Use of RJH Transfection Reagents to Deliver microRNAs into Leukemic Cells


Background

The convergence of genetic bioinformatics and nucleic acid chemistry along with sophisticated transfection systems have significantly expanded scientific and therapeutic applications of nucleic acids. The newly emerging non-coding RNAs, so called microRNAs (miRNAs), are gaining tremendous attention as regulators of a complex set of mediators affecting cell behavior. Unlike very specific siRNAs and shRNAs that regulate individual mRNAs (and hence their protein product), miRNAs appear to be regulating networks of signaling molecules that ultimately control a complex set of cellular behaviors (Nat. Rev. Drug Discov., 2017, 16: 203-222). Deregulation of miRNAs has been associated with numerous human diseases and significant attempts are now being made to deploy them in clinical settings as diagnostic and therapeutic agents. Being unable to penetrate the cell membrane on their own, a variety of transfection reagents have been used to deliver miRNAs to human cells. It has been possible to deploy the miRNAs on their own to correct the abnormal physiology or combine them with other nucleic acids (e.g., siRNA) to induce complementary effects and enhance any beneficial therapeutic effect.

Procedure

Human chronic myeloid leukemia K562 cells (drug-resistant K562-IMR phenotype) are passaged in 75 cm2 flasks and used at the exponential phase for transfection experiments described below.

  • • Complexes of nucleic acids and transfection reagent were prepared in RPMI without serum. The ratio of the total nucleic acid to transfection reagent was 1:10 (w/w). The volume of the complexes was typically 300 uL, where 100 uL was used for addition to each well of the 48-well plate (in triplicate).
  • • LeuFect A was used as the transfection reagent. To a LeuFect A solution in RPMI, a proprietary miRNA dissolved in nuclease-free water was added to form binary complexes of transfection reagent/miRNA. The complexes were incubated for 30 min at room temperature and then added to 48-well plates (100 uL/well in triplicate). The final concentrations of the nucleic acids were adjusted to be 40 or 80 nM.
  • • In the same way, complexes between the LeuFect A and a specific siRNA (targeting BCR-ABL fusion oncogene) was prepared in RPMI. Furthermore, the miRNA and BCR-ABL siRNA were mixed and used to prepare tertiary complexes with LeuFect A/miRNA/siRNA. The complexes were incubated for 30 min at room temperature and then added to the 48-well plates (100 uL/well in triplicate).
  • • After the complex addition, desired numbers of cells (30,000 cells/well in our case) were added to the complexes and the plate was incubated at 37oC/humidified atmosphere of 95/5 CO2/O2 for 3 days.
  • • The total cell activity was assessed by the MTT assay and used as a measure of cellular growth during the treatment period. The growth inhibition was calculated by normalizing the treated cells with non-treated (control) cells. As a treatment control, a scrambled (non-specific) double stranded RNA was used to make complexes and treat the cells in the same manner.
  • • Colony Forming Units (CFU) were determined by seeding 200 cells/well (24-well plates) in methylcellulose assays (in triplicate). After 2 weeks of growth, the number of CFU was counted under a microscope.

In this application note, we employ a transfection reagent developed by the RJH Biosciences to deliver a miRNA in leukemic cells. miRNAs are known to display altered activities in leukemic cells (Blood, 2017, 130: 1290-1301), acting as oncogenic stimuli on their own (so called oncomiRs) as well as supporting unchecked proliferation of cells in concert with other oncogenes. Here we describe the use of LeuFect A to undertake miRNA delivery and a specific siRNA against the BCR and ABL fusion oncogene. We describe a single transfection reagent that can deliver miRNA, siRNA and their combination, simplifying the delivery process.

Results and Discussion

The growth inhibitions by control RNA (C-RNA), miRNA, BCR-ABL specific siRNA and miRNA/siRNA complexes with LeuFect A are shown in Figure 1. There was no effect of C-RNA on cell growth, since relative cell viabilities were equivalent to non-treated (NT) cells. With BCR-ABL siRNA, significant retardation was seen at 80 nM dose, miRNA also gave an inhibition of cell growth at 80 nM. The combination treatment of Bcr-Abl siRNA and miRNA at 40 nM + 40 nM displayed synergistic activity in inhibiting cell growth. The summary of CFUs from the treated K562-IMR cells is shown in Figure 2. Note that control RNA treatment with LeuFect A did not affect CFU counts, but BCR-ABL siRNA reduced the CFU counts as expected. The miRNA treatment was equally effective in reducing cell growth, as well as the combination of siRNA and miRNA.
Picture1

Figure 1. Relative growth of K562-IMR cells

Relative growth of K562-IMR cells treated with the following nucleic acids: a control double-stranded RNA (C-RNA), a specific siRNA against BCR-ABL, and a proprietary miRNA (40 or 80 nM). Cells were treated with LeuFect A complexes of the indicated nucleic acids for 3 days and then the cell activity was determined by the MTT Assay. All cell activities were normalized with the cell activity from non-treated cells (taken as 100%).
Picture5

Figure 2. Colony Forming Unit (CFU) assay for K562-IMR cells

Colony Forming Unit (CFU) assay for K562-IMR cells treated with the following nucleic acids: a control double-stranded RNA (C-RNA), a specific siRNA against BCR-ABL oncogene, and a proprietary miRNA (40 or 80 nM, or 40+40 nM in case of dual nucleic acids). C-RNA was used with siRNA and miRNA where indicated. Cells were transfected with LeuFect A complexes of the indicated nucleic acids for 24 hours and then transferred to methyl-cellulose gels for further cultivation for 2 weeks. The number of CFUs were counted at the end of 2-week culture period (in triplicate).

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