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ShRNA lentivirus for inhibiting expression of long-chain non-coding RNA MALAT1 and application of shRNA lentivirus

A long-chain non-coding, lentiviral technology, applied in retro RNA viruses, DNA/RNA fragments, applications, etc., can solve the problems of affecting cells, not targeting MALAT1 to treat stress-induced cognitive dysfunction, etc., to achieve resistance Effects of neuronal cell damage, potential application value

Pending Publication Date: 2021-11-02
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are also studies suggesting that interfering with MALAT1 in nerve cells can affect the response of cells to GC stimulation, suggesting that MALAT1 may be involved in the regulation of stress response
However, the role of MALAT1 in stress-induced cognitive dysfunction has not been reported so far, and there is no related technology targeting MALAT1 to treat stress-induced cognitive dysfunction

Method used

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  • ShRNA lentivirus for inhibiting expression of long-chain non-coding RNA MALAT1 and application of shRNA lentivirus
  • ShRNA lentivirus for inhibiting expression of long-chain non-coding RNA MALAT1 and application of shRNA lentivirus
  • ShRNA lentivirus for inhibiting expression of long-chain non-coding RNA MALAT1 and application of shRNA lentivirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Malat1-shRNA SLR Works Construction and Identification

[0030] From NCBI to the transcription of Malat1 (NR_002847.3), designing the SHRNA interfering site for malaat1, filtering 4 sites with the highest score, chemical synthesis malat1-shRNA Oligo and negative control Shrna Oligo (Shanghai Jikai Biological Co., Ltd. Company), annealing to form a double strand, cloned to a slow viral expression vector, and obtained recombinant plasmid.

[0031] (1) Design of Malat1 interference target

[0032] From NCBI, the transcription of malaat1 (NR_002847.3) is designed to design a SHRNA interfering site for malaat1, filtering 4 sites with the highest score, and the target sequence is as follows:

[0033] 286-site: 5'-gcawtttaggagattgtaaaag-3 '(SEQ ID NO.1)

[0034] 743 sites: 5'-GGaagtgaaagacgaagaaga-3 '(SEQ ID NO.2)

[0035] 1048-site: 5'-gcAGTTTAGAGAGTTTTTTAG-3 '(SEQ ID NO.3)

[0036] 4807-site: 5'-gctcaggactttgcatataag-3 '(SEQ ID No.4)

[0037] (2) Construction of Malat1...

Embodiment 2

[0075] Example 2MALAT1-shRNA plasmid Interference Effect embodiment

[0076] The identified positive recombinant plasmid HT22 cells, cells were collected RNA was extracted and reverse transcribed to obtain the cDNA, the influence by real-time PCR assay The recombinant plasmid MALAT expression, select the most significant plasmid interference effect is further lentivirus packaging .

[0077] (A) transfection of cells

[0078] (1) take on HT22 cells in logarithmic growth phase, 50% were seeded into 6-well culture plates, placed in 5% CO 2 , 37 ℃ incubator until the culture reached a cell density of approximately 80%.

[0079] (2) instructions for use in accordance with jetPEI transfection reagent (Polyplus Inc.), to prepare a mixture of the plasmid transfection reagent, added to the cells dropwise.

[0080] After (3) 24hr observation cell state, replaced with fresh complete medium. 48hr later cells were harvested for RNA extraction experiments Real-time PCR.

[0081] (Ii) RNA extrac...

Embodiment 3

[0106] Example 3 Slow virus packaging and quality detection

[0107] MAlat1-shRNA interfering plasmid and VSVG, PSPAX2 plasmid were transfected with 293T cells, and the cell super clear after 48 hr was cultured, and the concentration and purification of slow virus were subjected to ultracentrifugation, and finally the viral titer was determined by fluorescence.

[0108] (1) Cultivation of 293T cells

[0109] 1, recovery 293T cells

[0110] (1) Configure a DMEM medium containing 10% FBS (referred to as complete medium) for the cultivation of 293T cells.

[0111] (2) 3 ml of complete medium was added to a 10 ml glass centrifuge tube.

[0112] (3) Take the cell from the liquid nitrogen tank or -80 ° C refrigerator, rapidly put into a 37 ° C water bath, gently shake 1 to 2 minutes to make it completely melted.

[0113] (4) Take the frozen storage tube to the ultra-clean station, and disinfect the surface with alcohol cotton ball. The cell suspension is added to the centrifuge tube pre...

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Abstract

The invention provides shRNA for inhibiting expression of long-chain non-coding RNA MALAT1. The shRNA is designed for sites 286, 743, 1048 and 4807 of a MALAT1 transcript sequence NR_002847.3 in Genbank or corresponding sites in other transcript sequences. The invention further provides a corresponding plasmid, a lentivirus, a pharmaceutical composition and application of the pharmaceutical composition in treatment of cognitive impairment. The MALAT1-shRNA interference plasmid disclosed by the invention can be used for remarkably and specifically inhibiting the expression of MALAT1 in hippocampal neurons, promoting the process of a neuronal cell cycle and inhibiting neuronal apoptosis and the expression of apoptosis key proteins, so that the effect of resisting stress-induced neuronal cell injury is achieved.

Description

Technical field [0001] The present invention belongs to the field of biotechnology, and more particularly to the SHRNA lentivirus and its application inhibiting long-chain non-coding RNA malat1 expression. Background technique [0002] With the accelerated rhythm of modern social life and the intensification of social competition, most people are under different levels of stress load, long-term excessive stress has been confirmed as a variety of major lethal diseases, and humanity The development of 70% of the disease is closely related to body stress loss. The brain is a body induction and should have a stress control center, and is also a target organ that should be excited, especially the cognitive function involving advanced brain activities, is particularly fragile in the face of the state. Epidemiological surveys show that people who have a stressed state in stressed states have twice as much as the prevalence of dementia. Among the diagnosed cognitive dysfunction patients,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/867C12N7/01A61K31/7088A61K48/00A61P25/00
CPCC12N15/1135C12N15/86C12N7/00A61K31/7088A61K48/005A61P25/00C12N2310/14C12N2320/30C12N2740/15021C12N2740/15043C12N15/113C12N2310/113A61K31/7105C12N2310/531
Inventor 王雪谢方钱令嘉王颖赵云
Owner ACADEMY OF MILITARY MEDICAL SCI
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