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ObjectiveTo verify the anti-oxidative stress effect of Huangqintang based on the nuclear factor E2-related factor 2 （Nrf2） signaling pathway by using Caco-2 cells as a carrier and RNA interference （RNAi） technology with in vitro experiments. MethodThe Caco-2 cells in the logarithmic growth phase were transfected with siRNA to construct siRNA Caco-2 cells. After normal Caco-2 cells and siRNA Caco-2 cells were incubated with Huangqintang of different doses， RNA and protein were extracted. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot were used to detect the mRNA and protein expression of heme oxygenase-1 （HO-1）， NAD（P）H quinone oxidoreductase 1 （NQO1）， glutathione S-transferase （GST）， Kelch-like ECH-associated protein 1 （Keap1）， and Nrf2. Meanwhile， the activities of superoxide dismutase （SOD） and GSH-Px， as well as the expression levels of malondialdehyde （MDA） and reactive oxygen species （ROS）， were detected by the colorimetric method and the probe method. ResultCompared with the results in the normal group， only the 400 mg·L-1 Huangqintang group and the sulforaphane （SFN） group could reduce the content of ROS and MDA in Caco-2 cells （P<0.01）， while the activities of SOD and GSH-Px in the cells of the Huangqintang groups and the SFN group showed an upward trend. Furthermore， there were significant differences in the 400 mg·L-1 Huangqintang group/the SFN group and the normal group （P<0.01）. Meanwhile， the protein and mRNA expression levels of HO-1， GST， Keap1， NQO1， and Nrf2 showed an upward trend in all groups （P<0.05， P<0.01）. After transfection， compared with the normal group， the model group showed increased content of MDA and ROS， blunted activities of GSH-Px and SOD， and reduced protein and mRNA expression of HO-1， GST， Keap1， and NQO1 （P<0.05， P<0.01）. After drug incubation， compared with the model group， the SFN group showed potentiated SOD activity， and the SFN group and the Huangqintang groups showed enhanced GSH-Px activity （P<0.01）. Moreover， the activities of SOD and GSH-Px in the 400 and 200 mg·L-1 Huangqintang groups and the SFN group showed an upward trend （P<0.01）， and the content of MDA in the 400 mg·L-1 Huangqintang group and the SFN group showed a downward trend. ROS decreased in all groups with drug intervention （P<0.01）， and the protein and mRNA expression of HO-1， GST， Keap1， NQO1， and Nrf2 increased to varying degrees （P<0.05， P<0.01）. ConclusionHuangqintang can play an anti-oxidative stress role by regulating the Nrf2 pathway.
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c-Myc protein encoded by c-Myc (cellular-myelocytomatosis viral oncogene) gene regulates the related gene expression through the Wnt/β-catenin signaling pathway, and has received extensive attention in recent years. The purpose of this study was to express Helicoverpa armigera c-Myc gene (Ha-c-Myc) by using prokaryotic expression system, prepare the polyclonal antibody, examine the spatio-temporal expression profile of Ha-c-Myc, and investigate the possible function of Ha-c-Myc in regulating H. armigera sterol carrier protein-2 (SCP-2) gene expression. The Ha-c-Myc gene was amplified by PCR and cloned into a prokaryotic expression plasmid pET-32a(+). The recombinant plasmid pET-32a-Ha-c-Myc was transformed into Escherichia coli BL21. IPTG was used to induce the expression of the recombinant protein. Protein was purified by Ni2+-NTA column and used to immunize New Zealand rabbits for preparing the polyclonal antibody. The Ha-c-Myc expression levels in different developmental stages (egg, larva, prepupa, pupa, and adult) of H. armigera and different tissues (midgut, fat body, head, and epidermis) of the prepupa were determined by real-time quantitative reverse transcription PCR (qRT-PCR). Ha-c-Myc siRNA was synthesized and transfected into H. armigera Ha cells. The relative mRNA levels of Ha-c-Myc and HaSCP-2 in Ha cells were detected by qRT-PCR. Results showed that the pET-32a-Ha-c-Myc recombinant plasmid was constructed. The soluble Ha-c-Myc protein of about 65 kDa was expressed in E. coli. The polyclonal antibody was prepared. Western blotting analysis suggested that the antibody had high specificity. Enzyme linked immunosorbent assay (ELISA) showed that the titer of the antibody was high. Ha-c-Myc gene expressed at all developmental stages, with high levels in the early and late instars of larva, and the prepupal stage. Tissue expression profiles revealed that Ha-c-Myc expressed in various tissues of prepupa, with high expression level in the midgut, but low levels in the epidermis and fat body. RNAi results showed that the knockdown of Ha-c-Myc expression significantly affected transcription of HaSCP-2, leading to a 50% reduction in HaSCP-2 mRNA expression level. In conclusion, the Ha-c-Myc was expressed through a prokaryotic expression system, and the polyclonal anti-Ha-c-Myc antibody was obtained. Ha-c-Myc may promote the expression of HaSCP-2 and play an important role in the lipid metabolism of H. armigera. These results may facilitate further study on the potential role and function mechanism of Ha-c-Myc in H. armigera and provide experimental data for exploring new targets of green pesticides.
Subject(s)
Animals , Rabbits , Escherichia coli/metabolism , Enzyme-Linked Immunosorbent Assay , Moths/genetics , Blotting, Western , Larva/genetics , Isoantibodies/metabolism , Antibody SpecificityABSTRACT
Exosome is an excellent vesicle for in vivo delivery of therapeutics, including RNAi and chemical drugs. The extremely high efficiency in cancer regression can partly be attributed to its fusion mechanism in delivering therapeutics to cytosol without endosome trapping. However, being composed of a lipid-bilayer membrane without specific recognition capacity for aimed-cells, the entry into nonspecific cells can lead to potential side-effects and toxicity. Applying engineering approaches for targeting-capacity to deliver therapeutics to specific cells is desirable. Techniques with chemical modification in vitro and genetic engineering in cells have been reported to decorate exosomes with targeting ligands. RNA nanoparticles have been used to harbor tumor-specific ligands displayed on exosome surface. The negative charge reduces nonspecific binding to vital cells with negatively charged lipid-membrane due to the electrostatic repulsion, thus lowering the side-effect and toxicity. In this review, we focus on the uniqueness of RNA nanoparticles for exosome surface display of chemical ligands, small peptides or RNA aptamers, for specific cancer targeting to deliver anticancer therapeutics, highlighting recent advances in targeted delivery of siRNA and miRNA that overcomes the previous RNAi delivery roadblocks. Proper understanding of exosome engineering with RNA nanotechnology promises efficient therapies for a wide range of cancer subtypes.
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Plant diseases and insect pests threaten the safety of crop production greatly. Traditional methods for pest management are challenged by the problems such as environmental pollution, off-target effects, and resistance of pathogens and insects. New biotechnology-based strategies for pest control are expected to be developed. RNA interference (RNAi) is an endogenous process of gene regulation, which has been widely used to study the gene functions in various organisms. In recent years, RNAi-based pest management has received increasing attention. The effective delivery of the exogenous interference RNA into the targets is a key step in RNAi-mediated plant diseases and pest control. Considerable advances were made on the mechanism of RNAi, and various RNA delivery systems were developed for efficient pest control. Here we review the latest advances on mechanisms and influencing factors of RNA delivery, summarize the strategies of exogenous RNA delivery in RNAi-mediated pest control, and highlight the advantages of nanoparticle complexes in dsRNA delivery.
Subject(s)
Animals , RNA Interference , Pest Control , Insecta/genetics , RNA, Double-Stranded , Gene Expression RegulationABSTRACT
AIM: To investigate the effect of the expression of miR-375 on the proliferation and invasion of choroidal melanoma(CM)MUM-2B cells.METHODS: MUM-2B cells were cultured and were transfected with miR-375 mimic sequence(mimic group), miR-375 inhibitor sequence(inhibitor group), negative control group and no treatment(blank group). The qRT-PCR, CCK-8, apoptosis and Transwell experiments were used respectively to detect the expression of miR-375, cell proliferation activity, apoptosis, cell migration and invasion.RESULTS: Compared with the negative control group(1.01±0.10)and the blank group(1.03±0.07), the expression level of miR-375 in the cells of the mimic group(2.65±0.15)was increased, while the expression level of miR-375 in the cells of the inhibitor group(0.28±0.06)was decreased(P<0.05). Compared with the blank group and negative control group, the OD values of the cells in the mimic group at 24, 48, 72, and 96h were decreased(P<0.05), while the OD values of the cells in the inhibitor group at 24, 48, 72, and 96h were increased(P<0.05). Compared with the apoptosis rates in the blank group and negative control group, which were(20.54±4.01)% and(22.80±4.28)%, the apoptosis rate in the mimic group(39.11±3.37)% was increased(P<0.05), while it was decreased in the inhibitor group(10.13±2.17)%(P<0.05). Compared with the blank group and negative control group, the number of migration cell and the number of invasion cell in the mimic group were decreased(P<0.05), while the number of migration cell and the number of invasion cell in the inhibitor group were increased(P<0.05). CONCLUSIONS: Up-regulating the expression of miR-375 in MUM-2B cells can reduce cell proliferation activity, accelerate cell apoptosis, and inhibit cell migration and invasion, while down-regulating the expression of miR-375 has the opposite effect. It indicates that miR-375 may play the function of tumor suppressor in the course of CM.
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Objective To investigate the role of angiotensin-converting enzyme (ACE) in the reproduction of Culex pipiens pallens, so as to provide insights into selection of targets for controlling mosquito vector populations. Methods Cx. pipiens pallens was collected from Tangkou County, Shandong Province in 2009. Female and male mosquitoes were selected at 72 hours post-eclosion, and quantitative real-time reverse transcription PCR (qPCR) assay was used to detect the expression of ACE gene in the whole body and reproductive tissues of male mosquitoes and fertilized female mosquitoes before (0 h) and after blood meals (24, 48, 72 h), respectively. Then, 150 female and 150 male mosquitoes at 0 to 4 hours post-eclosion were selected and divided into the wild-type group (WT group), small interfering RNA-negative control group (siNC group) and small interfering RNA-ACE group (siACE group), of 50 mosquitoes in each group. Mosquitoes in the WT group were given no treatment, and mosquitoes in the siNC and siACE groups were given microinjection of siNC and siACE into the hemolymph at a dose of 0.3 μg per mosquito. The knockdown efficiency was checked using qPCR assay, and the reproductive phenotype of mosquitoes was observed. Results The relative ACE gene expression was higher in the whole body of male mosquitoes (5.467 ± 1.006) relative to females (1.199 ± 0.241) (t = 5.835, P = 0.004) at 72 h post-eclosion, and the highest ACE expression was seen in reproductive tissues of male mosquitoes (199.100 ± 24.429), which was 188.3 times higher than in remaining tissues (1.057 ± 0.340) (t = 6.602, P = 0.002). Blood meal induced high ACE expression in all body tissues of fertilized female mosquitoes, with peak expression at 24 h after blood meals (14.957 ± 2.815), which was 14.8 times higher than that before blood meals (1.009 ± 0.139) (P = 0.002). The transcriptional level of ACEs continued to increase in the ovaries of female mosquitoes after blood meals during the vitellogenesis phase, peaking at 48 h after blood meals (5.500 ± 0.734), which was 5.1 times higher than that before blood meals (1.072 ± 0.178) (P = 0.002). Small RNA interference targeting ACE resulted in a 57.2% reduction in ACE expression in female mosquitoes in the siACE group (0.430 ± 0.070) relative to the siNC group (1.002 ± 0.070) (P = 0.001), and a 41.1% reduction in male mosquitoes in the siACE group (0.588 ± 0.067) relative to the siNC group (1.008 ± 0.131) (P = 0.016). Knockdown of ACE expression resulted in a 48.0% decrease in the number of eggs laid by female mosquitoes in the siACE group [(94.000 ± 27.386) eggs] relative to the siNC group [(180.800 ± 27.386)] (P < 0.001), and a 45.0% decrease in the number of eggs laid by wild female mosquitoes mated with males in the siACE group [(104.500 ± 20.965) eggs] relative to the siNC group [(190.050 ± 10.698) eggs] (P < 0.001). Conclusions Reduced ACE expression may inhibit the fecundity of male and female mosquitoes, and ACE may be as a potential target for mosquito vector population suppression.
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RNAs are involved in the crucial processes of disease progression and have emerged as powerful therapeutic targets and diagnostic biomarkers. However, efficient delivery of therapeutic RNA to the targeted location and precise detection of RNA markers remains challenging. Recently, more and more attention has been paid to applying nucleic acid nanoassemblies in diagnosing and treating. Due to the flexibility and deformability of nucleic acids, the nanoassemblies could be fabricated with different shapes and structures. With hybridization, nucleic acid nanoassemblies, including DNA and RNA nanostructures, can be applied to enhance RNA therapeutics and diagnosis. This review briefly introduces the construction and properties of different nucleic acid nanoassemblies and their applications for RNA therapy and diagnosis and makes further prospects for their development.
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Platinum-based chemotherapy resistance is a key factor of poor prognosis and recurrence in hepatocellular carcinoma (HCC). Herein, RNAseq analysis revealed that elevated tubulin folding cofactor E (TBCE) expression is associated with platinum-based chemotherapy resistance. High expression of TBCE contributes to worse prognoses and earlier recurrence among liver cancer patients. Mechanistically, TBCE silencing significantly affects cytoskeleton rearrangement, which in turn increases cisplatin-induced cycle arrest and apoptosis. To develop these findings into potential therapeutic drugs, endosomal pH-responsive nanoparticles (NPs) were developed to simultaneously encapsulate TBCE siRNA and cisplatin (DDP) to reverse this phenomena. NPs (siTBCE + DDP) concurrently silenced TBCE expression, increased cell sensitivity to platinum treatment, and subsequently resulted in superior anti-tumor effects both in vitro and in vivo in orthotopic and patient-derived xenograft (PDX) models. Taken together, NP-mediated delivery and the co-treatment of siTBCE + DDP proved to be effective in reversing chemotherapy resistance of DDP in multiple tumor models.
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Abstract Therapeutics that inhibit enzymes, receptors, ion channels, and cotransporters have long been the mainstay of cardiovascular medicine. Now, oligonucleotide therapeutics offer a modern variation on this paradigm of protein inhibition. Rather than target a protein, however, small interfering ribonucleic acids and antisense oligonucleotides target the messenger RNA (mRNA) from which a protein is translated. Endogenous, cellular mechanisms enable the oligonucleotides to bind a selected sequence on a target mRNA, leading to its degradation. The catalytic nature of the process confers an advantage over the stoichiometric binding of traditional small molecule therapeutics to their respective protein targets. Advances in nucleic acid chemistry and delivery have enabled development of oligonucleotide therapeutics against a wide range of diseases, including hyperlipidemias and hereditary transthyretin-mediated amyloidosis with polyneuropathy. While most of these therapeutics were initially designed for rare diseases, recent clinical trials highlight the potential impact of oligonucleotides on more common forms of cardiovascular disease.
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Resumo A aterosclerose é a causa mais comum de doença cardiovascular em todo o mundo, ela está associada a uma alta incidência de eventos clínicos. O acúmulo de evidências elucidou que os RNAs longos não codificantes (LncRNAs) são uma nova classe de transcritos com papéis críticos nos processos fisiopatológicos da aterosclerose. Nesta revisão, resumimos o progresso recente dos LncRNAs no desenvolvimento da aterosclerose. Descrevemos principalmente os diversos mecanismos regulatórios dos LncRNAs nos níveis transcricionais e pós-transcricionais. Este estudo pode fornecer informações úteis sobre os LncRNAs como alvos terapêuticos ou biomarcadores para o tratamento da aterosclerose.
Abstract Atherosclerosis is the most common cause of cardiovascular disease globally, associated with a high incidence of clinical events. Accumulating evidence has elucidated that long non-coding RNAs (lncRNAs) as a novel class of transcripts with critical roles in the pathophysiological processes of atherosclerosis. In this review, we summarize the recent progress of lncRNAs in the development of atherosclerosis. We mainly describe the diverse regulatory mechanisms of lncRNAs at the transcriptional and post-transcriptional levels. This study may provide helpful insights about lncRNAs as therapeutic targets or biomarkers for atherosclerosis treatment.
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Objective To investigate the effect of Sema6D knockdown on the proliferation, migration, invasion and angiogenesis-promoting ability of human osteosarcoma cell lines. Methods The expression of Sema6D in clinical tissues and cell lines of human osteosarcoma was detected. After the targeted siRNA transfection, the changes of proliferation, migration and invasion were measured by CCK-8, wound healing and Transwell experiments. HUVECs were co-cultured with tumor conditioned medium to detect their tube formation ability. And the expression of signal pathway proteins was detected by Western blot. Results Sema6D was highly expressed in human osteosarcoma tissues and cell lines(P < 0.05). After silencing Sema6D, the proliferation, migration and invasion of 143B and MG63 cells decreased significantly(P < 0.05), the angiogenesis ability of HUVECs decreased in vitro(P < 0.01), and the expression of PI3K/AKT/mTOR and ERK-related signal pathway proteins decreased(P < 0.05). Conclusion Sema6D is overexpressed in human osteosarcoma tissues and cell lines. Knockdown of Sema6D expression level could inhibit the proliferation, migration, invasion and angiogenesis-promoting ability of human osteosarcoma cells via reducing PI3K/AKT/mTOR and ERK signal pathway.
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Objective:To investigate the effect of a short hairpin RNA (shRNA) targeting epidermal growth factor receptor (EGFR) combined with sirolimus on proliferation and apoptosis of the human cutaneous squamous cell carcinoma cell line Colo-16, and to explore underlying mechanisms.Methods:Cultured Colo-16 cells were divided into 5 groups: normal cell group receiving conventional culture and treatment with phosphate-buffered saline (PBS) , negative control group transfected with a shRNA-NC-expressing plasmid and treated with PBS, sirolimus group receiving conventional culture and sirolimus treatment, EGFR shRNA group transfected with an EGFR shRNA-expressing plasmid and treated with PBS, and combined group transfected with an EGFR shRNA-expressing plasmid and treated with sirolimus. Methyl thiazol tetrazolium (MTT) assay was performed to evaluate cellular proliferative activity in the above groups from 24 to 96 hours, and flow cytometry to detect cell apoptosis after 48-hour treatment. Semiquantitative RT-PCR was conducted to determine the mRNA expression of Bcl-2 and Bax, and Western blot analysis to determine the expression of apoptosis-related proteins cleaved caspase-3, cleaved caspase-9, Bcl-2, Bax, cell proliferation-related proteins phosphorylated mammalian target of rapamycin (p-mTOR) , phosphorylated protein kinase B (p-AKT) , phosphorylated 70-kDa ribosomal protein S6 kinase (p-P70S6k) , and cyclin D1. Comparisons among groups were carried out by using one-way analysis of variance, and multiple comparisons between 2 groups by using Student-Newman-Keuls q test. Results:MTT assay showed that the proliferative activity of Colo-16 cells was significantly lower in the sirolimus group, EGFR shRNA group and combined group during 24 - 96 hours than in the normal cell group (all P < 0.05) , and higher in the combined group than in the sirolimus group and EGFR shRNA group at 24-96 hours (all P < 0.001) , and there was no significant difference in the cellular proliferative activity at any time points between the normal cell group and negative control group (all P > 0.05) . Flow cytometry showed that the apoptosis rate was significantly higher in the sirolimus group, EGFR shRNA group and combined group (9.52% ± 0.25%, 12.65% ± 0.23%, 19.81% ± 0.31%, respectively) than in the normal cell group (3.33% ± 0.18%, q = 60.07, 78.08, 122.81, respectively, all P < 0.001) and negative control group (3.42% ± 0.19%, q = 59.90, 77.91, 122.64, respectively, all P < 0.001) , and was highest in the combined group. As RT-PCR and Western blot analysis revealed, the sirolimus group, EGFR shRNA group and combined group showed significantly decreased mRNA expression of Bcl-2 and protein expression of cyclin D1, p-AKT, p-mTOR, p-P70S6K and Bcl-2, but significantly increased mRNA expression of Bax and protein expression of cleaved caspase-3, cleaved caspase-9 and Bax compared with the normal cell group (all P < 0.05) . Compared with the sirolimus group and EGFR shRNA group, the combined group showed significantly decreased mRNA expression of Bcl-2 and protein expression of cyclin D1, p-AKT, p-mTOR, p-P70S6K and Bcl-2 (all P < 0.05) , but significantly increased mRNA expression of Bax and protein expression of cleaved caspase-3, cleaved caspase-9 and Bax (all P < 0.01) . Conclusion:EGFR shRNA and sirolimus exerted a synergistic effect in inhibiting the proliferation and promoting the apoptosis of Colo-16 cells, which may be related to the inhibition of the phosphoinositide 3-kinase (PI3K) /AKT/mTOR pathway.
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As the discovery of RNA interference (RNAi) and the gradual conquering of a series of technical issues, a few of RNAi therapeutics have been approved in the non-tumor field abroad. With the advantages of high specificity, long duration of efficacy, and high success rate of development, RNAi therapeutics have become the emerging field globally. There are no RNAi therapeutics approved in oncology so far, and people are hoping a breakthrough in the field. In the present article, the characteristics and potential anti-tumor mechanism of RNAi therapeutics, difficulties in delivery system and progress in oncology are described, and the potential reasons why their success in non-tumor field is difficult to be simply replicated in tumor field are analyzed, providing reference for research and clinical transformation of RNAi therapeutics in oncology. .
Subject(s)
Humans , Lung Neoplasms/genetics , RNA Interference , RNA, Small Interfering/therapeutic useABSTRACT
OBJECTIVE@#To investigate the potential inhibitory effect of interference with PD-L1 on B cell lymphoma in mice.@*METHODS@#Three shRNA vectors for mouse CD274 (PD-L1) were constructed and transiently transfected into 293T cells. RT-qPCR was used to validate the interference efficiency of CD274. The shRNA vector that interfere efficiently with CD274 expression was packaged by using lentivirus packaging system to generate shRNA lentivirus, and then transfected into A20 lymphoma cell line. The methyl thiazol terazolium (MTT) assay was used to detect proliferation after 48 h culture of CD274-sh A20 cells. Meanwhile, BALB/c mice were hypodermically infected with CD274-sh A20 cells. Infected mice were observed daily and assessed to visualize tumor by in vivo fluorescence imaging.@*RESULTS@#The proliferation rate of CD274-sh A20 cells in vitro was significantly lower than that of A20 cells (P<0.05). The tumor size detected by in vivo fluorescence imaging showed a significant reduce in tumor bearing mice with CD274-sh compared with other tumor bearing mice. And the weight and size of tumor in CD274-sh group were also significantly reduced compared with other group (P<0.05). Moreover, the survival time of tumor bearing mice in CD274-sh group was longer than that of the PD-L1 high expression group.@*CONCLUSION@#PD-L1 plays an important role in the incidence and the progression of lymphoma, and the shRNA-based PD-L1 knockdown can inhibit cell proliferation of A20 cells and partly suppress tumor growth.
Subject(s)
Animals , Humans , Mice , B7-H1 Antigen/metabolism , Cell Line, Tumor , Lymphoma , Lymphoma, B-Cell , Mice, Inbred BALB C , RNA, Small Interfering/geneticsABSTRACT
OBJECTIVE To know about the development trend of small nucleic acid drugs in the world ，to provide reference for the research and development of small nucleic acid drug in China. METHODS By searching the academic literature and patents related to small nucleic acid drugs through the Web of Science literature database and PatSnap patent database from Jan. 1980 to Dec. 2021，research and development situation of small nucleic acid drugs were revealed comprehensively by analyzing research enthusiasm，R&D countries ，R&D institutions and technical topics of small nucleic acid drugs. RESULTS & CONCLUSIONS A total of 59 819 documents and 37 645 patent groups were included. The global trend of small nucleic acid drug literature publication and patent application could be divided into three stages. From 2003 to 2021，the research enthusiasm for small nucleic acid drugs continued to increase. The United States ，China，Japan and Germany were the main research and development countries for small nucleic acid drugs. The number of document publications （25 703，15 927 papers）and patent applications （14 240、8 937 groups） in the United States and China were ahead of other countries ，and the research and development activities were relatively strong. Moreover，the number of document publications and patent applications in China in this field had grown rapidly in recent years. The R&D institution with the largest number of publications was the University of California （2 499 papers），the R&D institution with the largest number of patent applications was the American Ionis Corporation （1 378 groups），and the Chinese Academy of Sciences （1 580 papers）had been shortlisted among the top 10 document producing institutions in the world. However ，our country ’s research and development in this field are mostly based on basic research ，and the research on industrial application is slightly insufficient. The research focus in the field of small nucleic acid drugs mainly focuses on nucleic acid sequences and their modification and improvement and drug loading technology. RNA interference technology has gradually become a hot technology for small nucleic acid drugs.
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It is known that low-frequency pulsed electromagnetic fields (PEMFs) can promote the differentiation and maturation of rat calvarial osteoblasts (ROBs) cultured in vitro. However, the mechanism that how ROBs perceive the physical signals of PEMFs and initiate osteogenic differentiation remains unknown. In this study, we investigated the relationship between the promotion of osteogenic differentiation of ROBs by 0.6 mT 50 Hz PEMFs and the presence of polycystin2 (PC2) located on the primary cilia on the surface of ROBs. First, immunofluorescence staining was used to study whether PC2 is located in the primary cilia of ROBs, and then the changes of PC2 protein expression in ROBs upon treatment with PEMFs for different time were detected by Western blotting. Subsequently, we detected the expression of PC2 protein by Western blotting and the effect of PEMFs on the activity of alkaline phosphatase (ALP), as well as the expression of Runx-2, Bmp-2, Col-1 and Osx proteins and genes related to bone formation after pretreating ROBs with amiloride HCl (AMI), a PC2 blocker. Moreover, we detected the expression of genes related to bone formation after inhibiting the expression of PC2 in ROBs using RNA interference. The results showed that PC2 was localized on the primary cilia of ROBs, and PEMFs treatment increased the expression of PC2 protein. When PC2 was blocked by AMI, PEMFs could no longer increase PC2 protein expression and ALP activity, and the promotion effect of PEMFs on osteogenic related protein and gene expression was also offset. After inhibiting the expression of PC2 using RNA interference, PEMFs can no longer increase the expression of genes related to bone formation. The results showed that PC2, located on the surface of primary cilia of osteoblasts, plays an indispensable role in perceiving and transmitting the physical signals from PEMFs, and the promotion of osteogenic differentiation of ROBs by PEMFs depends on the existence of PC2. This study may help to elucidate the mechanism underlying the promotion of bone formation and osteoporosis treatment in low-frequency PEMFs.
Subject(s)
Animals , Rats , Alkaline Phosphatase/metabolism , Electromagnetic Fields , Osteoblasts/metabolism , Osteogenesis/genetics , TRPP Cation Channels/physiologyABSTRACT
@#Trichinellosis is an important zoonotic parasitic disease worldwide and is principally caused by ingesting animal meat containing Trichinella infective larvae. Aspartyl aminopeptidase is an intracytoplasmic metalloproteinase that specifically hydrolyzes the N-terminus of polypeptides free of acidic amino acids (aspartic acid and glutamate), and plays an important role in the metabolism, growth and development of organisms. In this study, a novel T. spiralis aspartyl aminopeptidase (TsAAP) was cloned and expressed, and its biological properties and roles in worm growth and development were investigated. The results revealed that TsAAP transcription and expression in diverse T. spiralis stages were detected by RT-PCR and Western blotting, and primarily localized at cuticle, stichosome and intrauterine embryos of this nematode by immunofluorescence test. rTsAAP has the enzymatic activity of native AAP to hydrolyze the substrate H-Glu-pNA. There was a specific binding between rTsAAP and murine erythrocyte, and the binding site was localized in erythrocyte membrane proteins. Silencing of TsAAP gene by specific dsRNA significantly reduced the TsAAP expression, enzymatic activity, intestinal worm burdens and female fecundity. The results demonstrated that TsAAP participates in the growth, development and fecundity of T. spiralis and it might be a potential target molecule for anti-Trichinella vaccines.
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Objective @#To fabricate a co-delivery system of curcumin (CUR) and siRNA based on mesoporous silica nanoparticles (MSN) and investigate its potential application in inducing macrophage M2 polarization.@*Methods@# MSNs were synthesized using the conventional sol-gel method. The interior mesochannels were occupied by small-molecule CUR, while the exterior surface was adsorbed by cationic polymeric polyethyleneimine (PEI) to link the negatively charged siRNA molecules to formulate the (CUR@MSN)PEI/siRNA co-delivery system. The formulation process was monitored by transmission electron microscopy(TEM). The MTT assay was used to evaluate the cytotoxicity in RAW264.7 cells under various concentrations of nanoparticles. Confocal laser scanning microscopy and TEM were used to observe cell internalization using FAM-labeled siRNA. GAPDH-targeting siRNA was used to prepare nanoparticles and then was transfected into RAW264.7 cells to observe the silencing efficiency of target genes. The knockdown efficiency was examined by real-time quantitative PCR. The related control groups were untreated cells, CUR delivery only and the co-delivery of CUR and siRNA negative control. By loading miRNA-130a-3p antisense oligonucleotide (ASO) to transfect RAW264.7 cells, the effects on the polarization of macrophages were observed. The M2 polarization marker arginase 1 (Arg-1) was measured by western blotting. The related control groups were untreated cells, CUR delivery only and co-delivery of CUR and miRNA negative control. @* Results @# The (CUR@MSN)PEI/siRNA co-delivery system was successfully formulated. The nanoparticles exhibited dose-dependent cytotoxicity, and the cell viability was maintained over 90% when the nanoparticle concentration was less than 10 μg/mL. A high cell uptake efficiency was observed, and the target gene knockdown efficiency was greater than 80% (P < 0.05 vs. all the other groups). The CUR delivery-only group and co-delivery of the CUR-and miRNA-negative control group improved Arg-1 expression ~ 3-fold (P < 0.05 vs. untreated cells). Using the co-delivery of CUR and ASO, synergistic effects were obtained, and Arg-1 expression was increased ~ 8-fold (P < 0.05 vs. all the other groups).@*Conclusion @#The CUR-siRNA co-delivery system can effectively transfect macrophages and synergistically induce M2 polarization.
ABSTRACT
Objective:To treat mice with Alzheimer's disease （AD） with β-catenin RNA interference （RNAi） Huangjingwan （HW）， so as to explore the neuroprotective signal mechanism of its prevention and treatment of AD. Method:A total of 81 male Kunming mice were randomly divided into normal control group， sham model control group， AD model group， Donepezil group， HW+scrambled group， HW+RNAi group， HW group， with 8 mice in each of donepezil group and HW group， and 13 mice in each of other groups. The AD models were established through injection with D-galactose and scopolamine in the last 5 groups for 5 consecutive weeks. On the 1st day of the 4th week after modeling， 0.75 μL PEI-LMW/β-catenin siRNAs nano-complex was injected into the right lateral ventricle of each mouse in for one time to treat with β-catenin RNAi in mice brains of the HW+RNAi group. The 0.75 μL complex was injected into the right lateral ventricle of each mouse for one time as for β-catenin interference control of the HW+scrambled group. The 0.75 μL normal saline was injected into the right lateral ventricle of each mouse in one time of the sham control group. Two weeks after intracerebroventricular injection， β-catenin RNAi was confirmed to be successful， and Donepezil （6.5×10-4 g·kg-1） was intragastrically administered to each mouse of donepezil group. HW （2.5 g·kg-1） was intragastrically administered to each mouse of HW group， HW+RNAi group and HW+scrambled group. Normal saline （0.5 mL·d-1） was intragastrically administered to each mouse of the sham control group. All gastric perfusion lasted for 4 weeks. At the end of gavage， the difference in learning and memory ability of mice was evaluated by platform jumping test. Nissl staining was used to count the number of neurons in s1Tr area of cerebral cortex and CA1 and CA3 areas of hippocampus of each mouse in each group. The mRNA expressions of Wnt1， DVL2， GSK-3β， β-catenin and CyclinD1 in mice brain of each group were detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. Western blot was used to detect the expressions of Wnt1， DVL2， GSK-3β， β-catenin and CyclinD1 in mice brain of each group. Result:The expression of β-catenin could be significantly inhibited through the injection with PEI-LMW/β-catenin siRNAs nano-complex into the lateral ventricle of AD mice， and nearly no β-catenin expression could be detected， which successfully achieved gene silencing. Compared with the normal control group， mice in AD model group showed that the learning and memory performance decreased significantly， the number of jumping errors increased （P<0.01）， the number of neurons in S1Tr area of cerebral cortex and CA1， CA3 areas of hippocampus decreased significantly （P<0.01）， the mRNA and protein expressions of Wnt1， DVL2， β-catenin， CyclinD1 in brain decreased significantly （P<0.01）， while the mRNA and protein expressions of GSK-3β increased significantly （P<0.01）. Compared with the AD model group， mice in HW group showed that the learning and memory performance increased significantly， the number of jumping errors decreased， the number of neurons in S1Tr area of cerebral cortex and CA1， CA3 areas of hippocampus increased significantly， the mRNA and protein expressions of Wnt1， DVL2， β-catenin， CyclinD1 in brain increased significantly， while the mRNA and protein expression of GSK-3β decreased significantly （P<0.01）. Compared with the HW group， mice in HW+RNAi group showed that the learning and memory performance decreased significantly， the number of jumping errors increased significantly （P<0.01）， the number of neurons in S1Tr area of cerebral cortex and CA1， CA3 areas of hippocampus decreased significantly （P<0.01）， there was no significant change in mRNA and protein expressions of Wnt1， DVL2， GSK-3β in the brain， and the mRNA and protein expressions of β-catenin， CyclinD1 decreased significantly （P<0.01）. Conclusion:HW can treat and prevent AD by activating Wnt/β-catenin signal pathway.
ABSTRACT
This study cloned the transcription factor gene PnbHLH which held an open reading frame of 966 bp encoding 321 amino acids. This study constructed the overexpression vector of transcription factor PnbHLH of Panax notoginseng. The combination of PnbHLH overexpression and RNAi of the key enzyme gene PnCAS involved in the phytosterol biosynthesis was achieved in P. notoginseng cells, thus exploring the biosynthetic regulation of P. notoginseng saponins(PNS) by the synergistic effect of PnbHLH overexpression and PnCAS RNAi. The results showed that the PnbHLH transcription factor interacted with the promoters of key enzyme genes PnDS, PnSS and PnSE in the biosynthetic pathway of PNS, and then regulated the expression levels of key enzyme genes and affected the biosynthesis of saponins indirectly. Further study indicated that the synergistic effect of PnbHLH overexpression and PnCAS RNAi was a more effective approach to regulate the biosynthesis of saponins. Compared with the wild type and PnCAS RNAi cells of P. notoginseng, the contents of total saponins and monomeric saponins(Rd, Rb_1, Re, Rg_1 and R_1) were increased to some extent in the cell lines of PnbHLH overexpression and PnCAS RNAi. This indicated that the two ways of forward regulation and reverse regulation of saponin biosynthesis showed superposition effect. This study explored a more rational and efficient regulation strategy of PNS biosynthesis based on the advantages of multi-point regulation of transcription factors as well as the down-regulation of by-product synthesis of saponins.