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Compositions of mirna-17-3p and mirna-19b-1 and applications thereof

A technology of mir-19b-1 and rnamir-19b-1, applied in the fields of biotechnology and medicine, can solve the problems of incomplete understanding of biological functions and mechanisms of action, and in-depth research, so as to reduce the occurrence of drug resistance and the process of Simple and effective in reducing adverse reactions

Active Publication Date: 2018-12-18
NANTONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Both miR-17-3p and miR-19b-1 are miRNA small molecules known in the art, and are a member of the tumor-associated gene cluster miR-17-92, however, the prior art for miR-17-3p and The research on miR-19b-1 has not been in-depth, and its biological function and mechanism of action are not completely clear
Moreover, there are no related literature reports on the regulation of miR-17-3p and / or miR-19b-1 in tumor blood vessels and anti-tumor activity in vivo at home and abroad.

Method used

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  • Compositions of mirna-17-3p and mirna-19b-1 and applications thereof
  • Compositions of mirna-17-3p and mirna-19b-1 and applications thereof
  • Compositions of mirna-17-3p and mirna-19b-1 and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1. Analysis of microRNA expression in clinical samples of breast cancer

[0046] There are many expression changes of miRNAs in breast cancer clinical samples. This experiment uses TRIzol (provided by Invitrogen) to extract RNA from tissue samples obtained clinically (12 cases of breast cancer surgical resection samples from Nantong University Hospital from 2015 to February 2016), including tumor tissue and normal breast tissue. The steps follow the instructions of the reagents). And use quantitative PCR to compare the expression changes of miR-17-3p and miR-19b-1 in tumor tissue and normal breast tissue.

[0047] Real-time fluorescent quantitative PCR: RNA is extracted by conventional methods, and real-time fluorescent quantitative PCR detection is performed by microRNA chip (provided by QIAGEN) (the specific steps follow the chip instructions), and the double standard curve method is used to quantify, analyze the microRNA concentration of each sample, and pass the...

Embodiment 2

[0050] Example 2 Overexpression of miR-17-3p or / and miR-19b-1 inhibits breast cancer cells from inducing endothelial cell function

[0051] Method of overexpression of miR-17-3p or / and miR-19b-1: Use Lipofectamine 2000 Reagent to combine miR-17-3p, miR-19b-1, and miR-17-3p and miR-19b-1 according to the instructions The mimics (miR-17-3p, miR-19b-1mimics) were transfected into cultured breast cancer cells.

[0052] Tumor microenvironment preparation: digest the breast cancer cell MDA-MB-231 that up-regulated the expression of miR-17-3p, miR-19b-1, and the combination of miR-17-3p and miR-19b-1, and resuspend in complete medium Cells were added to the lower chamber of the transwell, 600μL was added to each well, and incubated overnight. After the cells adhere to the wall, change to fresh medium and culture for 48 hours.

[0053] Matrigel preparation: Place the matrigel frozen at -80°C in the refrigerator at 4°C overnight to become liquid; take 150μLMatrigel, coat the Transwell upper...

Embodiment 3

[0055] Example 3 Overexpression of miR-17-3p or / and miR-19b-1 inhibits angiogenesis in vivo

[0056] By establishing an in vivo angiogenesis model, the effect of overexpression of miR-17-3p or / and miR-19b-1 on endothelial cells' angiogenesis ability was studied.

[0057] The overexpression method of miR-17-3p, miR-19b-1, and the combination of miR-17-3p and miR-19b-1 is the same as in Example 2.

[0058] Establishment of an in vivo angiogenesis model: put matrigel stored in a refrigerator at -80℃ overnight at 4℃ to become liquid; overexpress miR-17-3p, miR-19b-1, miR-17-3p and miR-19b -1 HUVEC cells of the composition were digested and collected, and resuspended in PBS to 1×10 7 Cell suspension: Mix the cell suspension with 500 μL of matrigel containing heparin, and inoculate Balb / c nude mice subcutaneously. After 7 days, the cell tissue block was taken out and analyzed.

[0059] Analysis of the number of microvessels: the cell tissue block was wax-embedded, sectioned, and stained wi...

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Abstract

The invention belongs to the field of biotechnology and medical science and particularly relates to a miRNA-17-3p and miRNA-19b-1 combination and application thereof. The invention provides application of miRNA-17-3P, or a combination of its precursor and mimR-19b-1, or a combination of its precursor in the preparation of drugs for preventing or treating tumors. The invention discloses for the first time that miR-17-3p and miR-19b-1 can specifically inhibit the generation of tumor vessels and have significantly lower expression than normal tissues in breast cancer; in addition, the miR-17-3p and miR-19b-1 combination can inhibit the growth of cancer cells both in vitro and in vivo. The miR-17-3p and miR-19b-1 combination is useful in the preparation of drugs for preventing or treating solid tumors, particularly in the preparation of drugs for preventing or treating breast cancer.

Description

Technical field [0001] The present invention belongs to the fields of biotechnology and medicine, and specifically relates to a series of small RNA molecules-a combination of miR-17-3p and miR-19b-1 and its application in the preparation of drugs for preventing or treating tumors, miR-17- 3p and miR-19b-1 play an important role in tumor vascular regulation and anti-tumor activity in vivo. Background technique [0002] MicroRNA (microRNA, miRNA for short) is a type of non-coding single-stranded small RNA with a post-transcriptional regulatory function and a sequence length of about 18-24 nt. As early as 1993, Lee et al. reported an RNA molecule found in C.elegants that can regulate nematode development, namely lin-4 (Lee, RC, etc., The C.elegansheterochronic gene lin-4encodes small RNAs with antisense complementarity tolin-14. Cell. 1993, 75:843-854). By 2000, Reinhart et al. (Reinhart, B.J. et al., The21-nucleotide let-7RNA regulates developmental timing in Caenorhabditiselegan...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K31/7105A61K31/7115A61K31/7125A61K45/06A61K48/00A61P35/00
CPCA61K31/7105A61K31/7115A61K31/7125A61K45/06A61K48/0066A61K2300/00
Inventor 殷润婷张志强陈向凡罗琳
Owner NANTONG UNIVERSITY
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