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MiRNA-17-3p and miRNA-19b-1 combination and application thereof

A technology of mir-19b-1 and a composition, which is applied in the fields of biotechnology and medicine, can solve the problems of incompletely clear biological functions and mechanisms of action, and in-depth research, so as to reduce the occurrence of drug resistance, the process is simple, and the Effects of adverse reactions

Active Publication Date: 2016-07-06
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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  • MiRNA-17-3p and miRNA-19b-1 combination and application thereof
  • MiRNA-17-3p and miRNA-19b-1 combination and application thereof
  • MiRNA-17-3p and miRNA-19b-1 combination and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1. MicroRNA expression analysis in clinical samples of breast cancer

[0046] There are many miRNAs expression changes in clinical samples of breast cancer. In this experiment, TRIzol (provided by Invitrogen) was used to extract RNA from clinically obtained tissue samples (12 cases of breast cancer surgical resection samples from the Affiliated Hospital of Nantong University from 2015 to February 2016), including tumor tissues and normal breast tissues (specifically Follow the instructions of the reagents). And quantitative PCR was used 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 was extracted by conventional methods, real-time fluorescent quantitative PCR detection was performed through a microRNA chip (provided by QIAGEN) (the specific steps followed the chip instructions), quantified by double standard curve method, analyzed the microRNA concentrat...

Embodiment 2

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

[0051] The method of miR-17-3p or / and miR-19b-1 overexpression: use Lipofectamine2000Reagent to mix miR-17-3p, miR-19b-1, and the composition of miR-17-3p and miR-19b-1 according to the instructions Mimics (miR-17-3p, miR-19b-1mimics) were transfected into cultured breast cancer cells.

[0052] Tumor microenvironment preparation: Digest up-regulated breast cancer cell MDA-MB-231 expressing 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 transwell chamber, 600 μL per well, and incubated overnight. After the cells adhered to the wall, replace with fresh medium and culture for 48 h.

[0053] Matrigel preparation: put the matrigel frozen at -80°C refrigerator at 4°C overnight to become liquid; take 150 μL of Matrigel, coat the upper chamber of Transwell (operated on i...

Embodiment 3

[0055] Example 3 Inhibition of overexpression of miR-17-3p or / and miR-19b-1 on angiogenesis in vivo

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

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

[0058] Establishment of angiogenesis model in vivo: place matrigel frozen at -80°C at 4°C overnight to become liquid; overexpress miR-17-3p, miR-19b-1, and miR-17-3p and miR-19b -1 Composition of HUVEC cells was digested and collected, and resuspended with PBS to 1 x 10 7 Cell suspension; mix the cell suspension with 500 μL of heparin-containing matrigel, and inoculate Balb / c nude mice subcutaneously. After 7 days, cell tissue pieces were removed for analysis.

[0059] Analysis of the number of microvessels: the cell tissue blocks were wax-embedded, sectioned, st...

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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—compositions of miR-17-3p and miR-19b-1 and their application in the preparation of drugs for preventing or treating tumors, miR-17- 3p and miR-19b-1 play important roles in tumor vasculature regulation as well as antitumor activity in vivo. Background technique [0002] MicroRNA (microRNA, referred to as miRNA) is a kind of non-coding single-stranded small molecule RNA with a post-transcriptional regulation function and a sequence length of about 18-24nt. As early as 1993, Lee et al. reported a RNA molecule that could regulate the development of nematodes found in C. elegans (C. elegants), namely lin-4 (Lee, R.C. et al., The C. elegans sheterochronic gene lin-4 encodes small RNA with antisense complementarity tolin-14. Cell.1993 , 75:843-854). By 2000, Reinhart et al. (Reinhart, B.J. et al., The21-nucleotidelet-7RNAr...

Claims

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Application Information

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Patent Type & Authority Applications(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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