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Three microRNAs derived from tea tree and their applications

A technology for tea tree and diabetes, applied in the field of microRNA and its application, can solve the problem of detection of extremely low abundance

Active Publication Date: 2016-09-07
ZHEJIANG ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since sodium periodate is a strong oxidizing agent, it can oxidize the 2' and 3' free hydroxyl groups of animal-derived miRNAs, resulting in the animal-derived miRNAs not being detected or detected in extremely low abundance during miRNA sequencing and qPCR

Method used

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  • Three microRNAs derived from tea tree and their applications
  • Three microRNAs derived from tea tree and their applications
  • Three microRNAs derived from tea tree and their applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Screening and identification of miRNA

[0022] Fresh tea tree leaves were selected for feeding mice and miRNA sequencing (2 biological replicates). Two groups of ICR mice (18-20 g), 24 in total, were fasted for 12 hours respectively, the control group was fed mouse feed, and the experimental group was force-fed tea tree leaves. Liver tissue and serum of corresponding mice were collected at different time points, and total RNA was extracted respectively. Mix equal amounts of serum sample RNA at 0h and 3h in the mouse feed group for one miRNA sequencing, and mix equal amounts of serum sample RNA at 3h and 6h for one miRNA sequencing; One miRNA sequencing was performed by mixing, and equal amounts of liver sample RNAs from 3h and 6h were mixed for one miRNA sequencing. The sample collection and sequencing of the force-fed tea tree leaf group were as above. According to the sequencing results, it was selected to be found in the tea tree leaf miRNA sequencing a...

Embodiment 2

[0024] Example 2: Distribution of 3 miRNAs in plants

[0025] The improved CTAB method was used to extract and enrich small RNAs from tea tree leaves. The details are as follows: (1) Take 0.1 g of young leaves of tea tree from each sample, grind them quickly in liquid nitrogen, and transfer them to 600 µl CTAB buffer (2% CTAB; 100 mmol / L Tris-HCl, pH 8.0; 20 mmol / L EDTA; 1.4 mol / L NaCl; 2% β-mercaptoethanol) in a 1.5 ml centrifuge tube, mix well, and place in a 65°C water bath for 20 min. (2) Centrifuge at 8 000 r / min at 4°C for 10 min, transfer the supernatant to a new centrifuge tube, and extract twice with chloroform / isoamyl alcohol (24:1). (3) The supernatant was transferred to a new centrifuge tube again, 1 / 4 volume of 10 mol / L LiCl was added, mixed well, and precipitated at -20°C for 2 h. (4) Centrifuge at 10 000 r / min at 4°C for 10 min, discard the supernatant, dissolve the precipitate with 300 µl DEPC water, add 50 µl 50% PEG8000 and 50 µl 5 mol / L NaCl, mix well, and...

Embodiment 3

[0031] Embodiment 3: antitumor efficacy test

[0032] 1. In Vitro Antitumor Experiment

[0033] 1.1 The impact on the value-added of HepG2

[0034] Take a bottle of cultured cells in the exponential growth phase for 3 to 4 days, add an appropriate amount of 0.25% Trypsin-EDTA solution to make the adherent cells fall off, and use RPMI 1640 medium with 10% embryonic bovine serum to make 1×10 4 cells / ml suspension. Take a 96-well plate, add 100 μL of cell suspension to each well, and place at 37°C, 5% CO 2 and 100% humidity incubator for 24 hours, each well was added with different concentrations of csi-miR1, csi-miR2, and csi-miR3 synthesized by chemical methods, and each concentration was set to 5 duplicate wells, and solvent control group and positive Control group (paclitaxel). Continue to culture in the incubator for 48 h. For MTT, use serum-free RPMI1640 medium to make a 1 mg / ml solution, add 50 μl to each well, incubate at 37°C for 4 hours, remove the supernatant, add...

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Abstract

The invention discloses three microRNAs (ribose nucleic acids) from a tea tree and application thereof, belongs to the technical field of drugs, and provides three microRNA sequences and microRNA precursor sequences from the tea three as well as application thereof in treatment of tumor, diabetes mellitus, fatty liver and respiratory diseases.

Description

technical field [0001] The invention belongs to the technical field of medicines, and specifically relates to three microRNAs derived from tea trees and applications thereof. Background technique [0002] microRNA (miRNA) is a "star molecule" that has been studied more in recent years. Noncoding RNAs consisting of 18–25 nucleotides are first transcribed from the nuclear genome to form primary miRNAs (pri-miRNAs) with capped structures and polyadenosine tails. Pri-miRNA is treated with nuclease Drosha to form about 70 nt precursor miRNA (pre-miRNA) containing the stem link, which is then cleaved into mature miRNA by nuclease. miRNA binds to the 3'UTR sequence of target gene mRNA through incomplete base pairing, guides the silencing complex (RNA-induced silencing complex, RISC) to degrade mRNA or inhibits the translation of mRNA, thereby regulating gene expression. One miRNA can bind multiple target genes, and one mRNA can also be co-regulated by multiple miRNAs. This compl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00C12N15/11A61P35/00A61P3/10A61P1/16A61P11/00A61P31/00
Inventor 余陈欢应华忠钟宇森金晓音吕宇方杰
Owner ZHEJIANG ACAD OF MEDICAL SCI
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