MicroRNA inhibitor capable of inhibiting SOCS3 down-regulation and preparation method thereof

A technology of SOCS3 and inhibitors, applied in the direction of DNA preparation, biochemical equipment and methods, DNA/RNA fragments, etc., to achieve strong effectiveness, alleviate morphine tolerance, and high safety effects

Active Publication Date: 2021-10-22
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, extensive activation of AMPK will also bring many negative effects, such as the activation of autophagy after activation of AMPK, by providing the necessary nutrients for tumor cells, maintaining the survival of tumor cells, and promoting the growth of tumor cells[15]

Method used

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  • MicroRNA inhibitor capable of inhibiting SOCS3 down-regulation and preparation method thereof
  • MicroRNA inhibitor capable of inhibiting SOCS3 down-regulation and preparation method thereof
  • MicroRNA inhibitor capable of inhibiting SOCS3 down-regulation and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Based on bioinformatics theory and experimental verification, five microRNAs that may regulate SOCS3 are screened out, such as figure 1 As shown, including microRNA-30a-5p, microRNA-203-3p, microRNA-19a-3p, microRNA-455-5p and microRNA-218-5p, by transfecting these microRNAs mimics into microglial cells (BV-2 ), microRNAs mimics use double-stranded RNA as a negative control, miRNA-NC, we found that microRNA-30a-5p significantly inhibited the expression of SOCS3, and the screening results were as follows figure 2 , image 3 shown. also, Figure 4 Experimental data also showed that AMPK agonists (metformin, resveratrol and AICAR) could significantly inhibit the expression of microRNA-30a-5p, as shown in Figure 4 As shown, thereby up-regulating SOCS3 protein levels but not SOCS3 mRNA.

[0053] According to the natural sequence 5'-UGUAAACAUCCUCGACUGGAAG-3' of mmu-microRNA-30a-5p, its complementary strand, namely mmu-microRNA-30a-5p inhibitor, whose sequence...

Embodiment 2

[0054] Example 2: The lyophilized powder of the microRNA-30a-5p inhibitor prepared in Example 1 was prepared into a solution with DEPC water, and applied in The transfection reagent was used as the delivery medium of microRNA-30a-5p inhibitor, which contained 5 microliters of 10% glucose solution, 3 microliters of in 10 microliters system of 2 microliters of microRNA-30a-5pinhibitor, ensure to give each mouse 125pmol microRNA-30a-5p inhibitor, the Negative control of microRNA-30a-5p inhibitor is a single-stranded RNA scrambled sequence, the dosage is the same as the system microRNA-30a-5p inhibitor. Morphine (10 micrograms / 10 microliters) was administered for seven consecutive days to prepare a morphine tolerance model, and microRNA-containing microRNA- The above system of 30a-5pinhibitor. After 30 minutes of morphine administration every day, the effect of microRNA-30a-5pinhibitor on morphine tolerance was detected by tail-flick test, and the results were as follows: Fig...

Embodiment 3

[0055] Example 3: According to the method of Example 2, 125 pmol microRNA-30a-5pinhibitor was administered on the 0th, 3rd, and 6th days, and 10 micrograms / 10 microliters of morphine was given every day. After 7 days of continuous administration, the spinal cord was taken and passed Western blotting. (western blot) to detect the phosphorylation level of nuclear transcription factor p65 (NF-κB-p65), such as Figure 6 shown.

[0056] The experimental results showed that microRNA-30a-5p inhibitor could significantly inhibit the phosphorylation level of nuclear transcription factor p65 (NF-κB-p65) induced by morphine. *p<0.05versus the control group; ##p<0.01versusmorphine-treated group.

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Abstract

The invention discloses a microRNA (Ribonucleic Acid) inhibitor capable of inhibiting SOCS3 (Sequence of Coriolis 3) down-regulation and a preparation method of the micro RNA inhibitor. The micro RNA inhibitor comprises the following RNA sequence: 5 '-CUUCCAGUCGAGGAUGUUUUACA-3'. The microRNA inhibitor is an inhibitor of microRNA-30a-5p subjected to 2 '-Ome chemical modification, the microRNA-30a-5p is inhibited in a specific targeting manner, the gene silencing effect of endogenous microRNA-30a-5p is inhibited, the protein expression quantity of SOCS3 is increased, neuroinflammation is improved, morphine tolerance is relieved, and the microRNA inhibitor has the advantages of being high in targeting property, high in safety, high in effectiveness and high in stability.

Description

technical field [0001] The invention relates to the technical field of gene regulation, in particular to a microRNA inhibitor capable of inhibiting down-regulation of SOCS3 and a preparation method thereof. Background technique [0002] Morphine, as a representative drug of opioids, is the gold standard for the treatment of severe pain from various causes, but long-term exposure to morphine can cause serious side effects, such as tolerance, that is, with repeated use of the drug, the drug's efficacy decreases, It is necessary to increase the dosage or shorten the interval between administrations to maintain the original efficacy. Morphine tolerance is an extremely challenging clinical problem that limits its clinical application in pain management. There are a large number of reports on the mechanism of morphine tolerance, mainly including down-regulation of opioid receptors [1], changes in inhibitory neurons [2] and neuroinflammation [3]. A growing body of research sugges...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/10A61K31/7105A61P25/00
CPCC12N15/113A61K31/7105A61P25/00C12N2310/141
Inventor 姜春懿刘文涛韩园潘寅兵胡亮王玉田田万莉
Owner NANJING MEDICAL UNIV
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