The Ago2-miRNA-co-IP Assay to Study TGF- β1 Mediated Recruitment of miRNA to the RISC in CFBE Cells

Summary

Here, we describe the Ago2-miRNA-co-IP assay designed to quantify an active pool of specific miRNAs induced by TGF-β1 in human bronchial epithelial CFBE41o- cells. This assay provides functional information on the recruitment of miRNA to the RNA induced silencing complex, quantified by qRT-PCR using specific miRNA primers and TaqMan hydrolysis probes.

Abstract

Micro(mi)RNAs are short, non-coding RNAs that mediate the RNA interference (RNAi) by post-transcriptional mechanisms. Specific miRNAs are recruited to the cytoplasmic RNA induced silencing complex (RISC). Argonaute2 (Ago2), an essential component of RISC, facilitates binding of miRNA to the target-site on mRNA, followed by cleaving the miRNA-mRNA duplex with its endonuclease activity. RNAi is mediated by a specific pool of miRNAs recruited to RISC, and thus is referred to as the functional pool. The cellular levels of many miRNAs are affected by the cytokine Transforming Growth Factor-β1 (TGF-β1). However, little is known about whether the TGF-β1 affects the functional pools of these miRNAs. The Ago2-miRNA-co-IP assay, discussed in this manuscript, is designed to examine effects of TGF-β1 on the recruitment of miRNAs to RISC and it helps to determine whether changes in the cellular miRNA levels correlate with changes in the RISC-associated, functional pools. The general principles of the assay are as follows. Cultured cells treated with TGF-β1 or vehicle control are lysed and the endogenous Ago2 is immunoprecipitated with immobilized anti-Ago2 antibody, and the active miRNAs complexed with Ago2 are isolated with a RISC immunoprecipitation (RIP) assay kit. The miRNAs are identified with quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) using miRNA-specific stem-looped primers during reverse transcription, followed by PCR using miRNA-specific forward and reverse primers, and TaqMan hydrolysis probes.

Introduction

Transforming Growth Factor-β1 (TGF-β1) is a multifunctional cytokine that can change the expression of many micro(mi)RNAs^1,2,3. The total cellular level of a particular miRNA does not correlate with its inhibitory potential because only a specific fraction of the miRNA is incorporated into RNA induced silencing complex (RISC) to perform RNA interference (RNAi)³. Only up to 10% of each miRNA is RISC-associated and participates in RNAi^4,5. Next, the RNAi process involves binding of the RISC-associated miRNA to the target mRNA recognition sequence(s)⁶. The RISC association is influenced by the availability of the target mRNA and the miRNA complementarity to the binding site, usually present at 3’ untranslated region (UTR) of the mRNA⁴. The Argonaute2-miRNA-co-immunoprecipitation (Ago2-miRNA-co-IP) assay, described in this manuscript, is designed to examine the effect of TGF-β1 on the recruitment of specific miRNAs to RISC by detecting differences in the RISC-associated miRNAs after TGF-β1 treatment, compared to the vehicle control. Examining the RISC-associated functional pool of a specific miRNA is much more informative about the miRNA effects than examining the total cellular level of the miRNA. RISC consists of proteins that scan the binding site on the target mRNA and cleave the miRNA-mRNA duplex. Argonaute2 (Ago2) is the main component of RISC. Out of the five Ago isoforms (Ago1-Ago5), Ago2 is the only one that has endonuclease activity and participates in RNAi in human cells^7,8,9,10. The Ago2-miRNA-RISC complex is the functional unit for miRNA-mediated post-transcriptional mRNA repression¹¹. The Ago2-associated miRNA represents the native state of miRNA in response to intracellular or extracellular signaling. Thus, immunoprecipitation of the endogenous Ago2 provides an excellent opportunity to detect the active, RISC-associated fraction of a specific miRNA as well as the functional assessment of its targets. This assay is superior to the pull-down of endogenous target mRNA with biotinylated miRNA mimics because of unpredictable efficiency of the cellular uptake of biotinylated nucleic acid molecules and their off-target effects.

The Ago2-miRNA-co-IP assay, discussed in this manuscript, was optimized to determine the effects of TGF-β1 on RISC recruitment of miRNAs in immortalized human bronchial epithelial CFBE41o- cells³. Components of the RIP assay kit were used to perform Ago2-miRNA-co-IP assay with modifications in the protocol provided by the manufacturer. A separation method was used to isolate small and large RNA, in which small RNA was used to quantify miRNA with the help of quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) using miRNA-specific stem-looped primers during reverse transcription, followed by PCR using miRNA-specific forward and reverse primers, and TaqMan hydrolysis probes.

Protocol

1. Preparation before experiment

1. Seeding cells
   1. Prepare 10% collagen I solution in Minimal Essential Medium (MEM) and add 500 µL to each 24 mm cell culture filter in a 6-well plate. Distribute to cover the entire surface of the filter by rotating gently by hand. Incubate filters under the UV light in the laminar flow hood at room temperature for 30 minutes (min), followed by incubation in cell culture incubator at 37 °C for 1 h.
   2. Prepare cell culture medium (MEM gassed with CO₂ for 20 min, 10% Fetal Bovine Serum (FBS), 50 U/mL penicillin, 50 U/mL streptomycin, 2 mM L-glutamine, 0.5 μg/mL puromycin).
   3. Bring the collagen-coated filters (step 1.1.1) from the incubator and suction off the excess collagen. Add 1.5 mL of the cell culture medium (step 1.1.2) to the basolateral side and 0.5 mL to the apical side (on to the filter) in the 6-well plate.
   4. Seed 1 x 10⁶ of CFBE41o- cells¹² suspended in 500 µL of the cell culture medium. Rotate the plate gently by hand to distribute the cells evenly on the filters and incubate in the cell culture incubator at 37 °C with 5% CO₂.
   5. Remove medium from the apical side one day after seeding cells and continue culturing cells in air-liquid-interface (no medium on the apical side). Change the basolateral medium daily and perform the experiment on the 8^th day.
2. Treatment of Cells with TGF-β1 or Vehicle Control
   1. Prepare FBS-free cell culture medium (FBS-free medium): Gas MEM with 5% CO₂ for 20 min, add 50 U/mL penicillin, 50 U/mL streptomycin, and 2 mM L-glutamine and filter sterilize the medium.
   2. Prepare vehicle control: Add 20 µL of 1 M HCl and 5 mg of BSA to 5 mL of double distilled water (4 mM HCl with 1 mg/mL BSA) to obtain 1 ng/µL stock solution. Filter sterilize the mixture before use.
   3. Prepare TGF-β1 solution: Reconstitute 1 µg of lyophilized TGF-β1 in 1 mL of sterile-filtered vehicle (4 mM HCl and 1 mg/mL BSA) to obtain 1 ng/µL stock solution. Aliquot in small volumes and store at -20 °C.
   4. Prepare the working concentration of TGF-β1 or vehicle control at 15 ng/mL of FBS-free medium (add 15 µL of the TGF-β1 or vehicle control stock solution per mL of FBS-free medium). Suction off the cell culture medium from the basolateral side and add the TGF-β1 or vehicle control containing medium 24 h before the experiment.
3. Preparation of buffers for cell lysis and immunoprecipitation
   1. Prepare mi-Lysis buffer (+) for cell lysis. Add 1 tablet of protease inhibitor mini and 15 µL of 1 M DTT per 10 mL of volume into the mi-Lysis buffer provided in the RIP-Assay Kit.
   2. Prepare mi-Wash buffer (+) for washing protein G agarose beads. Add 52.5 µL of 1 M DTT per 35 mL of volume into the mi-Wash buffer provided in the RIP-Assay Kit.
4. Conjugation of Anti-Ago2 antibody with protein G agarose beads
   1. Wash separately two 30 µL aliquots of protein G agarose bead slurry (50%) by repeating the following procedure three times: add 100 µL of ice-cold PBS, mix gently, centrifuge at 2,000 x g for 1 min, and suction off the supernatant with 200 µL pipette.
   2. Wash the beads once with 100 µL of ice-cold mi-Wash Buffer (+), suction off the supernatant with 200 µL pipette and add 500 µL of mi-Wash Buffer (+) to the beads and mix gently. Keep the tubes on ice.
   3. Add 7.5 µg of mouse monoclonal (IgG2) anti-human EIF2C2 (Ago2) antibody (see Table of Materials) or non-specific mouse IgG2 (negative control) to the protein G beads slurry in the mi-Wash Buffer (+).
   4. Incubate the tubes overnight, rotating at 8 rotations per minute (rpm) in a cold room.

2. Immunoprecipitation of Ago2

1. Lysis of cells
   1. Bring the plate containing cells cultured on 24 mm filters from the cell culture incubator and quickly transfer the filters to a plate on ice filled with ice-cold PBS. Allow PBS to overflow into the apical side of the filters to cover cells.
   2. One side at a time, suction off PBS and wash the apical and basolateral side with ice-cold PBS twice.
   3. Suction off all PBS, add 300 µL of ice-cold mi-Lysis buffer (+) to the cells, scrape the cells and transfer to 1.5 mL tube marked for respective treatment condition (TGF-β1 or vehicle control).
   4. Add 200 µL of mi-Lysis buffer (+) to each tube and vortex thoroughly.
   5. Incubate the tubes on ice for 15 min.
   6. Centrifuge the tube at 14,000 x g for 10 min at 4 °C, collect the supernatant as the cell lysate, and keep on ice.
2. Preclearing of cell lysate with unconjugated protein G agarose beads
   1. Wash 30 µL of fresh protein G agarose bead slurry (50%) in a 1.5 mL tube 3 times with 100 µL of PBS (step 1.4.1) and remove excess PBS.
   2. Wash beads with 500 µL of mi-Wash Buffer (+) once, and once with 500 µL of mi-Lysis Buffer (+). Remove the excess buffer.
   3. Add cell lysate from step 2.1.6 to the tubes containing unconjugated protein G beads.
   4. Rotate (8 rpm) the tubes for 1 h in the cold room to preclear the cell lysates.
   5. After preclearing, centrifuge the tubes at 2,000 x g for 1 min at 4 °C. Collect the precleared lysate as supernatant in fresh tubes and keep on ice.
   6. Prepare pre-immunoprecipitation whole cell lysate (WCL) for detection of Ago2 protein by western blotting. Take 10 µL of precleared lysates and add 10 µL of sample buffer (with 10% DTT), heat at 85 °C for 4 min, cool down on the bench, and store at -20 °C.
3. Immunoprecipitation of Ago2 complexes with anti-Ago2 antibody immobilized on protein G agarose beads
   1. Centrifuge the anti-Ago2 antibody conjugated to protein G beads prepared in step 1.4.4 at 2,000 x g for 1 min at 4 °C, remove the supernatant and discard. Wash beads once with 500 µL of mi-Lysis Buffer (+), centrifuge as before, and discard the supernatant again.
   2. Mix 500 µL of the precleared cell lysates from step 2.2.6 with anti-Ago2 antibody conjugated with protein G beads and incubate for 3 h rotating (8 rpm) in the cold room.
   3. Bring tubes from cold room on ice to the bench top and centrifuge at 2,000 x g for 1 min at 4 °C. Then remove and discard the supernatant.
   4. Wash the protein G agarose beads with immobilized anti-Ago2 antibody-Ago2 complexes containing the co-immunoprecipitated miRNAs with 1 mL of mi-Wash buffer (+), centrifuge at 2,000 x g for 1 min at 4 °C, and discard the supernatant. Repeat this process twice.
   5. Resuspend the beads with 1 mL of mi-Wash Buffer (+) and take 100 µL of the slurry to a new tube for the post-immunoprecipitation protein samples for western blotting.
   6. Centrifuge the tubes at 2,000 x g for 1 min at 4 °C and discard the supernatant.
   7. Add 20 µL of sample buffer (10% DTT), heat for 5 min at 85 °C, mix, and centrifuge at 2,000 x g for 1 min at room temperature. Store samples at -20 °C.
   8. Centrifuge the tubes with the remaining 900 µL of immobilized anti-Ago2 antibody-Ago2 complexes from step 2.3.5 at 2,000 x g for 1 min at 4 °C, suction off and discard the supernatant. Keep the samples on ice.

3. RNA Isolation

1. Label two 1.5 mL tubes per sample for isolation of small RNA and large RNA in upcoming steps and add 2 µL of mi-solution IV to each tube, to be used in step 3.5 and 3.8.
2. Prepare the master mix solution by adding 10 µL of mi-solution I and 240 µL of mi-solution II from the RIP-assay kit, per sample, to a 1.5 mL tube and keep the tube at room temperature.
3. Add 250 µL of the master mix to each tube containing immobilized anti-Ago2 antibody-Ago2 complexes (prepared in step 2.3.8), vortex, and centrifuge at 2,000 x g for 1 min at room temperature.
4. Add 150 µL of mi-solution III in the same tube and mix well. Then centrifuge at 2,000 x g for 2 min at room temperature. The supernatant at this step contains large RNA in addition to small RNA (i.e., total RNA).
5. Carefully transfer the supernatant to the tube containing 2 µL of mi-solution IV, prepared in step 3.1. Avoid contaminating the supernatant with beads to interfere with qRT-PCR.
6. Add 300 µL of ice-cold 2-popanol to each tube containing total RNA, vortex, spin down, and incubate at -20 °C for 2 h for optimal precipitation of large RNA.
7. After the incubation at -20 °C, centrifuge the samples at 12,000 x g for 10 min at 4 °C to separate the small and large RNAs, contained in the supernatant and pellet, respectively. Keep the pellets on ice until step 3.11.
8. Transfer the supernatant with small RNAs to the tube prepared in step 3.1 (containing 2 µL of mi-solution IV) and add 500 µL of ice-cold 2-propanol, vortex, and spin down.
9. Incubate the supernatants overnight at -20 °C for optimal precipitation of small RNAs.
10. The next day, take out the tubes containing small RNAs from -20 °C freezer and centrifuge at 12,000 x g for 10 min at 4 °C. Aspirate the supernatant, without disturbing the pellet, and wash the pellet containing most of the small RNA following step 3.11.
11. Rinse the pellet containing large RNA (step 3.7) and small RNA (step 3.10) with 500 µL of ice-cold 70% ethanol, each. Slowly mix the pellet with ice-cold 70% ethanol and then centrifuge at 12,000 x g for 3 min at 4 °C. Carefully aspirate the supernatant, without disturbing the pellet. Repeat the process of washing once again.
12. Aspirate remaining ethanol with pipette of smaller volumes (10 µL or 20 µL) and allow to air dry for 30 min at room temperature in RNase free environment.
13. Reconstitute the small RNA and large RNA pellets by adding 50 µL of nuclease free water and heating at 65 °C for 5 min. Then store the RNA at -80 °C till further use after checking the quality and concentration of RNA by nanodrop.

4. Quantification of miRNA by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR)

1. cDNA Preparation through Reverse Transcription of miRNA with miRNA Specific Stem-looped Primers
   NOTE: RNA detection by standard qRT-PCR requires a template of at least two times the length of the forward or reverse primer, each approximately 20 nucleotides long. Thus, the minimum length is at least 40 nucleotides, making small RNAs too short for detection. The protocol describes miRNA-specific RT-primers (Table of Materials) containing highly stable stem-loop structure that lengthens the target cDNA. The forward PCR primer adds additional length with nucleotides that optimizes its melting temperature and enhances assay specificity. The reverse primer disrupts the stem loop¹³. The following protocol describes RT of RNA to cDNA for miR-145-5p, miR-143-5p, and miR-154-5p using the miRNA reverse transcription Kit (Table of Materials).
   1. Prepare a master mix for each miRNA by adding 100 mM dNTPs (0.3 µL), Reverse Transcriptase (2 µL), 10x RT buffer (3 µL), RNase Inhibitor (0.38 µL), miRNA-specific looped primer (6 µL), and nuclease free water (8.32 µL). The combined volume of master mix is 20 µL.
   2. Add 10 µL of small RNA from step 3.13 (50-100 ng) to three separate 200 µL tubes for each cDNA of miR-145-5p, miR-143-5p, and miR-154-5p.
   3. Add the master mix to small RNA, mix gently, and spin to the bottom of tubes. Keep the tubes on ice until loading into the thermal cycler.
   4. Set the program in thermal cycler for single cycle as hold at 16 °C for 30 min, hold at 42 °C for 30 min, hold at 85 °C for 5 min, and hold at 4 °C. Set the reaction volume to 30.0 µL. See step 4.1.6.1.
   5. Load the reaction tubes into the thermal cycler. Start the RT run. See step 4.1.6.2.
   6. Switch ON the thermal cycler and place the tubes into the tray. It will show that “System is booting, please wait”. Click on Browse/New Methods. Save the method and use in another experiment by browsing through the menu. Either select from the list of methods saved before and start run or create “new” method. The “edit run” method or “new” method screen will appear.
      1. Edit temperature and cycles as stated in step 4.1.4. Add or remove stages and steps by clicking on particular stage or step and then clicking on add or delete button. Click on Save or directly run the program. In Save Run Method, name the “run” method, set the reaction volume (30 µL), set cover temperature to 105 °C, save, and exit.
      2. When “browse run method” screen appears, select the saved method from the “run method” list. Click Start Run and again confirm the reaction volume and cover temperature. Click on Start Run Now. The screen will show sample temperature, cover temperature, and time remaining. Record the start and stop time after completion and remove the tubes from the thermal cycler.
         NOTE: The tubes containing cDNA can be stored in -20 °C till their further use for qPCR of miRNA.
2. qPCR with the miRNA-specific forward/reverse primers and hydrolysis probes
   NOTE: The following protocol is for qPCR of miR-145-5p, miR-143-5p, and miR-154-5p using miRNA-specific cDNAs prepared in step 4.1. The single tube miRNA Assay (20x) contains miRNA specific forward/reverse primers and hydrolysis probe (Table of Materials). The forward primer adds nucleotide to increase length and optimize melting temperature, while the reverse primer disrupts the stem loop of cDNA¹³. The Universal PCR Master Mix (Table of Materials) provides additional components required for qPCR. The reaction (20 μL) is done in triplicate for each miRNA.
   1. Prepare separate master mix for miR-145-5p, miR-143-5p, and miR-154-5p in triplicates using respective miRNA assay (20x) (1 μL), 2x Universal PCR Master Mix (No AmpErase UNG; 10 μL), and nuclease free water (7.5 μL). The total volume for one reaction is 18.5 μL.
   2. Add miRNA-specific cDNA (1.5 μL) prepared in 4.1 to to each well of the PCR plate in triplicates.
   3. Add the miRNA-specific master mix (18.5 μL) prepared in step 4.2.1 to the wells of PCR plate containing respective cDNA (1.5 μL).
   4. Seal plate with appropriate sealer and run qPCR for 45 cycles on a qPCR thermocycler (Table of Materials). Set cycle temperature as hold at 95 °C for 10 min, followed by 45 repeats of 95 °C for 15 seconds (s) and 60 °C for 60 s. See step 4.2.6.
   5. Set the qPCR curve baselines manually per plate and standardized thresholds for all qPCR runs at a point at which amplification becomes logarithmic for all samples.
   6. Switch ON the thermal cycler and place the PCR plate into the tray. The thermal cycler machine operates with the software installed on the accompanying computer. Click on the icon of the system software.
      1. Click on Create New Document or Open Existing Document (if there is a previously saved document). New document wizard will appear. The assay will be “standard curve (Absolute quantification)”. Click on Next.
      2. Add a new detector. Name the detector as miR-143, miR-145, and miR-154 one by one by clicking Create Another. Select reporter dye as “FAM” for all, and click Okay. Then find the name as miR-143, miR-145, and miR-154 and add them into detector’s document by clicking Add. Then click Next.
      3. “Set up sample plate” will appear. Select the wells for a detector and then click on Use to select the corresponding detector. Then click on Finish. The system initializing will appear.
      4. Go to the Instrument and set stage, temperature and time as mentioned in step 4.2.4. Set the sample volume to 20 μL. Then start the run by clicking Start. Then save the plate with a name in a desired location. The estimated time will show on the screen near the start button. Note down the time.
      5. After completion, go to File | Export | Results, and save results as CT values in desired location. The CT values will be in .csv files. Open it and copy the values into a spreadsheet file.
   7. Subtract average of triplicate threshold cycle (Ct) of the vehicle control-treated samples from average Ct of TGF-β1-treated samples to calculate ΔCt. Finally, calculate fold change (FC) of miRNA levels using formula 2^-ΔCt. The log2 transformation of the FC value can be used for graphical representation.

Results

We have previously shown that TGF-β1 increased the total cellular levels of miR-145-5p, miR-143-5p, and miR-154-5p miRNAs in CFBE41o- cells³. Next, we employed the Ago2-miRNA-co-IP assay to elucidate the functional effects of TGF-β1 on these miRNAs. The RISC recruitment of miR-145-5p, miR-143-5p, and miR-154-5p was studied in CFBE41o- cells stably expressing the wild type (WT)-cystic fibrosis transmembrane conductance regulator (CFTR) or mutant CFTR with the deletion of phenylalanin...

Discussion

The Ago2-miRNA-co-IP assay is designed to investigate the active pool of miRNAs in response to TGF-β1 treatment. The active or RISC-associated miRNAs are important to understand their inhibitory potential for the target mRNA⁴. Panshin et al. recently showed that the immunoprecipitation efficiency of Ago2 and miRNAs may depend on the protocol¹⁶. There are several differences between the protocol here and the above published data. The protocol here was optimized for CFBE...

Materials

Name	Company	Catalog Number	Comments
100 mM dNTPs (with dTTPs)	Applied Biosystems	4366596
10x Reverse Transcription Buffer (RT Buffer)	Applied Biosystems	4366596
2-propanol	Fisher BioReagents	BP2618-1
2x Laemmli Sample Buffer	Bio-Rad	1610737
7300 Real Time PCR System	Applied Biosystems	4345240
Anti-Ago2 antibody (anti-EIF2C2), mouse monoclonal against human Ago2	Medical & Biological Laboratories Co. Ltd	RN003M
Bovine Albumin Fraction V (7.5% solution)	Thermo Scientific	15260037
Collagen I (Purecol-Type I Bovie collagen solution)	Advanced Biometrix	50005-100mL
DL-Dithiothreitol (DTT)	Sigma	646563-.5ML
DTT	Sigma-Aldrich	646563
Ethanol	Deacon Laboratories	64175
Fetal Bovine Serum	ATLANTA Biologicals	S10350
Goat Anti-Mouse IgG	Bio-Rad	1706516
L-glutamine (200 mM Solution; 29.20 mg/mL)	Corning	25-005-Cl
Mini cell scrapers United Biosystems	Thermo Fisher	MCS-200
Minimal Essential Medium	Thermo Fisher Scientific	11095-080
miRNA specific stem looped RT primers	Applied Biosystems	4427975
Mouse IgG2 control	Dako, Glostrup, Denmark	A0424
MultiScribe Reverse Transcriptase, 50 U/µL	Applied Biosystems	4366596
Nano Drop ND-1000 Spectrophotometer	NanoDrop Technologies, Inc.	E112352
Nuclease-free water	Ambion	AM9937
Opti-MEM (1x) Reduced Serum Medium	Gibco by Life Technologies	11058-021
PBS	Gibco	14190250
Penicillin-streptomycin, Sterile	Sigma-Aldrich	P0781
Pierce Protease Inhibitor Tablets, EDTA-Free	Thermo Scientific	A32955
Protein G agarose beads (Pierce Protein G Plus Agarose)	Thermo Scientific	22851
Puromycin	InvivoGen	ant-pr-1
RiboCluster Profiler RIP-Assay Kit for microRNA	Medical & Biological Laboratories Co. Ltd	RN1005
RNase Inhibitor, 20 U/µL	Applied Biosystems	4366596
TaqMan 2x Universal PCR master mix without AmpErase UNG	Applied Biosystems	4427975
TaqMan miRNA single tube Assay (20x) containing miRNA specific forward/reverse primers and probe	Applied Biosystems	4427975 (assay ID #002278, #002146, and #000477)
TGF-beta1	Sigma	T1654
Transwell filters (24 mm)	Corning Life Sciences Plastic	3412
Veriti 96 Well Thermal Cycler (Model:9902)	Applied Biosystems	4375786