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General expression framework of artificial circular RNA targeting to inhibit miRNA-34a, and expression method and application thereof

A mir-34a-5p, circular technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, biochemical equipment and methods, can solve problems such as obstacles to the development of analogue or antagonist drugs

Inactive Publication Date: 2019-04-02
浙江自贸区锐赛生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, limited by the physical and chemical properties of nucleic acid drugs, there are many obstacles in the development of microRNA mimetic or antagonist drugs, the most important of which are the targeted delivery and half-life of drug molecules in vivo.
Although the chemical modification technology of nucleic acid molecules and the delivery technology of nucleic acid drugs have been greatly improved, the development of microRNA mimetic or antagonistic molecular drugs still needs to solve the problems of delivery efficiency and in vivo stability more effectively.

Method used

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  • General expression framework of artificial circular RNA targeting to inhibit miRNA-34a, and expression method and application thereof
  • General expression framework of artificial circular RNA targeting to inhibit miRNA-34a, and expression method and application thereof
  • General expression framework of artificial circular RNA targeting to inhibit miRNA-34a, and expression method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: The involvement and synthesis of the artificial circular RNA expression framework sequence and the corresponding linear RNA control expression framework sequence.

[0038] 1. Use the miRbase database to query the sequence of human miR-34a-5p as "uggcagugucuuagcugguugu", and its reverse complementary DNA sequence is "ACAACCAGCTAAGACACTGCCA", replace the general framework formula I with this sequence

[0039] "34inh". The LS sequence is "GTGTGT" and the IS sequence is

[0040] "ACAAAACAGCCACGCTTCGAGCACGAATCGCCAACTCACGAACG", natural number n=5, combined with the US and DS sequence elements in the general framework formula I shown in SEQ ID NO.1 and SEQ ID NO.2, to obtain the general expression framework of the artificial circular RNA molecule rs-ciR34-10, The base sequence is shown in SEQ ID NO.6.

[0041] 2. Randomly scramble the reverse complementary DNA sequence of the above-mentioned miR-34a-5p, and replace the general purpose of the artificial circular RN...

Embodiment 2

[0044] Example 2: Lentiviral Packaging

[0045] 1. 24 hours before transfection, digest 293T cells in the logarithmic growth phase with trypsin, transfer to 10cm cell culture dish, 37°C, 5% CO 2 Cultured in an incubator. After 24 hours, when the cell density reaches 70%-80%, it can be used for transfection. Cell state is critical for virus packaging, so good cell state and low passage times need to be guaranteed.

[0046] 2. Replace the cell culture medium with serum-free medium before transfection.

[0047] 3. Add the prepared plasmid DNA solutions (lentiviral plasmid 10 μg, helper plasmid pLP1, pLP2, pLP / VSVG each 5 μg) into the sterilized centrifuge tube, mix with the corresponding volume of Opti-MEM, and adjust the total volume to 1.5ml.

[0048] 4. Shake the Lipofectamine 2000 reagent gently, take 60 μl Lipofectamine 2000 reagent and mix it with 1.5ml Opti-MEM in another tube, and incubate at room temperature for 5 minutes.

[0049] 5. Mix the diluted DNA with the di...

Embodiment 3

[0061] Example 3: RT-PCR detection of circular RNA molecule expression in LO2 cells infected with lentivirus

[0062] 1. Resuscitate LO2 cells, according to the corresponding culture conditions, at 37°C, 5% CO 2 Incubator cultivation.

[0063] 2. Inoculate the cell suspension in a 6-well plate (400,000 / well), 37°C, 5% CO 2 Incubator cultivation.

[0064] 3. Add appropriate amount of artificial circular RNA overexpression lentivirus, linear control sequence lentivirus and negative control lentivirus to the cells according to the MOI of colon cancer cells and the titer of each virus, and at the same time add Polybrene with a final concentration of 8ug / ml to enhance Infect.

[0065] 4. After 24 hours of infection, replace the complete medium without lentivirus to continue the culture.

[0066] 5. After 72 hours of infection, add 1ml Trizol to each well of the 6-well plate, repeatedly pipette 10 times with a 1ml pipette tip, and collect it into an EP tube; centrifuge at 12000g...

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Abstract

The invention relates to a general expression framework of artificial circular RNA targeting to inhibit miRNA-34a, and an expression method and an application thereof. The general expression frameworkconsists of three parts: an upstream sequence, an intermediate sequence and a downstream sequence. The general expression framework of the artificial circular RNA targeting to inhibit miRNA-34a can be mediated in cells by plasmids or viral tools to express artificial circular RNA molecules targeting to inhibit miRNA-34a, and has the ability to inhibit the biological function of miRNA-34a-5p in cells. The invention also relates to the general expression framework and the application of eukaryotic expression plasmids, lentiviruses, adenoviruses, adeno-associated viruses and retroviruses carrying the expression framework in preparation of drugs for obesity, fatty liver, hyperlipidemia and diabetes.

Description

[technical field] [0001] The invention relates to a general expression framework and an expression method of an artificial circular RNA targeting and inhibiting miR-34a and its application in preparing medicines for obesity, fatty liver, hyperlipidemia and diabetes. [Background technique] [0002] In recent years, with the improvement of people's living standards, the prevalence of obesity, fatty liver, hyperlipidemia and diabetes related to excess energy intake has been increasing. According to the 2018 China National Health and Nutrition Big Data Report, there may be more than 200 million overweight or obese citizens, including 160 million patients with dyslipidemia (including hyperlipidemia), 120 million patients with fatty liver, and more than 90 million patients with diabetes. Metabolic abnormal diseases seriously affect people's health. Therefore, the development of drugs that can effectively regulate glucose and lipid metabolism and maintain the stable state of glucos...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/63A61K48/00A61K31/7088A61P3/04A61P3/06A61P3/10A61P1/16
CPCA61K31/7088A61K48/005A61P1/16A61P3/04A61P3/06A61P3/10C12N15/113C12N15/63C12N2310/50
Inventor 张腾罗卫峰岳颖赵玉晓余卫国
Owner 浙江自贸区锐赛生物医药科技有限公司
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