (mRNA) pairs that function in a cis-acting manner. Subsequently, enrichment analysis showed that the genes targeted by aluminum-responsive lncRNAs were involved in diterpenoid biosynthesis, peroxisome function, and starch/sucrose metabolism. Further analysis of genotype differences in the transcriptome led to the identification of 15 aluminum-responsive lncRNAs specifically altered by aluminum stress in XZ16. The RNA sequencing data were further validated by RT-qPCR. The functional roles of lncRNA-mRNA interactions demonstrated that these lncRNAs are involved in the signal transduction of secondary messengers, and a disease resistance protein, such as RPP13-like protein 4, is probably involved in aluminum tolerance in XZ16. The current findings significantly contribute to our understanding of the regulatory roles of lncRNAs in aluminum tolerance and extend our knowledge of their importance in plant responses to aluminum stress.

Heart proteomic profiling discovers MYH6 and COX5B as biomarkers for sudden unexplained death.

Sudden unexplained death (SUD) is not uncommon in forensic pathology. Yet, diagnosis of SUD remains challenging due to lack of specific biomarkers. This study aimed to screen differentially expressed proteins (DEPs) and validate their usefulness as diagnostic biomarkers for SUD cases. We designed a three-phase investigation, where in the discovery phase, formalin-fixed paraffin-embedded (FFPE) heart specimens were screened through label-free proteomic analysis of cases dying from SUD, mechanical injury and carbon monoxide (CO) intoxication. A total of 26 proteins were identified to be DEPs for the SUD cases after rigorous criterion. Bioinformatics and Adaboost-recursive feature elimination (RFE) analysis further revealed that three of the 26 proteins (MYH6, COX5B and TNNT2) were potential discriminative biomarkers. In the training phase, MYH6 and COX5B were verified to be true DEPs in cardiac tissues from 29 independent SUD cases as compared with a serial of control cases (n = 42). Receiver operating characteristic (ROC) analysis illustrated that combination of MYH6 and COX5B achieved optimal diagnostic sensitivity (89.7 %) and specificity (84.4 %), with area under the curve (AUC) being 0.91. A diagnostic software based on the logistic regression formula derived from the training phase was then constructed. In the validation phase, the diagnostic software was applied to eight authentic SUD cases, seven (87.5 %) of which were accurately recognized. Our study provides a valid strategy towards practical diagnosis of SUD by integrating cardiac MYH6 and COX5B as dual diagnostic biomarkers.

fMRI signals in white matter rewire gray matter community organization.

Human brain gray matter (GM) has usually been clustered into multiple functional networks. The white matter (WM) fiber bundles are known to interconnect these networks simultaneously, engaging in numerous cognitive functions. However, the exact interconnections between GM and WM are still unclear, whether functional signals in WM rewires GM community organization remains to be explored. In this study, we divided brain functional connections into three types by using edge-centric method, including intra-GM, intra-WM and GM-WM connections, and calculated the edge community evaluation indexes for quantifying GM community engagement. The results showed that the involvement of WM significantly enhanced community entropy in the heteromodal system, while the sensory-attention system remained barely changed. In addition, delta community entropy showed a significant correlation with clinical cognitive scale. Our results suggested that WM rewired GM community organization, enhancing the community engagement of brain regions in the heteromodal system. This involvement was observed to be disrupted in disease groups. Our study revealed that considering the functional signals of GM and WM simultaneously could better understand the brain's functional organization.

Copper Nanodrugs with Controlled Morphologies through Aqueous Atom Transfer Radical Polymerization.

Copper (Cu) nanodrugs can be facilely prepared through atom transfer radical polymerization (ATRP) in an aqueous medium. However, it is difficult to control the morphology of Cu nanodrugs and thereby optimize their anticancer activity. In this work, aqueous ATRP was combined with polymerization-induced self-assembly (PISA) to prepare Cu nanodrugs with various morphologies. We mapped the relationship between polymerization condition and product morphology in which each morphology shows a wide preparation window. Decreasing the reaction temperature and feeding more Cu catalysts can improve the mobility of chains, facilitating the morphology evolution from sphere to other high-order morphologies. The resultant Cu nanodrugs with high monomer conversion and high Cu loading efficiency could be easily taken by cancer cells, showing excellent anticancer efficacy in vitro. This work proposed a potential strategy to prepare Cu nanodrugs with a specific morphology in batches, providing the method to optimize the anticancer efficacy through morphology control.

Dual-Energy-Barrier Stable Superhydrophobic Structures for Long Icing Delay.

Using superhydrophobic surfaces (SHSs) with the water-repellent Cassie-Baxter (CB) state is widely acknowledged as an effective approach for anti-icing performances. Nonetheless, the CB state is susceptible to diverse physical phenomena (e.g., vapor condensation, gas contraction, etc.) at low temperatures, resulting in the transition to the sticky Wenzel state and the loss of anti-icing capabilities. SHSs with various micronanostructures have been empirically examined for enhancing the CB stability; however, the energy barrier transits from the metastable CB state to the stable Wenzel state and thus the CB stability enhancement is currently not enough to guarantee a well and appliable anti-icing performance at low temperatures. Here, we proposed a dual-energy-barrier design strategy on superhydrophobic micronanostructures. Rather than the typical single energy barrier of the conventional CB-to-Wenzel transition, we introduced two CB states (i.e., CB I and CB II), where the state transition needed to go through CB I and CB II then to Wenzel state, thus significantly improving the entire CB stability. We applied ultrafast laser to fabricate this dual-energy-barrier micronanostructures, established a theoretical framework, and performed a series of experiments. The anti-icing performances were exhibited with long delay icing times (over 27,000 s) and low ice-adhesion strengths (0.9 kPa). The kinetic mechanism underpinning the enhanced CB anti-icing stability was elucidated and attributed to the preferential liquid pinning in the shallow closed structures, enabling the higher CB-Wenzel transition energy barrier to sustain the CB state. Comprehensive durability tests further corroborated the potentials of the designed dual-energy-barrier structures for anti-icing applications.

Copper Nanodrugs by Atom Transfer Radical Polymerization.

In this study, some copper catalysts used for atom transfer radical polymerization (ATRP) were explored as efficient anti-tumor agents. The aqueous solution of copper-containing nanoparticles with uniform spheric morphology was in situ prepared through a copper-catalyzed activator generated by electron transfer (AGET) ATRP in water. Nanoparticles were then directly injected into tumor-bearing mice for antitumor chemotherapy. The copper nanodrugs had prolonged blood circulation time and enhanced accumulation at tumor sites, thus showing potent antitumor activity. This work provides a novel strategy for precise and large-scale preparation of copper nanodrugs with high antitumor activity.

Influencing Factors of Massive Hemorrhage and High-Grade Renal Vascular Injury after PCNL: A Retrospective Comparative Study.

Purpose: Severe hemorrhage after percutaneous nephrolithotomy (PCNL) is a rare but alerting event. In this study, we report the factors affecting massive hemorrhage after PCNL, various levels of vascular damage during renal angiography, and the therapeutic effect of superselective renal artery embolization (SRAE). Patients and Methods. A retrospective analysis was performed on the data of 69 patients with postoperative PCNL hemorrhage who underwent SRAE from January 2010 to March 2021. Inclusion criteria for all cases were failure of conservative treatment for severe renal hemorrhage after surgery and then treatment with SRAE. In addition, 98 patients without significant hemorrhage after PCNL were randomly selected as the control group. All clinical data are confirmed by imaging and laboratory examinations. We performed univariate and multivariate analyses to find risk factors of massive hemorrhage and high-grade renal vascular injury after PCNL. Results: A total of 69 patients underwent angiography, 64 of which received SRAE due to positive hemorrhages detected by angiography. Urinary tract infection (OR (95% CI) = 11.214 (2.804∼44.842)), high blood pressure (OR (95% CI) = 5.686 (1.401∼23.083)), and no hydronephrosis (OR (95% CI) = 0.189 (0.049∼0.724)) are the most important factors leading to massive hemorrhage after PCNL. In patients who need SRAE after hemorrhage, high-grade vascular injury (grade III) is related to advanced age and decreased hemoglobin. Conclusion: During the perioperative period of PCNL, patients with a risk of hypertension, urinary tract infection, and no hydronephrosis should be strengthened to monitor their high risk of postoperative hemorrhage. For patients with postoperative hemorrhage, we can use the patient's age and decreased hemoglobin before and after operation for analysis. In this way, individualized assessment can greatly improve the efficiency of SRAE treatment.

NEAT1 Regulates Calcium Oxalate Crystal-Induced Renal Tubular Oxidative Injury via miR-130/IRF1.

Aims: Calcium oxalate (CaOx) crystal deposition induces damage to the renal tubular epithelium, increases epithelial adhesion, and contributes to CaOx nephrocalcinosis. The long noncoding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) is thought to be involved in this process. In this study, we aimed to investigate the mechanism by which NEAT1 regulates renal tubular epithelium in response to inflammatory and oxidative injury triggered by CaOx crystals. Results: As CaOx crystals were deposited in mouse kidney tissue, the expression of NEAT1 was significantly elevated and positively correlated with interferon regulatory factor 1 (IRF1), Toll-like receptor 4 (TLR4), and NF-κB. NEAT1 targets and inhibits miR-130a-3p as a competitor to endogenous RNA. miR-130 binds to and exerts inhibitory effects on the 3'-untranslated region of IRF1. After transfected with silence-NEAT1, IRF1, TLR4, and NF-κB were also variously inhibited, and oxidative damage in renal calcinosis was subsequently attenuated. When we simultaneously inhibited NEAT1 and miR-130, renal tubular injury was exacerbated. Innovation and Conclusion: We found that the lncRNA NEAT1 can enhance IRF1 signaling through targeted repression of miR-130a-3p and activate TLR4/NF-κB pathways to promote oxidative damage during CaOx crystal deposition. This provides an explanation for the tubular epithelial damage caused by CaOx crystals and offers new ideas and drug targets for the prevention and treatment of CaOx nephrocalcinosis. Antioxid. Redox Signal. 38, 731-746.

Floating Ag-NPs@Cu-NW bundles fabricated on copper mesh for highly sensitive SERS detection of uric acid in pretreatment-free urine.

The rapid and sensitive detection of ultra-trace marker molecules from biological samples is of great significance for the wide application of surface-enhanced Raman spectroscopy (SERS) methods in clinical diagnosis and disease monitoring. However, the cumbersome biological sample processing procedures and the poor enrichment of target analytes in hot spots hinder the practical applications of SERS methods. In this paper, we synthesized a novel floating SERS substrate by a simple one-step oxidation process, annealing and in situ chemical etching to form Ag-NPs@Cu-NW bundles on copper mesh (CM). In particular, under spontaneous bottom-up capillary action, the pressure difference at different nanogaps drives uric acid molecules to actively enter hot spots, so that the Ag-NPs@Cu-NW bundle nanostructure with the advantages of a light weight CM is capable of preventing the common coffee-ring effect and enhancing the spatial enrichment of analytes. Therefore, this SERS substrate realizes highly sensitive detection of uric acid at a level of 50 nM in pretreatment-free urine. Currently, this portable, flexible, simple, fast and cost-effective SERS substrate has great potential for early screening and clinical diagnosis of diseases in different biofluids.

Sortilin 1 Promotes Hepatocellular Carcinoma Cell Proliferation and Migration by Regulating Immune Cell Infiltration.

Objectives: Recent evidence suggests that Sort1 promotes carcinogenesis and tumor progression in multiple types of cancers. This study investigates the role of Sort1 in hepatocellular carcinoma (HCC). Methods: The differentially expressed gene was screened through GEO and TCGA databases. The Sort1 gene was identified and its expression was then verified by TCGA and HCCDB (a database of hepatocellular carcinoma expression atlas) databases. The Human Protein Atlas database was used to assess the gene expression in tissues. The TCGA and KM-plotter databases were used to study the relationship between Sort1 and HCC. The correlation between Sort1 and immune cells was evaluated through the TIMER database. GO and KEGG enrichment analysis was used to investigate the possible mechanism. The role of Sort1 in cell proliferation and invasion of HCC was further explored through in vitro experiments. Result: The differentially expressed molecule obtained from database screening was Sort1. Its expression was higher in cancer tissues than in paracancerous ones, and it was mainly located in the cytoplasm. The TCGA, KM-plotter databases, and our study data showed that low expression of Sort1 in HCC patients had better overall survival (OS), progression-free survival (PFI), and disease-specific survival (DSS). Further analysis indicated a significant correlation between Sort1 expression and immune cell infiltration. The gene set enrichment analysis (GSEA) analysis showed that Sort1 affected the biological events of HCC by participating in the WNT, TGF-BETA, JAK, STAT, and CALCIUM signaling pathways. In vitro, cytological experiments demonstrated reduced expression of PCNA, Ki-67, Vimentin, N-cadherin, and MMP-9 mRNA after knocking down Sort1, although E-cadherin expression was promoted. Overall, these processes reduced the ability of proliferation and invasion of HCC cells. Conclusion: Downregulation of Sort1 can prolong the OS, PFI, and DSS of HCC patients. Furthermore, due to its link with immune cell infiltration, the Sort1 gene represents a potentially novel predictive biomarker of HCC. The growth of HCC can be significantly inhibited by interfering with Sort1; therefore, these results provide a potential target for developing anticancer strategies for HCC.

Renal puncture access via a nonpapillary track in percutaneous nephrolithotomy: an in vitro porcine kidney experience.

Recently, an increasing number of investigators have debated the wide rule of the puncture to renal papilla in PCNL. We evaluated the effect of renal papillary and nonpapillary puncture on bleeding in an in vitro porcine kidney experience, with the aim of determining the safe puncture sites of collecting system in PCNL. A total of 70 fresh porcine kidneys were selected and subjected to nephrostomy. We performed a puncture through a renal papilla, infundibulum, renal column, or minor calyceal neck (including the front, back, up, and down). The primary outcome was the amount of bleeding. The results showed that the papillary puncture group yielded minimal bleeding (1.59 ± 1.01 ml/min) compared with the infundibular puncture group (6.25 ± 4.46 ml/min, P < .001), renal column puncture group (4.24 ± 3.79 ml/min, P = 0.001), and minor calyceal neck puncture group (2.27 ± 1.35 ml/min, P = 0.011). However, after stratifying by orientation, the up (1.75 ± 0.80 ml/min, P = 0.501) or down (1.77 ± 0.72 ml/min, P = 0.437) minor calyceal neck puncture group and papillary puncture group yielded comparable bleeding. In summary, nonpapillary puncture must be carefully considered. Infundibular and renal column punctures were inferior to papillary puncture, and up or down minor calyceal neck puncture may be a prudent choice in specific situations.

Renal puncture access using a blunt needle: proposal of the blunt puncture concept.

PURPOSE: Severe haemorrhage in percutaneous nephrolithotomy (PCNL) is an alarming event, and preventing injury to renal major vessels is a challenge. We evaluated the efficiency of a blunt needle in renal puncture procedures. METHODS: We first retrospectively reviewed the embolization images of post-PCNL patients to analyse the types of arteries injured, which were considered target arteries. Then, either a blunt needle or a conventional needle was used to directly puncture target arteries in ex vivo porcine kidneys and to establish renal access ex vivo and in vivo. The primary outcome was the incidence of target artery injuries, which were observed by digital subtraction angiography, nephroscopy and 3-dimensional endocasts. The secondary outcome was the rate of excreted fluid per access. RESULTS: The segmental and interlobar arteries were the most common types of injured arteries that needed to be embolized after PCNL. When these arteries were punctured directly, blunt needles reduced injury (1/20 vs. 16/20; OR 4.750; 95% CI 1.966-11.478; P < .001) by 76% compared to injuries induced by conventional needles. Moreover, the blunt needle group also had a significantly lower incidence of these arteries' injuries ex vivo due to renal puncture and yielded a lower rate of excreted fluid in ex vivo and in vivo renal puncture procedures. CONCLUSION: A blunt needle for renal puncture can be effective in reducing injury to renal major arteries and the accompanying haemorrhage. We propose the concept of blunt puncture, which may be a promising method for achieving safe renal access in PCNL.

lncRNA OGFRP1 promotes tumor progression by activating the AKT/mTOR pathway in human gastric cancer.

As biomolecules of great clinical value, lncRNAs play a crucial role as regulators in the processes of tumor origin, metastasis, and recurrence. Thus, lncRNAs are urgently needed for research in gastric cancer. We elucidated the specific function of OGFRP1, both in vitro and in vivo. OGFRP1 was expressed at abnormally high levels in gastric cancer samples (n = 408) compared to normal samples (n = 211). Similar results were obtained in 30 clinical case samples. Interference of OGFRP1 markedly blocked cell proliferation and migration, and it induced cell cycle arrest and the apoptosis of gastric cancer cells in vitro. Phosphorylation of AKT was inhibited in cells transfected with OGFRP1 siRNA, as compared to their control cells. The in vivo results further confirmed the antitumor effects of OGFRP1 knockdown on gastric cancer. Decreases in tumor volume (104.23±62.27 mm3) and weight (0.1006±0.0488 g) in nude mice were observed during the OGFRP1 interference, as compared with the control group (418.96±211.96 mm3 and 0.2741±0.0769 g). OGFRP1 promotes tumor progression through activating the AKT/mTOR pathway. Our findings provide a new potential target for the clinical treatment of human gastric cancer.

Cellular Toxicity Study of Silicon Nanowires.

The objective of this study was to achieve the practical bioapplications of silicon nanowires (SiNWs). In this study, the tumor and normal cell lines were used as models to systematically investigate the cytotoxicity of SiNWs synthesized by HF-assisted etching methods. Morphology observation, Cell Counting Kit 8, real-time polymerase chain reaction, and flow cytometry analysis were used to elucidate the cytotoxicity of SiNWs. The results showed that the cytotoxicity of SiNWs is greatly dependent on cell lines, SiNWs concentration, and incubation time. Particularly, SiNWs show better biocompatibility with tumor cell lines (eg, human epithelial cervical cancer [Hela] cells and human hepatocellular liver carcinoma [HepG2] cells) than normal cell lines (eg, human normal liver [HL-7702] cells and human embryonic kidney [HEK293T] cells). The reasons may be that SiNWs could tightly attach to the cell membrane in the cell medium, which obviously affects cell adhesion and inhibits their cell viability, especially for normal cell lines. From systematical analysis and comparison, we obtain the concentration limits of SiNWs, which may advance SiNWs applications and its toxicological study in vitro.

Contrast sensitivity visual acuity in REM sleep behavior disorder: a comparison with and without Parkinson disease.

STUDY OBJECTIVES: REM sleep behavior disorder (RBD) is characterized by dream enactment behavior and is a premotoric sign associated with parkinsonism and dementia. We previously found contrast sensitivity visual acuity (CSVA) deficiencies in earliest stages of Parkinson disease (PD), plausibly associated with alpha-synuclein deposits in the inner retinal layers. We speculated that individuals with REM sleep behavior without clinical signs of parkinsonism might also show similar deficiencies. METHODS: Twenty-three patients with RBD and 28 healthy control patients. Eleven with PD and 12 with idiopathic RBD (iRBD). Twelve patients with RBD were re-evaluated after 1 year. Evaluations consisted of CSVA SLOAN low contrast acuity charts, optical coherence topography, Unified Parkinson's Disease Rating Scale (UPDRS), and general neurologic and ophthalmologic examinations. Data analyzed between groups using a one-way analysis of variance, and a paired samples t test for returning patients. RESULTS: Participants were classified into three groups: controls (n = 28), iRBD (n = 12), and RBD+PD (n = 11). Analysis of variance revealed CSVA scores were statistically significantly different between the three groups F2, 50 = 7.037, P = .002. Longitudinal analysis of RBD group showed CSVA decreased significantly at 1 year (P = .0141). To date, PD has developed in three individuals with iRBD based on progression of their UPDRS scores. CONCLUSIONS: CSVA is reduced in individuals with RBD and declines over time. It is plausible that patients with iRBD may show early loss in dopaminergic lateral inhibition in the retina, evidenced by their progressive loss of CSVA. This may represent a global loss of dopaminergic neurons similar to PD.

Sh-MARCH8 Inhibits Tumorigenesis via PI3K Pathway in Gastric Cancer.

BACKGROUND/AIMS: To identify new treatment strategies for gastric cancer and to elucidate the mechanism underlying its pathophysiology, we transfected sh-MARCH8 into the human gastric cancer cell lines MKN-45 and AGS to investigate the roles of MARCH8 in gastric cancer. METHODS: We used genetic engineering to construct the sh-MARCH8 interference plasmid and transfected it into gastric cancer cells. Colony formation assays and cell viability measurements were performed to detect the viability and proliferation of cancer cells. Wound healing assays were performed to estimate the migration and proliferation rates of the cells. Cell invasion assays were used to estimate the invasive abilities of the cells. Cell apoptosis analysis was performed by using flowing cytometry. Western blot analysis was performed to estimate the expression levels of proteins. Statistical analysis was performed using the SPSS 18.0 software. Student's t-test was used to determine the significance of all pairwise comparisons of interest. RESULTS: We observed that the transfection of sh-MARCH8 inhibited the survival and proliferation of MKN-45 and AGS cells. The migration and invasion of the MKN-45 and AGS cells were significantly decreased, and apoptosis was induced in comparison with the control cells. These results were further confirmed by data showing that sh-MARCH8 increased the BAX/BCL2 ratio in MKN-45 and AGS cells. We also observed that sh-MARCH8 inactivated the PI3K and ß-catenin stat3 signaling pathways by changing protein expression levels or the phosphorylation of related proteins. CONCLUSION: These data suggested that sh-March8 reduced viability and induced apoptosis of the MKN-45 and AGS cells through the PI3K and ß-catenin stat3 signaling pathways. Taken together, our data revealed that transfection of sh-MARCH8 into the MKN-45 and AGS gastric cancer cell lines inhibited their growth, and this approach may be useful as a novel strategy for gastric cancer therapy.

Long non-coding RNA NNT-AS1 affects progression of breast cancer through miR-142-3p/ZEB1 axis.

Some evidences have been provided to verify the effects of lncRNA NNT-AS1 on cancer progression. However, the crucial impacts of NNT-AS1 on the malignancy of breast cancer have not been elaborated. This study aims to detect the expression pattern and functional effects of NNT-AS1 in breast cancer. qRT-PCR analysis was applied to detect the expression of NNT-AS1 in both BC tissues and matched normal tissues. Loss of function assay was carried out to detect the effects of silenced NNT-AS1 on proliferation, metastasis and EMT process of BC cells. To understand the functional mechanism of NNT-AS1, mechanism assays were designed and performed in BC cells. Subcellular fractionation assay demonstrated that NNT-AS1 was located in the cytoplasm of BC cells. Therefore, NNT-AS1 might exert ceRNA functions in BC cells. To validate this hypothesis, we found the combination between NNT-AS1 and miR-142-3p through conducting bioinformatics analysis, RIP and luciferase reporter assays. Similarly, the combination between miR-142-3p and ZEB1 was verified. Finally, the recue assays were carried out to demonstrate the effects of NNT-AS1/miR-142-3p/ZEB1 axis on the biological behaviors of BC cells. All the above findings revealed a fact that NNT-AS1 affects breast cancer progression through modulating miR-142-3p/ZEB1 axis.

Strengthening Gastric Cancer Therapy by Trastuzumab-Conjugated Nanoparticles with Simultaneous Encapsulation of Anti-MiR-21 and 5-Fluorouridine.

BACKGROUND/AIMS: MicroRNA-21 is an oncogenic miR (oncomiR) frequently elevated in gastric cancer (GC). Overexpression of miR-21 decreases the sensitivity of GC cells to 5-fluorouridine (5-Fu) and trastuzumab, a humanized monoclonal antibody targeting human epidermal growth factor receptor 2 (HER2). Receptor-mediated endocytosis plays a crucial role in the delivery of biotherapeutics including anti-miRNA oligonucleotides (AMOs). This study is a continuation of earlier findings involving poly(ε-caprolactone) (PCL)-poly (ethylene glycol) (PEG) nanoparticles (PEG-PCL NPs), which were coated with trastuzumab to target GC with HER2 receptor over-expression using anti-miRNA-21 (AMO-21) and 5-Fu. METHODS: HER-PEG-PCL NPs were prepared by one-step carbodiimide coupling using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAc) and Sulfo-NHS in aqueous phase. Covalent coupling of amino groups at the surface of PEG-PCL with the carboxyl groups of trastuzumab was analyzed by X-ray photoelectron spectroscopy (XPS). AMO-21/5-Fu NPs were formulated by a double-emulsion solvent evaporation technique. The cell line specificity, cellular uptake and AMO-21 delivery were investigated through the rhodamine-B-labeled 6-carboxyfluorescein (FAM)-AMO-21-PEG-PCL NPs coated with or without the antibody in both Her2-positive (NUGC4) and negative GC cells (SGC7901) visualized by fluorescence microscopy. The cytotoxicity of the HER-PEG-PCL NPs encapsulating AMO-21 was evaluated by MTT and apoptosis. Real-time reverse-transcription polymerase chain reaction (RT-PCR) was used to examine miR-21 and phosphatase and tensin homolog (PTEN) and Sprouty2 expression in GC cell lines. The antitumor effects of AMO-21/5-Fu NPs were compared with other groups in xenograft gastric cancer mice. RESULTS: The antibody conjugates significantly enhanced the cellular uptake of NPs. The AMO-21/5-Fu NPs effectively suppressed the target miRNA expression in GC cells, which further up-regulated PTEN and Sprouty2. As a result, the sensitivity of HER2-expressing gastric cancer to trastuzumab and 5-Fu were enhanced both in vitro and in vivo. The approach enhanced the targeting by trastuzumab as well as antibody-dependent cellular cytotoxicity (ADCC) of immune effector cells Conclusions: Taken together, the results provide insight into the biological and clinical potential of targeted AMO-21 and 5-Fu co-delivery using modified trastuzumab for GC treatment.

Enhanced antiproliferative activity of antibody-functionalized polymeric nanoparticles for targeted delivery of anti-miR-21 to HER2 positive gastric cancer.

MiR-21 is an oncogenic miR frequently elevated in gastric cancer. Overexpression of miR-21 decreases the sensitivity of gastric cancer cells to trastuzumab, which is a humanized monoclonal antibody targeting human epidermal growth factor receptor 2. However, optimization of miRNA or its anti-miRNA oligonucleotides (AMOs) for delivery is a challenge. Receptor-mediated endocytosis plays a crucial role in the delivery of biotherapeutics including AMOs. This study is a continuation of our earlier findings involving poly(ε-caprolactone) (PCL)-poly (ethylene glycol) (PEG) nanoparticles (PEG-PCL NPs), which were coated with trastuzumab to target gastric cancer cells with HER2 receptor over-expression using anti-miRNA-21 antisense oligonucleotides (AMO-21). The antibody conjugates (HER-PEG-PCL NPs) act against target cells via antibody-dependent mechanisms and also based on encapsutalated AMO-21. X-ray photoelectron spectroscopy validated the presence of trastuzumab on NP surface. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a stable antibody expression. The cell line specificity, cellular uptake, AMO-21 delivery, and cytotoxicity of the HER-PEG-PCL NPs were investigated. We found that the antibody conjugates significantly enhanced the cellular uptake of NPs. The HER-PEG-PCL NPs effectively suppressed the target miRNA expression in gastric cancer cells, which further up-regulated phosphatase and tensin homolog (PTEN). As a result, the sensitivity of HER2-expressing gastric cancer cells to trastuzumab was enhanced. The approach enhances the targeting by trastuzumab as well as antibody-dependent cellular cytotoxicity of immune effector cells. The antitumor effects of AMO-21-HER-PEG-PCL NPs were compared with trastuzumab in xenograft gastric cancer mice. The results provide insight into the biological and clinical potential of targeted AMO-21 delivery using modified trastuzumab for gastric cancer treatment.