This study investigates the use of miRNA inhibitors to suppress tumor growth in a mouse model of cancer. By utilizing lipid nanoparticles for delivery, the research demonstrates a method for targeting oncogenic miRNAs effectively.
miRNA inhibitors are small, chemically modified single-stranded RNA molecules that bind to endogenous miRNA and repress its activity.
To model the effect of intratumoral treatment using miRNA inhibitors, begin by taking a suspension of bioluminescent enzyme-tagged cancer cells. Add a liquified basement membrane matrix to the cancer cell suspension.
Load the resulting mixture into a syringe. Inject it subcutaneously into the left flank of an immunodeficient nude mouse. Allow the cancer cells to grow and form a tumor.
Concomitantly, prepare a treatment mix by adding lipid nanoparticles - an in-vivo delivery agent - to the tube containing miRNA-inhibitor solution. Incubate the treatment mix to allow the encapsulation of miRNA-inhibitors into the lipid nanoparticles.
Inject the treatment mix directly into the tumor. Lipid nanoparticles deliver miRNA-inhibitors inside the tumor cell. Once inside, miRNA-inhibitors bind to specific oncogenic miRNAs, preventing them from hybridizing to their target mRNAs. This leads to the death of cancer cells.
Finally, inject a bioluminescent substrate solution subcutaneously into the mouse. In the presence of bioluminescence enzymes, the substrate in cancer cells oxidizes with subsequent light emission.
Visualize the mouse using a bioluminescence imaging chamber. Intratumorally injected miRNA-inhibitors significantly suppress tumor growth in the mouse.
After establishing a Cal62 human thyroid cancer cell line in complete medium, suspend a 1 times 10 to the sixth cell aliquot in 50 microliters of PBS at 4 degrees Celsius and mix the cells with an equal volume of basement membrane matrix.
Load the cells into a 1-milliliter syringe equipped with a 27G 1/2" needle and subcutaneously inject 100 microliters of the sample into the left flank of a 6-week-old immunodeficient BALB/c nude mouse. Two weeks after the injection, add the antagomiR or control treatment buffer solution to 160 microliters of room temperature in vivo delivery reagent in a 1.5-milliliter tube and immediately vortex the solution for 10 seconds to ensure complexation of the mixture.
Incubate the treatment solution for 30 minutes at 50 degrees Celsius, followed by a brief centrifugation in a microcentrifuge. Then, dilute the sedimented treatment complex six-fold with fresh PBS and thorough mixing, and inject the entire 200-microliter volume of the treatment directly into the tumor.
Inject 50 microliters of a 40 milligrams per milliliter D-luciferin substrate solution subcutaneously two times a week into each experimental animal, making sure to confirm a lack of response to toe pinch after isoflurane anesthesia. Place the animal in the chamber of an in vivo bioluminescence imaging system and image the bioluminescent signal with the in vivo imaging software according to standard protocols.
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