Modelling Intratumoral Treatment with miRNA Inhibitors: A Method to Analyze the Effect of miRNA Inhibitors on Tumor Growth in Murine Model

Abstract

Source: Ramirez-Moya, J. et al. In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice. J. Vis. Exp. (2019)

This video demonstrates the in vivo delivery of miRNA inhibitors as therapeutic agents against tumor growth in orthotopic mouse models. The intratumoral administration of synthetic miRNA inhibitors or antagomiRs allows them to bind and inhibit endogenous miRNAs, thereby causing mRNA upregulation and suppression of tumor growth.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Flank Inoculation of Cells and Intratumoral antagomiR Treatment

1. Cell preparation
   1. Engineer a Cal62 human thyroid cancer cell line (KRASG12R and p53A161D mutations) to overexpress a transgenic construct that constitutively expresses GFP and luciferase (CMV-Firefly Luc-IRES-eGFP). Select the transgenic cells by antibiotic resistance or by sorting the GFP-positive cells with flow cytometry.
   2. Grow the cells in Dulbecco's modified Eagle's medium (DMEM....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Basement Membrane Matrix: Matrigel Basement Membrane Matrix High Concentration	Corning	#354248
AntagomiR: mirVana miRNA inhibitor	Thermo Fisher	4464088	In vivo ready
In vivo delivery reagent: Invivofectamine 3.0 Reagent	Thermo Fisher	IVF 3005
Negative control: mirVana miRNA Inhibitor, Negative Control #1	Thermo Fisher	4464077	In vivo ready
XenoLight D-Luciferin - K+ Salt Bioluminescent Substrate	PerkinElmer	122799	Diluted in PBS
In vivo imaging software: IVISLumina II Imaging System	Caliper Life Sciences