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W tym Artykule

  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.

Streszczenie

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3' untranslated region (UTR) of target mRNA to inhibit protein translation or in some instances cause mRNA degradation. The binding of the miRNA to the 3' UTR of the target mRNA is mediated by a 2–8 nucleotide seed sequence at the 5' end of miRNA. While the role of miRNAs as cellular regulatory molecules is well established, identification of the target mRNAs with functional relevance remains a challenge. Bioinformatic tools have been employed to predict sequences within the 3' UTR of mRNAs as potential targets for miRNA binding. These tools have also been utilized to determine the evolutionary conservation of such sequences among related species in an attempt to predict functional role. However, these computational methods often generate false positive results and are limited to predicting canonical interaction between miRNA and mRNA. Therefore, experimental procedures that measure direct binding of miRNA to its mRNA target are necessary to establish functional interaction. In this report, we describe a sensitive method for validating direct interaction between the cellular miRNA miR-125b and the 3' UTR of PARP-1 mRNA. We elaborate a protocol in which synthetic biotinylated-miRNA mimics were transfected into mammalian cells and the miRNA-mRNA complex in the cellular lysate was pulled down with streptavidin-coated magnetic beads. Finally, the target mRNA in the pulled-down nucleic acid complex was quantified using a qPCR-based strategy.

Wprowadzenie

MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate protein expression1. Precursors of miRNA reside in clusters through many regions of the genome, most frequently within the intergenic regions and introns of protein-coding genes2,3. Biogenesis of miRNAs involve transcription of the pri-miRNAs from miRNA-encoding genes4. The pri-miRNAs undergo sequential processing, first in the nucleus and then in the cytoplasm to generate single stranded mature miRNAs4,5. Subsequently, the mature miRNAs are incorporated into the RNA-induced silencing complex (RISC): a multimeric protein-RNA complex that includes a member of the Argonaute family of proteins for target recognition4,5,6,7. Mature miRNAs in the RISC complex predominantly bind to the 3' UTR of target mRNAs8,9,10 but can also bind occasionally in the coding and the 5'UTR region of the mRNA10,11. The binding of miRNA to the mRNA sequences results in translational silencing12,13,14 and in some cases mRNA destabilization15. Since a single miRNA can target many mRNAs, these regulatory molecules are involved in almost every cellular process and have been implicated in various disease conditions16,17,18.

Detailed understanding of how miRNAs regulate cellular pathways requires identification of the target mRNAs. Multiple bioinformatics platforms are available to predict putative miRNA:mRNA interactions19,20. These predictions rely on perfect Watson-Crick base pairing between the 2–8 nucleotide seed sequence of the miRNA and a complementary sequence within the target mRNA4,21. Additionally, these tools generate secondary structure of the miRNA:mRNA duplex, calculate thermodynamic parameters of this molecular interaction and show conservation of the binding sites across species to enhance functional relevance of the target prediction. Unfortunately, these tools also have the limitation of predicting false positive targets at a very high rate (~27–70%)15,22. Most importantly, these in silico platforms fail to recognize the non-canonical interactions of miRNAs with their targets23. Therefore, such predictive analyses are often combined with experimental methods to validate functionally relevant targets.

Multiple approaches have been developed to experimentally validate miRNA:mRNA interaction. Genetic experiments using miRNA mimics, sponges and inhibitors that alter the levels of miRNAs in the cell provide clues for its regulatory effect on the target gene expression24,25,26. Additionally, reporter based assays via co-transfection of a clone containing the 3' UTR region of the target mRNA and miRNA mimics or inhibitors into cells provide evidence of the regulatory function of miRNAs26. While these methods are crucial to study post-transcriptional regulation of gene expression by miRNAs, transfection efficiency and pleotropic effects of alterations in cellular miRNA levels are major limitations of these genetic approaches23,26. Therefore, complementary biochemical methods that probe direct interaction between miRNA and its target are employed to better understand the cellular function of miRNAs.

One widely-used method to study direct interaction between miRNA and its target is immunoprecipitation of the RISC complex followed by the detection of mRNA target within the complex27,28,29,30. Recently, an improved RISC trap method was also utilized to identify miRNA targets; it couples stabilization of targets within the RISC-miRNA-mRNA intermediates with the purification of mRNA targets. However, these methodsface the inherent challenges of non-specific interactions between RNA and RNA-binding proteins that are usually segregated by cellular compartments31,32. In addition, these assays are dependent on the presence of AGO2 protein for immunoprecipitation of RISC complex33. Given that AGO2 is not the only argonaute to mediate efficient miRNA:mRNA interactions, exclusion of other argonautes could lead to biased results34. Therefore, alternative strategies are needed to study direct binding of miRNA to mRNA.

In this report, we elaborate a one-step approach for probing direct interaction between a miRNA and its target mRNA. First, 3' biotinylated locked nucleic acid (LNA) miRNA mimics are transfected into mammalian cells. Then, the miRNA:mRNA complex in the cellular lysate is captured using streptavidin coated magnetic beads. The mRNA target bound to its complementary miRNA is quantified using qPCR.

Protokół

1. Transfection of 3'-biotinylated miRNA

NOTE: Perform the following steps inside a sterile laminar flow hood.

  1. Seed 4 x 105–5 x 105 HEK-293T cells per well in 2 mL of complete Dulbecco's Modified Eagle Medium (DMEM) [DMEM supplemented with 10% Fetal Bovine serum (FBS) and 1x Pen-strep antibiotic]. Culture the cells in an incubator set at 37 °C, 5% CO2 overnight.
  2. The next day, check the health and adherence of the plated cells under a light microscope.
    NOTE: Make sure the cells have adhered and attained appropriate morphology before proceeding to the next step. The HEK-293T cells are typically ready for transfection 18–24 h post plating.
  3. In a sterile 1.7 mL microfuge tube, dilute 75 picomoles of biotinylated miRNA in 200 µL of minimal essential media. Transfer this mix to another 1.7 mL microfuge tube containing Liposome based transfection reagent (8 µL/well) diluted in 200 µL of minimal essential media. Mix thoroughly, but gently, by pipetting and incubate for 20–25 min at room temperature to allow formation of the transfection complexes.
  4. Remove the old media from the 6 well culture plate by gentle pipetting and replenish each well with 1.6 mL of freshly prepared complete DMEM (without 1x Pen-strep antibiotics).
  5. Add the transfection complexes (from 1.3) drop-wise to the cells in the 6-well plate using a well-calibrated pipette. Swirl the plate gently while adding the complexes to ensure their uniform distribution across the plate.
    NOTE: This step is very critical for achieving high-efficiency transfection and must be performed with care and patience.
  6. Culture the cells at 37 °C and 5% CO2 for at least 36 h.

2. Preparation of Streptavidin Coated Magnetic Beads — I

  1. Resuspend the streptavidin magnetic beads thoroughly by vortexing. Transfer 30 µL of bead suspension (per sample) to a 2 mL nuclease-free microfuge tube.
  2. Place the tube containing the bead suspension on the magnetic bead separator stand ("magnet" hereafter) for 2 min. After ensuring that the beads are drawn to the side of the tube in contact with the magnet, carefully remove the supernatant using a micropipette.
  3. Add 100 µL of bead wash buffer (10 mM Tris-Cl pH 7.5, 0.5 mM EDTA, 1 M NaCl) to the beads. Remove the tube from the magnet. Vortex for 15 s at room temperature to wash the beads.
  4. Place the tube containing beads on the magnet for 2 min, and carefully remove and discard the supernatant.
  5. Repeat steps 2.3 and 2.4 for a total of three washes. After the final wash step, remove the tube from the magnet.
  6. Add 100 µL of RNase freeing solution (0.1 M NaOH, 0.05 M NaCl) to the beads, mix well by vortexing for 15 s, and incubate at room temperature for 5 min. Place the tube containing beads on the magnet for 2 min, and carefully remove and discard the supernatant.
  7. Repeat step 2.6 for a total of three times.
  8. Add bead resuspension solution (0.5 M NaCl) to the beads, vortex 15 s, and incubate at room temperature for 5 min. Place the resuspended beads on the magnet for 2 min, and carefully remove the supernatant.
  9. Add 200 µL of bead blocking solution (1 µg/µL BSA, 2 µg/µL Yeast tRNA) to the beads. Mix by gentle vortexing for 15 s at room temperature. Incubate at 4 °C for 16 h (over-night) on a multi-tube rotator.

3. Preparation of Cell Lysates

  1. Harvest the transfected HEK-293T cells by gentle scraping using a sterile cell scraper inside the laminar hood, then transfer each sample into a sterile 2 mL microfuge tube.
  2. Pellet the scraped cells by centrifugation at 1,500 x g for 5 min. Resuspend the pellet in 1x sterile PBS (Phosphate Buffer Saline), pH 7.2. Centrifuge again at 1,500 x g for 5 min to obtain a pellet free of residual media. Immediately plunge the tube containing pellet into ice.
  3. Prepare fresh complete cell lysis buffer (150 mM NaCl, 25 mM Tris-Cl, pH-7.5, 5mM DTT, 0.5% IGEPAL, 60 U/mL Superase, 1x Protease Inhibitor).
    NOTE: A stock of cell lysis buffer lacking IGEPAL, Superase, and Protease inhibitor cocktail may be prepared in advance and stored at room temperature. Prepare a fresh batch of complete cell lysis buffer by adding the above supplements at the recommended final concentrations to the stock cell lysis buffer and storing it on ice.
  4. Add 260 µL of ice-cold complete cell lysis buffer to each sample in the microfuge tube (i.e., each microfuge tube contains cells harvested from one well of the 6-well plate) and resuspend the cell pellet into a homogenous suspension by pipetting.
  5. Lyse the cells using the freeze-thaw method: Incubate the tubes at -80 °C for 10–15 min. Then, allow the cells to thaw out on ice.
  6. Centrifuge the resulting cell lysate at 16,000 x g for 5 min in a refrigerated benchtop centrifuge set at 4 °C.
  7. Transfer the cleared cell lysate to a sterile 1.7 mL microfuge tube on ice. Discard the pellet.
    NOTE: The final volume of cleared lysate should be ~240–250 µL.
  8. Add 5M NaCl to the cleared lysate to a final concentration of 1M and maintain the samples on ice.

4. Preparation of Streptavidin Magnetic Beads — II

  1. Prepare fresh complete pull-down wash buffer (10 mM KCl, 1.5 mM MgCl2, 10 mM Tris-Cl pH 7.5, 5 mM DTT, 1 M NaCl, 0.5% IGEPAL, 60U/mL Superase, and 1x Protease Inhibitor cocktail)
    NOTE: A stock of pull-down wash buffer lacking IGEPAL, Superase, and Protease inhibitor cocktail may be prepared in advance and stored at room temperature. Prepare a fresh batch of complete cell lysis buffer by adding the above supplements at the recommended final concentrations to the stock cell lysis buffer and storing it on ice.
  2. Place the tube containing the prepared beads (from step 2.9) on the magnet for 2 min. Carefully remove and discard the supernatant.
  3. Add 150 µL of ice-cold complete pull-down wash buffer to the beads. Vortex for 15 s and incubate at room temperature for 30–60 s. Place the tubes on the magnet for 2 min. Carefully remove and discard the supernatant.
  4. Repeat step 4.3 for a total of 3 times.
  5. Resuspend the beads in 300 µL of complete pull-down wash buffer.

5. Pull-down of Target mRNA-miRNA Complexes

  1. Transfer 300 µL of the cell lysate (from step 3.8) to the microfuge tube containing 300 µL of beads (from step 4.5).
  2. Incubate the mixture on a nutating mixer for 1 h at room temperature.
  3. Place the tube on the magnet for 5 min. Carefully remove and discard the supernatant.
  4. Add 300 µL of ice-cold complete pull-down wash buffer to the beads. Vortex for 15 s at room temperature and place the tube on the magnet for 5 min. Carefully remove and discard the supernatant.
  5. Repeat step 5.4 two more times.
  6. Resuspend the beads in 100 µL of nuclease-free water and incubate on ice.

6. Total RNA Extraction, cDNA Synthesis, and qPCR

  1. Extract total RNA from the resuspended beads (from step 5.6) using an appropriate method (see Table of Materials). Resuspend the extracted total RNA in 25 µL of nuclease-free water. Determine the RNA concentration and quality using a spectrophotometer (absorbance at 260/280). Prepare a working stock of RNA at 50 ng/µL.
  2. Perform cDNA synthesis, in triplicates, using 50 ng of total RNA (from step 6.1) using an appropriate cDNA synthesis kit (see Table of Materials) using oligo dT primers in a final volume of 20 µL (Table 1). Then, load PCR tubes into the qPCR instrument to perform thermal cycling as per the conditions described in Table 2.
  3. Perform qPCR on the CDNA (from step 6.2) using SYBR Green chemistry (see Table of Materials):
    1. Perform all qPCR reactions in triplicates in a 96 well clear bottom plate in sterile conditions.
    2. Aliquot 9 µL of the reaction mixture from the qPCR master mix (see Table 3) into each well of the plate. Then, add 1 µL of the cDNA to each well to achieve a final volume of 10 µL. Seal the PCR plate using heat resistant PCR plate sealer and load into the qPCR instrument
    3. Perform thermal cycling as per the conditions described in Table 4.
    4. Normalize the expression levels (Ct values) of each sample to the expression levels of the scrambled control. Use these values to calculate fold changes in expression.

Wyniki

MiRNAs regulate cellular processes by binding to target mRNAs. Therefore, identifying mRNA targets are a key to understand miRNA function. Here we elaborate a method for identifying mRNA target of cellular miRNA. This protocol is adapted from Wani et al.35 with the modification of utilizing biotinylated LNA-based miRNA mimics to pulldown target mRNA. The use of LNA-based oligonucleotide mimics enhances specificity of target binding owing to the higher stab...

Dyskusje

Identification of mRNA targets of cellular miRNAs is important for understanding their regulatory function. Several computational tools are employed to predict targets based on seed sequence complementarity and conservation of target sequences19,20. Although these tools are valuable, they can generate high levels of both false positives and false negatives. Therefore, these predictions are coupled with experimental methods such as immunoprecipitation of RISC comp...

Ujawnienia

The authors declare no competing financial and non-financial interests.

Podziękowania

This work was partly supported by National Institutes of Health (NIH) Grants DA037779 (to J.P.), DA024558, DA30896, DA033892, DA021471, AI22960 and MD007586 (to C.D.). The work was also supported by the RCMI Grant G12MD007586, the Vanderbilt CTSA Grant UL1RR024975, the Meharry Translational Research Center (MeTRC) CTSA grant (U54 RR026140 from NCRR/NIH, the U54 Grant MD007593 from NIMHD/NIH, and the Tennessee Center for AIDS Research (P30 AI110527).

Materiały

NameCompanyCatalog NumberComments
Cell line- HEK293T cellsATCCCRL-3216
Heat-inactivated Fetal bovine serum (Hi-FBS)GIBCO/Thermofisher10438-026
Dulbecco’s modified Eagle’s medium (DMEM)GIBCO/Thermofisher11995-065
Phosphate buffered saline (PBS) (1x)GIBCO/Thermofisher20012-027
Trypsin-EDTA (0.25%) GIBCO/Thermofisher25200-056
Penicillin-Streptomycin solution (100x)Cellgro/Mediatech30-002-CI
DNase AmbionAM2238
Opti-MEM Reduced serum mediaGIBCO/Thermofisher3198-088
Liopfectamine 2000 Transfection reagentInvitrogen/ Thermofisher11668-019
Biotinylated hsa-miR-125bExiqon339178/ID-27281622
Biotinylated scrambled controlExiqon479997-671/ID-714884
IGEPALSigma-Aldrich18896
Streptavidin Magnetic beadsPierce88816
Yeast tRNAInvitrogen/Thermofisher15401-011
Bovine Serum Albumin (BSA)Sigma-AldrichA2153
Potassium chloride (KCl) solutionSigma-Aldrich60142
Magnesium chloride (MgCl2) solutionSigma-AldrichM1028
Sodium hydroxide (NaOH)Sigma-Aldrich795429
Ethylenediamine tetraacetic acid, disodium salt (EDTA) solution, 0.5 M, pH 8.0Sigma-AldrichE7889
Dithiothreitol (DTT)Sigma-Aldrich43815
SuperaseAmbion/ThermofisherAM2694
Protease Inhibitor cocktailSigma-AldrichP8340
RNAse/DNAse free waterGIBCO/Thermofisher10977-015
RNeasy extraction kitQiagen74104
iScript-Select cDNA synthesis kitBiorad170-8897
qPCR Primers Invitrogen/Thermofisher
iTaq-Universal SYBR green supermixBiorad172-5120
DynaMag-Spin MagnetInvitrogen/Thermofisher12320D

Odniesienia

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