siRNA targeting PSMC5 gene and application of PSMC5
阅读说明：本技术 靶向PSMC5基因的siRNA及PSMC5的应用 (siRNA targeting PSMC5 gene and application of PSMC5 ) 是由 朱丽红 毕伟 陆大祥 肖书 赵佳仪 于 2018-07-17 设计创作，主要内容包括：本发明提供了靶向PSMC5基因的siRNA及PSMC5的应用。本发明提供了26S蛋白酶体调节亚基8(PSMC5)作为治疗阿尔茨海默病的新靶点的技术方案,通过抑制PSMC5的表达实现阿尔茨海默病的有效治疗,可为筛选研发抗AD药物提供新的思路和新的方法。本发明还提供了一种靶向PSMC5基因的siRNA,可通过抑制TOLL样受体激活介导的信号通路,抑制小胶质细胞的过度激活,从而保护海马神经元,有效改善和治疗AD；还可对潜在的小胶质细胞过度激活诱导的学习记忆、认知功能障碍类疾病人群的保护治疗作用。本发明可在神经变性疾病及老年医学领域得到广泛的应用,并产生巨大的社会与经济效益。(The invention provides siRNA targeting PSMC5 gene and application of PSMC 5. The invention provides a technical scheme that 26S proteasome regulatory subunit 8(PSMC5) is used as a new target for treating Alzheimer disease, realizes effective treatment of Alzheimer disease by inhibiting expression of PSMC5, and can provide a new idea and a new method for screening and developing anti-AD drugs. The invention also provides a PSMC5 gene targeted siRNA which can inhibit excessive activation of microglia by inhibiting a signal channel mediated by TOLL-like receptor activation, thereby protecting hippocampal neurons and effectively improving and treating AD; and also has protective and therapeutic effects on learning and memory induced by excessive activation of potential microglia and cognitive dysfunction. The invention can be widely applied to the fields of neurodegenerative diseases and old-age medicine, and can generate great social and economic benefits.)
Application of PSMC5 protein as a target in screening or preparing medicines for inhibiting excessive activation of microglia.
Application of PSMC5 protein as a target in screening or preparing medicines for preventing and/or treating Alzheimer disease, Parkinson disease or multiple sclerosis.
3. An siRNA targeting PSMC5 gene, wherein the sense strand or antisense strand of said siRNA is as follows:
sense strand: 5'-GCAGUGGACUCCGUCAAUATT-3' the flow of the air in the air conditioner,
antisense strand: 5'-UAUUGACGGAGUCCACUGCTT-3' are provided.
4. A recombinant expression vector, a transgenic cell line or a recombinant bacterium comprising a nucleotide sequence encoding the siRNA targeting PSMC5 gene of claim 3.
5. Use of the siRNA targeting PSMC5 gene of claim 3 or the recombinant expression vector comprising the nucleotide sequence encoding the siRNA targeting PSMC5 gene of claim 4 for the preparation of a medicament for inhibiting the activation of microglia.
6. Use of the siRNA targeting PSMC5 gene of claim 3 or the recombinant expression vector comprising the nucleotide sequence encoding the siRNA targeting PSMC5 gene of claim 4 for the preparation of a medicament for preventing and/or treating alzheimer's disease, parkinson's disease or multiple sclerosis.
7. A medicament for preventing and/or treating alzheimer's disease, characterized in that:
the medicine takes PSMC5 gene as a target spot and inhibits the expression level of PSMC5 gene.
8. The agent for the prophylaxis and/or treatment of alzheimer's disease according to claim 7, wherein:
the active component of the medicine is the siRNA targeting PSMC5 gene or a recombinant expression vector containing nucleotide sequence encoding the siRNA targeting PSMC5 gene.
9. The agent for the prophylaxis and/or treatment of alzheimer's disease according to claim 7 or 8, characterized in that:
the medicament contains one or more pharmaceutically acceptable carriers.
10. The agent for the prophylaxis and/or treatment of alzheimer's disease according to claim 7 or 8, characterized in that:
the medicine can be further prepared into tablets, granules, capsules, oral liquid or injections.
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to siRNA targeting PSMC5 gene and application of
Background
Alzheimer's Disease (AD) is the most common type of senile dementia, and is a neurological degenerative disease with major clinical manifestations of cognitive decline, decline of life functions and abnormal mental behaviors. At present, there are 460 million new cases per year (1 new case every 7 seconds) worldwide, and by 2040 years worldwide AD patients will reach 8110 million. Next to heart disease, cancer and stroke, AD has become the main cause of disability and inability to live independently in the elderly, causing serious harm and heavy burden to families and the whole society, and having severe prevention and treatment situation. Therefore, intensive research on the pathogenesis of AD and discussion on feasible treatment means have great significance for improving the health level of the nation and reducing the financial burden of the nation.
AD is primarily characterized pathologically by amyloid aggregation in brain tissue, formation of neurofibrillary tangles (NFTs) by intracellular abnormally phosphorylated tau protein aggregation, neuronal loss, and microglial activation. The etiology of AD is not clear so far, and the pathogenesis is complicated. To date, various clinical treatment strategies for AD, including cholinesterase inhibitors, improving cerebral blood flow to promote cerebral metabolism, anti-oxidation, estrogen therapy, etc., have failed to halt the progression of the disease process and address the progressive decline of central cholinergic neurons. Therefore, all current clinical treatments are basically ineffective or very slight, and effective prevention and treatment of AD is in trouble.
Microglial activation plays a very important role in the pathogenesis of AD. Microglia are widely distributed in the central nervous system, serve as main immune cells in the central nervous system, play important roles in supporting, nourishing, protecting and repairing the survival and the whole life activities of neurons, and are indispensable components of the nervous system. Under normal conditions, microglia are in a resting state and can be activated when the nervous system is damaged and can be further activated into macrophage-like microglia. The A beta activated microglial cell mediated immune inflammatory reaction can cause apoptosis of hippocampal neurons of AD patients, and further gradually appears cognitive function decline. The main role of a β oligomers in the early stages of AD onset, while the persistent inflammatory response in the brain of AD-maintaining patients is mainly a β in the form of fibers. The reason for the differences in the effects of the different aggregated forms may be their structural differences, while the structure required for activation of microglia may be the beta turn structure rather than the beta fiber. Microglia are also closely related to the formation of age spots in AD patients. In senile plaques, aggregated A beta is located at a core position, activated microglia are found around A beta deposition, and the formation of the senile plaques can be accelerated by the activated microglia for a long time through various secreted cytokines, so that the neurofibrillary tangles are promoted. Epidemiological studies have found that the incidence of AD in elderly people taking NSAIDs for a long period of time is significantly reduced. Animal experiment results show that after the NSAIDs are administered, the quantity of A beta deposition and activated microglia in the brain of the AD model animal is obviously reduced, and the inflammatory response in the brain is obviously relieved. Therefore, the method takes the inhibition of microglial activation as a target point, inhibits/alleviates neuroinflammatory reaction, regulates and controls the balance of hippocampal neuron-microglial neural network, thereby improving and blocking the progress of AD, and is an effective strategy for preventing and treating AD at present.
PSMC5 is also called S8, and is 26S proteasome regulatory subunit 8(26S proteasome regulation subunit 8) which is an important regulatory subunit of ubiquitin-proteasome pathway and has chaperone-like activity. In 1995, Lee isolated and identified PSMC5 in a yeast two-hybrid test, in 1997, Hoyle located the same by using a fluorescence in situ hybridization technology, PSMC5 consists of 406 amino acids, has a 845.6KD size, is defined as 17q24-q25 in a chromosome, encodes a subunit of ATPase enzyme, and is a member of ATPase multi-A family.
There are no reports on the influence of PSMC5 on microglia activation, improvement and blocking of AD progression.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides application of 26S proteasome regulatory subunit 8(PSMC5) serving as a target point in screening or preparing medicines for inhibiting excessive activation of microglia.
The invention also aims to provide application of 26S proteasome regulatory subunit 8(PSMC5) as a target in screening or preparing medicines for preventing and/or treating Alzheimer disease, Parkinson disease or multiple sclerosis.
Still another object of the present invention is to provide an siRNA targeting PSMC5 gene.
The invention also aims to provide application of the siRNA or the recombinant expression vector targeting the PSMC5 gene.
The purpose of the invention is realized by the following technical scheme:
the application of 26S proteasome regulatory subunit 8(PSMC5 protein) as a target in screening or preparing medicines for inhibiting excessive activation of microglia. The medicine is a medicine for inhibiting the expression level of PSMC5, and the inventor firstly discovers that the activation of microglia can be effectively inhibited by down-regulating
The application of 26S proteasome regulatory subunit 8(PSMC5 protein) as a target in screening or preparing medicaments for preventing and/or treating Alzheimer disease, Parkinson disease or multiple sclerosis; preferably the application in screening or preparing medicaments for preventing and/or treating Alzheimer disease. By down-regulating the expression level of PSMC5, the activation-mediated signal pathway of TOLL-like receptor is inhibited, the release and expression of inflammatory factor are inhibited, the neuroinflammation reaction is inhibited/relieved, and the balance of hippocampal neuron-microglia neural network is regulated, so that the learning memory and cognitive dysfunction is corrected, the motor coordination ability is improved, and the progress of diseases caused by the excessive activation of microglia is improved and blocked.
An siRNA targeting PSMC5 gene, wherein the sense strand or antisense strand of said siRNA is as follows:
sense strand: 5'-GCAGUGGACUCCGUCAAUATT-3' (SEQ ID NO.1),
antisense strand: 5'-UAUUGACGGAGUCCACUGCTT-3' (SEQ ID NO. 2).
The siRNA is searched by Blast Search and confirmed to have no homology with a human known gene sequence except
The target sequence for siRNA interference is located between 56 th and 76 th bases of the mRNA coding sequence of PSMC5 gene, namely 5'-GCAGUGGACUCCGUCAAUATT-3'.
The siRNA targeting the PSMC5 gene can be synthesized by biotechnology companies.
Contains a recombinant expression vector, a transgenic cell line or a recombinant bacterium of the nucleotide sequence of the siRNA of the targeting PSMC5 gene.
The PSMC5 gene targeting siRNA or the recombinant expression vector containing the nucleotide sequence encoding the PSMC5 gene targeting siRNA is applied to the preparation of the drug for inhibiting the activation of microglia.
The PSMC5 gene targeting siRNA or the recombinant expression vector containing the nucleotide sequence encoding the PSMC5 gene targeting siRNA is applied to the preparation of medicines for preventing and/or treating Alzheimer disease, Parkinson disease or multiple sclerosis; preferably the application in the preparation of the medicament for preventing and/or treating Alzheimer disease.
The medicine takes PSMC5 gene as a target point and inhibits the expression level of PSMC5 gene.
The medicinal active component is preferably the siRNA targeting PSMC5 gene or a recombinant expression vector containing nucleotide sequence encoding the siRNA targeting PSMC5 gene.
The medicament may contain one or more pharmaceutically acceptable carriers.
The carrier is preferably a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, an adsorption carrier, a surfactant or a lubricant, etc.
The medicine can be further prepared into various forms such as tablets, granules, capsules, oral liquid or injection, and the medicines of various formulations can be prepared according to the conventional method in the pharmaceutical field.
Compared with the prior art, the invention has the following advantages and effects:
(1) the invention provides a novel target for treating Alzheimer disease, namely 26S proteasome regulatory subunit 8(PSMC5), and realizes effective treatment of Alzheimer disease by inhibiting expression of
(2) The invention also provides a siRNA interference fragment for down-regulating PSMC5 expression, the siRNA has obvious interference effect on PSMC5, and inhibits the release and expression of inflammatory factors, inhibits/alleviates neuroinflammatory reaction and regulates the balance of hippocampal neuron-microglia neural network by inhibiting the activation of a signal channel mediated by a TOLL-like receptor, thereby correcting learning and memory dysfunction, improving motor coordination ability and improving and blocking the progress of AD.
(3) The PSMC5siRNA can also be applied to the protection and treatment effects on potential microglia over-activation induced learning and memory and cognitive dysfunction disease populations.
(4) The PSMC5siRNA can be used as a biological agent for preventing and/or treating AD, can be widely applied to the fields of neurodegenerative diseases and senile medicine, and can generate huge social and economic benefits.
Drawings
FIG. 1 is a graph of interference efficiency analysis of different PSMC5 siRNAs.
FIG. 2 is a graph showing the analysis of the result of PSMC5siRNA inhibiting LPS-induced NO production in BV2 cells.
FIG. 3 is a graph showing the analysis of the results of the reduction of IL-1. beta. production by BV2 cells induced by LPS by PSMC5 siRNA.
FIG. 4 is a graph showing the results of analysis of the inhibition of LPS-induced BV2 cell PSMC5 protein expression by different PSMC5 siRNAs.
FIG. 5 is a graph showing the analysis of the results of the effect of intraperitoneal LPS injection on learning and memory ability of mice.
FIG. 6 is a graph showing the analysis of the results of the effect of intraperitoneal LPS injection on mice in the darkness avoidance experiment.
FIG. 7 is a graph showing the analysis of the results of the effect of intraperitoneal LPS injection on the mouse climbing rod experiment.
FIG. 8 is a graph showing the results of Western blot of the expression level of PSMC5 at various time points by lateral ventricle injection of
FIG. 9 is a graph showing the effect of PSMC5siRNA on I- κ Ba protein levels.
FIG. 10 is a graph showing the results of analysis of the effect of PSMC5siRNA on LPS-induced BV2 microglia Toll-
FIG. 11 is a graph of the analysis of the results of the effect of PSMC5siRNA on LPS-induced BV2 microglial inflammatory factor.
Fig. 12 is a schematic of BV2 microglial coculture with primary hippocampal neurons.
Fig. 13 is a graph of the analysis of the results of PSMC5siRNA inhibition of BV2 activation-mediated damage to primary hippocampal neurons.
Fig. 14 is a photomicrograph of a cell morphology microscope showing that PSMC5siRNA inhibits BV2 activation-mediated apoptosis of hippocampal neurons.
FIG. 15 is a graph showing the analysis of the results of the effect of PSMC5siRNA on LPS-induced learning and memory impairment in the localization experiment.
FIG. 16 is a graph showing the analysis of the results of the effect of PSMC5siRNA on LPS-induced learning and memory impairment in the pole-climbing experiment.
FIG. 17 is a graph showing the analysis of the results of the effect of PSMC5siRNA on LPS-induced learning and memory impairment in the darkening-avoidance experiment in mice.
FIG. 18 is a graph showing the analysis of the results of PSMC5siRNA on the effect of proinflammatory factors released in the serum of LPS-induced learning memory impairment mice.
FIG. 19 is a graph showing the analysis of the effect of PSMC5siRNA on the release of anti-inflammatory factors in the serum of mice with learning and memory impairment induced by LPS.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The reagents used in the following examples were all purchased from commercial sources.
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