LncRNA DCRT Protects Against Dilated Cardiomyopathy by Preventing NDUFS2 Alternative Splicing by Binding to PTBP1.

BACKGROUND: Dilated cardiomyopathy is characterized by left ventricular dilation and continuous systolic dysfunction. Mitochondrial impairment is critical in dilated cardiomyopathy; however, the underlying mechanisms remain unclear. Here, we explored the cardioprotective role of a heart-enriched long noncoding RNA, the dilated cardiomyopathy repressive transcript (DCRT), in maintaining mitochondrial function. METHODS: The DCRT knockout (DCRT-/-) mice and DCRT knockout cells were developed using CRISPR-Cas9 technology. Cardiac-specific DCRT transgenic mice were generated using α-myosin heavy chain promoter. Chromatin coimmunoprecipitation, RNA immunoprecipitation, Western blot, and isoform sequencing were performed to investigate the underlying mechanisms. RESULTS: We found that the long noncoding RNA DCRT was highly enriched in the normal heart tissues and that its expression was significantly downregulated in the myocardium of patients with dilated cardiomyopathy. DCRT-/- mice spontaneously developed cardiac dysfunction and enlargement with mitochondrial impairment. DCRT transgene or overexpression with the recombinant adeno-associated virus system in mice attenuated cardiac dysfunction induced by transverse aortic constriction treatment. Mechanistically, DCRT inhibited the third exon skipping of NDUFS2 (NADH dehydrogenase ubiquinone iron-sulfur protein 2) by directly binding to PTBP1 (polypyrimidine tract binding protein 1) in the nucleus of cardiomyocytes. Skipping of the third exon of NDUFS2 induced mitochondrial dysfunction by competitively inhibiting mitochondrial complex I activity and binding to PRDX5 (peroxiredoxin 5) and suppressing its antioxidant activity. Furthermore, coenzyme Q10 partially alleviated mitochondrial dysfunction in cardiomyocytes caused by DCRT reduction. CONCLUSIONS: Our study revealed that the loss of DCRT contributed to PTBP1-mediated exon skipping of NDUFS2, thereby inducing cardiac mitochondrial dysfunction during dilated cardiomyopathy development, which could be partially treated with coenzyme Q10 supplementation.

LncRNA CHKB-DT Downregulation Enhances Dilated Cardiomyopathy Through ALDH2.

BACKGROUND: Human cardiac long noncoding RNA (lncRNA) profiles in patients with dilated cardiomyopathy (DCM) were previously analyzed, and the long noncoding RNA CHKB (choline kinase beta) divergent transcript (CHKB-DT) levels were found to be mostly downregulated in the heart. In this study, the function of CHKB-DT in DCM was determined. METHODS: Long noncoding RNA expression levels in the human heart tissues were measured via quantitative reverse transcription-polymerase chain reaction and in situ hybridization assays. A CHKB-DT heterozygous or homozygous knockout mouse model was generated using the clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9 system, and the adeno-associated virus with a cardiac-specific promoter was used to deliver the RNA in vivo. Sarcomere shortening was performed to assess the primary cardiomyocyte contractility. The Seahorse XF cell mitochondrial stress test was performed to determine the energy metabolism and ATP production. Furthermore, the underlying mechanisms were explored using quantitative proteomics, ribosome profiling, RNA antisense purification assays, mass spectrometry, RNA pull-down, luciferase assay, RNA-fluorescence in situ hybridization, and Western blotting. RESULTS: CHKB-DT levels were remarkably decreased in patients with DCM and mice with transverse aortic constriction-induced heart failure. Heterozygous knockout of CHKB-DT in cardiomyocytes caused cardiac dilation and dysfunction and reduced the contractility of primary cardiomyocytes. Moreover, CHKB-DT heterozygous knockout impaired mitochondrial function and decreased ATP production as well as cardiac energy metabolism. Mechanistically, ALDH2 (aldehyde dehydrogenase 2) was a direct target of CHKB-DT. CHKB-DT physically interacted with the mRNA of ALDH2 and fused in sarcoma (FUS) through the GGUG motif. CHKB-DT knockdown aggravated ALDH2 mRNA degradation and 4-HNE (4-hydroxy-2-nonenal) production, whereas overexpression of CHKB-DT reversed these molecular changes. Furthermore, restoring ALDH2 expression in CHKB-DT+/- mice alleviated cardiac dilation and dysfunction. CONCLUSIONS: CHKB-DT is significantly downregulated in DCM. CHKB-DT acts as an energy metabolism-associated long noncoding RNA and represents a promising therapeutic target against DCM.

LncRNA MIR217HG aggravates pressure-overload induced cardiac remodeling by activating miR-138/THBS1 pathway.

AIM: Cardiac hypertrophy and fibrosis are associated with cardiac remodeling and heart failure. We have previously shown that miRNA-217, embedded within the third intron of MIR217HG, aggravates pressure overload-induced cardiac hypertrophy by targeting phosphatase and tensin homolog. However, whether the MIR217HG transcript itself plays a role in cardiac remodeling remains unknown. METHODS: Real-time PCR assays and RNA in situ hybridization were performed to detect MIR217HG expression. Lentiviruses and adeno-associated viruses with a cardiac-specific promoter (cTnT) were used to control MIR217HG expression in vitro and in vivo. Transverse aortic constriction (TAC) surgery was performed to develop cardiac remodeling models. Cardiac structure and function were analyzed using echocardiography and invasive pressure-volume analysis. KEY FINDINGS: MIR217HG expression was increased in patients with heart failure. MIR217HG overexpression aggravated pressure-overload-induced myocyte hypertrophy and fibrosis both in vivo and in vitro, whereas MIR217HG knockdown reversed these phenotypes. Mechanistically, MIR217HG increased THBS1 expression by sponging miR-138. MiR-138 recognized the 3'UTR of THBS1 and repressed THBS1 expression in the absence of MIR217HG. Silencing THBS1 expression reversed MIR217HG-induced cardiac hypertrophy and remodeling. CONCLUSION: MIR217HG acts as a potent inducer of cardiac remodeling that may contribute to heart failure by activating the miR-138/THBS1 pathway.

A Kaposi's sarcoma-associated herpes virus-encoded microRNA contributes to dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is the leading cause of heart transplantation. By microRNA (miRNA) array, a Kaposi's sarcoma-associated herpes virus (KSHV)-encoded miRNA, kshv-miR-K12-1-5p, was detected in patients with DCM. The KSHV DNA load and kshv-miR-K12-1-5p level in plasma from 696 patients with DCM were measured and these patients were followed-up. Increased KSHV seropositivity and quantitative titers were found in the patients with DCM compared with the non-DCM group (22.0% versus 9.1%, p < 0.05; 168 versus 14 copies/mL plasma, p < 0.05). The risk of the individual end point of death from cardiovascular causes or heart transplantation was increased among DCM patients with the KSHV DNA seropositivity during follow-up (adjusted hazard ratio 1.38, 95% confidence interval 1.01-1.90; p < 0.05). In heart tissues, the KSHV DNA load was also increased in the heart from patients with DCM in comparison with healthy donors (1016 versus 29 copies/105 cells, p < 0.05). The KSHV and kshv-miR-K12-1-5p in DCM hearts were detected using immunofluorescence and fluorescence staining in situ hybridization. KSHV itself was exclusively detectable in CD31-positive endothelium, while kshv-miR-K12-1-5p could be detected in both endothelium and cardiomyocytes. Moreover, kshv-miR-K12-1-5p released by KSHV-infected cardiac endothelium could disrupt the type I interferon signaling pathway in cardiomyocytes. Two models of kshv-miR-K12-1-5p overexpression (agomiR and recombinant adeno-associated virus) were used to explore the roles of KSHV-encoded miRNA in vivo. The kshv-miR-K12-1-5p aggravated known cardiotropic viruses-induced cardiac dysfunction and inflammatory infiltration. In conclusion, KSHV infection was a risk factor for DCM, providing developmental insights of DCM involving virus and its miRNA ( https://clinicaltrials.gov . Unique identifier: NCT03461107).

LncRNA ZNF593-AS alleviates diabetic cardiomyopathy via suppressing IRF3 signaling pathway.

Diabetes could directly induce cardiac injury, leading to cardiomyopathy. However, treatment strategies for diabetic cardiomyopathy remain limited. ZNF593-AS knockout and cardiomyocyte-specific transgenic mice were constructed. In addition, high-fat diet (HFD)-induced diabetic mouse model and db/db mice, another classic diabetic mouse model, were employed. ZNF593-AS was silenced using GapmeR, a modified antisense oligonucleotide, while overexpressed using a recombinant adeno-associated virus serotype 9-mediated gene delivery system. Transcriptome sequencing, RNA pull-down assays, and RNA immunoprecipitation assays were also performed to investigate the underlying mechanisms. ZNF593-AS expression was decreased in diabetic hearts. ZNF593-AS attenuated the palmitic acid-induced apoptosis of cardiomyocytes in vitro. In HFD-induced diabetic mice, ZNF593-AS deletion aggravated cardiac dysfunction and enhanced cardiac apoptosis and inflammation. In contrast, HFD-induced cardiac dysfunction was improved in ZNF593-AS transgenic mice. Consistently, ZNF593-AS exerted the same cardioprotective effects in db/db mice. Mechanistically, ZNF593-AS directly interacted with the functional domain of interferon regulatory factor 3 (IRF3), and suppressed fatty acid-induced phosphorylation and activation of IRF3, contributing to the amelioration of cardiac cell death and inflammation. In conclusion, our results identified the protective role of ZNF593-AS in diabetic cardiomyopathy, suggesting a novel potential therapeutic target.

Overexpression of cytosolic long noncoding RNA cytb protects against pressure-overload-induced heart failure via sponging microRNA-103-3p.

Long noncoding RNAs (lncRNAs) play crucial roles in cardiovascular diseases. To date, only limited studies have reported the role of mitochondria-derived lncRNAs in heart failure (HF). In the current study, recombinant adeno-associated virus 9 was used to manipulate lncRNA cytb (lnccytb) expression in vivo. Fluorescence in situ hybridization (FISH) assay was used to determine the location of lnccytb, while microRNA (miRNA) sequencing and bioinformatics analyses were applied to identify the downstream targets. The competitive endogenous RNA (ceRNA) function of lnccytb was evaluated by biotin-coupled miRNA pull-down assays and luciferase reporter assays. Results showed that lnccytb expression was decreased in the heart of mice with transverse aortic constriction (TAC), as well as in the heart and plasma of patients with HF. FISH assay and absolute RNA quantification via real-time reverse transcription PCR suggested that the reduction of the lnccytb transcripts mainly occurred in the cytosol. Upregulation of cytosolic lnccytb attenuated cardiac dysfunction in TAC mice. Moreover, overexpression of cytosolic lnccytb in cardiomyocytes alleviated isoprenaline-induced reactive oxidative species (ROS) production and hypertrophy. Mechanistically, lnccytb acted as a ceRNA via sponging miR-103-3p, ultimately mitigating the suppression of PTEN by miR-103-3p. In summary, we demonstrated that the overexpression of cytosolic lnccytb could ameliorate HF.

miR-320a induces pancreatic ß cells dysfunction in diabetes by inhibiting MafF.

A variety of studies indicate that microRNAs (miRNAs) are involved in diabetes. However, the direct role of miR-320a in the pathophysiology of pancreatic ß cells under diabetes mellitus remains unclear. In the current study, islet transplantation and hyperglycemic clamp assays were performed in miR-320a transgenic mice to explore the effects of miR-320a on pancreatic ß cells in vivo. Meanwhile, ß cell-specific overexpression or inhibition of miR-320a was delivered by adeno-associated virus (AAV8). In vitro, overexpression or downregulation of miR-320a was introduced in cultured rat islet tumor cells (INS1). RNA immunoprecipitation sequencing (RIP-Seq), luciferase reporter assay, and western blotting were performed to identify the target genes. Results showed that miR-320a was increased in the pancreatic ß cells from high-fat-diet (HFD)-treated mice. Overexpression of miR-320a could not only deteriorate the HFD-induced pancreatic islet dysfunction, but also initiate pancreatic islet dysfunction spontaneously in vivo. Meanwhile, miR-320a increased the ROS level, inhibited proliferation, and induced apoptosis of cultured ß cells in vitro. Finally, we identified that MafF was the target of miR-320a that responsible for the dysfunction of pancreatic ß cells. Our data suggested that miR-320a could damage the pancreatic ß cells directly and might be a potential therapeutic target of diabetes.

The double face of miR-320: cardiomyocytes-derived miR-320 deteriorated while fibroblasts-derived miR-320 protected against heart failure induced by transverse aortic constriction.

MicroRNAs (miRNAs) are aberrantly expressed in the pathophysiologic process of heart failure (HF). However, the functions of a certain miRNA in different cardiac cell types during HF are scarcely reported, which might be covered by the globe effects of it on the heart. In the current study, Langendorff system was applied to isolate cardiomyocytes (CMs) and cardiac fibroblasts (CFs) from transverse aortic constriction (TAC)-induced mice. Slight increase of miR-320 expression was observed in the whole heart tissue of TAC mice. Interestingly, miR-320 was significantly elevated in CMs but decreased in CFs from TAC mice at different time points. Then, recombinant adeno-associated virus 9 with cell-type-specific promoters were used to manipulate miR-320 expressions in vivo. Both in vitro and in vivo experiments showed the miR-320 overexpression in CMs exacerbated cardiac dysfunction, whereas overexpression of miR-320 in CFs alleviated cardiac fibrosis and hypertrophy. Mechanically, downstream signaling pathway analyses revealed that miR-320 might induce various effects via targeting PLEKHM3 and IFITM1 in CMs and CFs, respectively. Moreover, miR-320 mediated effects could be abolished by PLEKHM3 re-expression in CMs or IFITM1 re-expression in CFs. Interestingly, miR-320 treated CFs were able to indirectly affect CMs function, but not vice versa. Meanwhile, upstream signaling pathway analyses showed that miR-320 expression and decay rate were rigorously manipulated by Ago2, which was regulated by a cluster of cell-type-specific TFs distinctively expressed in CMs and CFs, respectively. Together, we demonstrated that miR-320 functioned differently in various cell types of the heart during the progression of HF.

A Key GWAS-Identified Genetic Variant Contributes to Hyperlipidemia by Upregulating miR-320a.

It has been unclear whether the elevated levels of the circulating miR-320a in patients with coronary artery disease is due to environmental influence or genetic basis. By recombinant adeno-associated virus (rAAV)-mediated loss- and gain-of-function studies in the mouse liver, we revealed that elevated miR-320a is sufficient to aggravate diet-induced hyperlipidemia and hepatic steatosis. Then, we analyzed the data from published genome-wide association studies and identified the rs12541335 associated with hyperlipidemia. We demonstrated that the rs13282783 T allele indeed obligated the silencer activity by preventing the repressor ZFP161 and co-repressor HDAC2 from binding to DNA that led to miR-320a upregulation. We further confirmed this genetic connection on an independent population and through direct genome editing in liver cells. Besides environmental (diet) influence, we established a genetic component in the regulation of miR-320a expression, which suggest a potential therapeutic avenue to treat coronary artery disease by blocking miR-320a in patient liver.

Nuclear miR-665 aggravates heart failure via suppressing phosphatase and tensin homolog transcription.

Although numerous miRNAs have been discovered, their functions in the different subcellular organelles have remained obscure. In this study, we found that miR-665 was enriched in the nucleus of cardiomyocytes, and then investigated the underlying role of nuclear miR-665 in heart failure. RNA fluorescence in situ hybridization assays in human heart tissue sections and primary cardiomyocytes showed that miR-665 was localized in the nucleus of cardiomyocytes. Increased expression of nuclear miR-665 was observed not only in the cardiomyocytes isolated from the heart of mice treated in vivo by transverse aortic constriction (TAC), but also in phenylephrine (PE)-treated cultured cardiomyocytes in vitro. To further explore the role of miR-665 in heart failure, a type 9 recombinant adeno-associated virus (rAAV) system was employed to manipulate the expression of miR-665 in mice. Overexpression of miR-665 aggravated TAC-induced cardiac dysfunction, while down-expression of miR-665 showed opposite effects. Bioinformatic prediction and biological validation confirmed that the PTEN (phosphatase and tensin homolog) gene was one of the targets of miR-665 in the nucleus. Furthermore, restoring PTEN expression significantly eliminated the destructive effects of miR-665 over-expression in TAC-induced cardiac dysfunction. Our data showed that nuclear miR-665 aggravates heart failure via inhibiting PTEN expression, which provided a therapeutic approach for heart failure.

The Different Roles of miRNA-92a-2-5p and let-7b-5p in Mitochondrial Translation in db/db Mice.

Excessive reactive oxygen species (ROS) generated in mitochondria is known to be a causal event in diabetic cardiomyopathy. Recent studies suggest that microRNAs (miRNAs) are able to translocate to mitochondria to modulate mitochondrial activities, but the roles of such miRNAs in diabetic cardiomyopathy remain unclear. We observed a marked reduction of mitochondrial gene cytochrome-b (mt-Cytb) in the heart of db/db mice compared with controls. Downregulation of mt-Cytb by small interfering RNA (siRNA) recaptured some key features of diabetes, including elevated ROS production. Microarray revealed that none of the miRNAs were upregulated, but 14 miRNAs were downregulated in mitochondria of db/db heart. miR-92a-2-5p and let-7b-5p targeted mt-Cytb and positively modulated mt-Cytb expression. Re-expression of miR-92a-2-5p and let-7b-5p into cardiomyocytes led to reduced ROS production. Furthermore, recombinant adeno-associated virus (rAAV)-mediated delivery of miR-92a-2-5p, but not let-7b-5p, was sufficient to rescue cardiac diastolic dysfunction in db/db heart. Let-7b-5p not only upregulated mt-Cytb in mitochondria, but also downregulated insulin receptor substrate 1 in cytosol and finally lead to no efficiency for improvement of diastolic dysfunction in db/db mice. Our findings demonstrate that reduced mitochondrial miRNAs contribute to impaired mitochondrial gene expression and elevated ROS production. Re-expression of miR-92a-2-5p enhances mitochondrial translation and reduces ROS production and lipid deposition, which finally rescues diabetic cardiomyopathy.

MiR-665 aggravates heart failure via suppressing CD34-mediated coronary microvessel angiogenesis.

BACKGROUND: Heart failure (HF) is a major public health problem worldwide. The development of HF was related to coronary microvessel dysfunction. Whether miRNAs participate in HF by regulating coronary microvessel function remain unclear. METHODS: The potential targets of miR-665 were predicted by rnahybrid software, then verified through anti-Ago2 co-immunoprecipitation, Western blotting and luciferase reporter assays. rAAV9 system was used to manipulate the expression of miR-665 in vivo. RESULTS: Significant increase of miR-665 was observed in endothelial cells of human heart with heart failure. In vitro over-expression of miR-665 in endothelial cells resulted in decreased proliferation but enhanced apoptosis. rAAV-mediated delivery of miR-665 reduced coronary microvessel angiogenesis and cardiac microvessel density, then further impaired cardiac function in vivo. Furthermore, CD34 was confirmed as one of the miR-665 targets. Consistently, re-expression of CD34 attenuated miR-665-mediated damage effects in vitro and in vivo. We also found that Sp1 regulated miR-665 expression in endothelial cells. CONCLUSION: Our findings demonstrated that miR-665 played an important role in heart failure via damaging coronary microvessel angiogenesis, and suggested that miRNA-based therapeutics may protect against coronary microvessel dysfunction and heart failure.

miR-217 Promotes Cardiac Hypertrophy and Dysfunction by Targeting PTEN.

Previously, we found that the miR-217 expression level was increased in hearts from chronic heart failure (CHF) patients by using miRNA profile analysis. This study aimed to explore the role of miR-217 in cardiac dysfunction. Heart tissue samples from CHF patients were used to detect miR-217 expression levels. A type 9 recombinant adeno-associated virus (rAAV9) was employed to manipulate miR-217 expression in mice with thoracic aortic constriction (TAC)-induced cardiac dysfunction. Cardiac structure and function were measured by echocardiography and invasive pressure-volume analysis. The expression levels of miR-217 were increased in hearts from both CHF patients and TAC mice. Overexpression of miR-217 in vivo aggravated pressure overload-induced cardiac hypertrophy, fibrosis, and cardiac dysfunction, whereas miR-217-TUD-mediated downregulation of miR-217 reversed these effects. PTEN was predicted and validated as a direct target of miR-217, and re-expression of PTEN attenuated miR-217-mediated cardiac hypertrophy and cardiac dysfunction. Importantly, cardiomyocyte-derived miR-217-containing exosomes enhanced proliferation of fibroblasts in vitro. All of these findings show that miR-217 participates in cardiac hypertrophy and cardiac fibrosis processes through regulating PTEN, which suggests a promising therapeutic target for CHF.

MiR-21 protected against diabetic cardiomyopathy induced diastolic dysfunction by targeting gelsolin.

BACKGROUND: Diabetes is a leading cause of mortality and morbidity across the world. Over 50% of deaths among diabetic patients are caused by cardiovascular diseases. Cardiac diastolic dysfunction is one of the key early signs of diabetic cardiomyopathy, which often occurs before systolic dysfunction. However, no drug is currently licensed for its treatment. METHODS: Type 9 adeno-associated virus combined with cardiac Troponin T promoter were employed to manipulate miR-21 expression in the leptin receptor-deficient (db/db) mice. Cardiac structure and functions were measured by echocardiography and hemodynamic examinations. Primary cardiomyocytes and cardiomyocyte cell lines were used to perform gain/loss-of-function assays in vitro. RESULTS: We observed a significant reduction of miR-21 in the diastolic dysfunctional heart of db/db mice. Remarkably, delivery of miR-21 efficiently protected against the early impairment in cardiac diastolic dysfunction, represented by decreased ROS production, increased bioavailable NO and relieved diabetes-induced cardiomyocyte hypertrophy in db/db mice. Through bioinformatic analysis and Ago2 co-immunoprecipitation, we identified that miR-21 directly targeted gelsolin, a member of the actin-binding proteins, which acted as a transcriptional cofactor in signal transduction. Moreover, down-regulation of gelsolin by siRNA also attenuated the early phase of diabetic cardiomyopathy. CONCLUSION: Our findings reveal a new role of miR-21 in attenuating diabetic cardiomyopathy by targeting gelsolin, and provide a molecular basis for developing a miRNA-based therapy against diabetic cardiomyopathy.

MiR-30c-5p ameliorates hepatic steatosis in leptin receptor-deficient (db/db) mice via down-regulating FASN.

Approximately 15-40% of the general adult population suffers from non-alcoholic fatty liver disease (NAFLD) worldwide. However, no drug is currently licensed for its treatment. In this study, we observed a significant reduction of miR-30c-5p in the liver of leptin receptor-deficient (db/db) mice. Remarkably, recombinant adeno-associated virus (rAAV)-mediated delivery of miR-30c-5p was sufficient to attenuate triglyceride accumulation and hepatic steatosis in db/db mice. Through computational prediction, KEGG analysis and Ago2 co-immunoprecipitation, we identified that miR-30c-5p directly targeted fatty acid synthase, a key enzyme in fatty acid biosynthesis. Moreover, down-regulation of FASN by siRNA attenuated some key features of NAFLD, including decreased triglyceride accumulate and lipid deposition. Our findings reveal a new role of miR-30c-5p in counterbalancing fatty acid biosynthesis, which is sufficient to attenuate triglyceride accumulation and hepatic steatosis in db/db mice.

PEGylated niosomes-mediated drug delivery systems for Paeonol: preparation, pharmacokinetics studies and synergistic anti-tumor effects with 5-FU.

This work describes the preparation of a PEGylated niosomes-mediated drug delivery systems for Paeonol, thereby improving the bioavailability and chemical stability of Paeonol, prolonging its cellular uptake and enhancing its synergistic anti-cancer effects with 5-Fu. PEGylated niosomes, which are prepared from biocompatible nonionic surfactant of Spans 60 and cholesterol, and modified with PEG-SA. Pae-PEG-NISVs were evaluated in vitro and in vivo. The cytotoxicity of Pae-PEG-NISVs was investigated against HepG2 cells. Fluorescence microscope was used to detect the apoptotic morphological changes. Growth inhibition assays were carried out to investigate whether Pae-PEG-NISVs could enhance the antiproliferative effects of Pae co-treated with 5-FU on HepG2 cells. The optimized Pae-PEG-NISVs had mean diameters of approximately 166 nm and entrapment efficiency (EE) of 61.8%. Furthermore, the in vitro release study of Paeonol from PEGylated niosomes exhibited a relatively prolonged release profile for 12 h. Pharmacokinetic studies in rats after i.v. injection showed that Pae-PEG-NISVs had increased elimination half-lives (t1/2, 87.5 versus 17.0 min) and increased area under the concentration-time curve (AUC0-t, 38.0 versus 19.48 µg/ml*min) compared to Paeonol solution. Formulated Paeonol had superior cytotoxicity versus the free drug with IC50 values of 22.47 and 85.16 µg/mL at 24 h on HepG2 cells, respectively, and we found that low concentration of Pae-PEG-NISVs and 5-Fu in conjunction had obviously synergistic effect. Our results indicate that the PEG-NISVs system has the potential to serve as an efficient carrier for Paeonol by effectively solubilizing, stabilizing and delivering the drug to the cancer cells.

MicroRNA-21 Lowers Blood Pressure in Spontaneous Hypertensive Rats by Upregulating Mitochondrial Translation.

BACKGROUND: Excessive reactive oxygen species generated in mitochondria has been implicated as a causal event in hypertensive cardiomyopathy. Multiple recent studies suggest that microRNAs (miRNAs) are able to translocate to mitochondria to modulate mitochondrial activities, but the medical significance of such a new miRNA function has remained unclear. Here, we characterized spontaneous hypertensive rats (SHRs) in comparison with Wistar rats, finding that micro RNA-21 (miR-21) was dramatically induced in SHRs relative to Wistar rats. We designed a series of experiments to determine whether miR-21 is involved in regulating reactive oxygen species generation in mitochondria, and if so, how induced miR-21 may either contribute to hypertensive cardiomyopathy or represent a compensatory response. METHODS: Western blotting was used to compare the expression of key nuclear genome (nDNA)-encoded and mitochondrial genome (mtDNA)-encoded genes involved in reactive oxygen species production in SHRs and Wistar rats. Bioinformatics was used to predict miRNA targets followed by biochemical validation using quantitative real-time polymerase chain reaction and Ago2 immunoprecipitation. The direct role of miRNA in mitochondria was determined by GW182 dependence, which is required for miRNA to function in the cytoplasm, but not in mitochondria. Recombinant adeno-associated virus (type 9) was used to deliver miRNA mimic to rats via tail vein, and blood pressure was monitored with a photoelectric tail-cuff system. Cardiac structure and functions were assessed by echocardiography and catheter manometer system. RESULTS: We observed a marked reduction of mtDNA-encoded cytochrome b (mt-Cytb) in the heart of SHRs. Downregulation of mt-Cytb by small interfering RNA in mitochondria recapitulates some key disease features, including elevated reactive oxygen species production. Computational prediction coupled with biochemical analysis revealed that miR-21 directly targeted mt-Cytb to positively modulate mt-Cytb translation in mitochondria. Circulating miR-21 levels in hypertensive patients were significantly higher than those in controls, showing a positive correlation between miR-21 expression and blood pressure. Remarkably, recombinant adeno-associated virus-mediated delivery of miR-21 was sufficient to reduce blood pressure and attenuate cardiac hypertrophy in SHRs. CONCLUSIONS: Our findings reveal a positive function of miR-21 in mitochondrial translation, which is sufficient to reduce blood pressure and alleviate cardiac hypertrophy in SHRs. This observation indicates that induced miR-21 is part of the compensatory program and suggests a novel theoretical ground for developing miRNA-based therapeutics against hypertension.

Food wastes as fish feeds for polyculture of low-trophic-level fish: bioaccumulation and health risk assessments of heavy metals in the cultured fish.

The major purpose of this study was to use different types of food wastes which serve as the major sources of protein to replace the fish meal used in fish feeds to produce quality fish. Two types of food waste-based feed pellets FW A (with cereals) and FW B (with cereals and meat products) and the commercial feed Jinfeng® were used to culture fingerlings of three low-trophic-level fish species: bighead carp, grass carp, and mud carp (in the ratio of 1:3:1) for 1 year period in the Sha Tau Kok Organic Farm in Hong Kong. Heavy metal concentrations in all of the fish species fed with food waste pellets and commercial pellets in Sha Tau Kok fish ponds were all below the local and international maximum permissible levels in food. Health risk assessments indicated that human consumption of the fish fed with food waste feed pellets was safe for the Hong Kong residents. The present results revealed that recycling of food waste for cultivating low-trophic-level fish (mainly herbivores and detritus feeders) is feasible, and at the same time will ease the disposal pressure of food waste, a common problem of densely populated cities like Hong Kong.

Fabrication of graphene-isolated-Au-nanocrystal nanostructures for multimodal cell imaging and photothermal-enhanced chemotherapy.

Using nanomaterials to develop multimodal systems has generated cutting-edge biomedical functions. Herein, we develop a simple chemical-vapor-deposition method to fabricate graphene-isolated-Au-nanocrystal (GIAN) nanostructures. A thin layer of graphene is precisely deposited on the surfaces of gold nanocrystals to enable unique capabilities. First, as surface-enhanced-Raman-scattering substrates, GIANs quench background fluorescence and reduce photocarbonization or photobleaching of analytes. Second, GIANs can be used for multimodal cell imaging by both Raman scattering and near-infrared (NIR) two-photon luminescence. Third, GIANs provide a platform for loading anticancer drugs such as doxorubicin (DOX) for therapy. Finally, their NIR absorption properties give GIANs photothermal therapeutic capability in combination with chemotherapy. Controlled release of DOX molecules from GIANs is achieved through NIR heating, significantly reducing the possibility of side effects in chemotherapy. The GIANs have high surface areas and stable thin shells, as well as unique optical and photothermal properties, making them promising nanostructures for biomedical applications.

Replacing fish meal by food waste in feed pellets to culture lower trophic level fish containing acceptable levels of organochlorine pesticides: health risk assessments.

The present study used food waste (collected from local hotels and restaurants) feed pellets in polyculture of low-trophic level fish [bighead (Aristichtys nobilis), grass carp (Ctenopharyngodon idellus), and mud carp (Cirrhina molitorella)] aiming at producing safe and quality products for local consumption. The results indicated that grass carp (hexachlorocyclohexanes (HCHs) <0.03; dichlorodiphenyltrichloroethanes (DDTs) 1.42-3.34 ng/g ww) and bighead carp (HCHs<0.03; DDTs 1.55-2.56 ng/g ww) fed with food waste feed pellets were relatively free of organochlorine pesticides (OCPs). The experimental ponds (water and sediment) were relatively free of OCPs, lowering the possibility of biomagnification of OCPs in the food chains within the ponds. The raw concentrations of OCPs extracted from the fish were not in the bioavailable form, which would ultimately reach bloodstream and exert adverse effects on human body. Health risk assessments based on digestible concentrations are commonly regarded as a more accurate method. The results of health risk assessments based on raw and digestible concentrations showed that the fish fed with food waste feed pellets were safe for consumption from the OCP perspective.