Insulin-like growth factor-1 prevents miR-122 production in neighbouring cells to curtail its intercellular transfer to ensure proliferation of human hepatoma cells

miRNAs are 20–22 nt long post-transcriptional regulators in metazoan cells that repress protein expression from their target mRNAs. These tiny regulatory RNAs follow tissue and cell-type specific expression pattern, aberrations of which are associated with various diseases. miR-122 is a liver-specific anti-proliferative miRNA that, we found, can be transferred via exosomes between human hepatoma cells, Huh7 and HepG2, grown in co-culture. Exosomal miR-122, expressed and released by Huh7 cells and taken by miR-122 deficient HepG2 cells, was found to be effective in repression of target mRNAs and to reduce growth and proliferation of recipient HepG2 cells. Interestingly, in a reciprocal process, HepG2 secretes Insulin-like Growth Factor 1 (IGF1) that decreases miR-122 expression in Huh7 cells. Our observations suggest existence of a reciprocal interaction between two different hepatic cells with distinct miR-122 expression profiles. This interaction is mediated via intercellular exosome-mediated miR-122 transfer and countered by a reciprocal IGF1-dependent anti-miR-122 signal. According to our data, human hepatoma cells use IGF1 to prevent intercellular exosomal transfer of miR-122 to ensure its own proliferation by preventing expression of growth retarding miR-122 in neighbouring cells.

SiControl siRNA sequence not targeting any nuclear encoded genes -------  fractions. Out of 10ml of total Huh7 CM, RNA was extracted from 500 µl of post exosomal fraction. RNA was also extracted from the pellet which precipitated at 10,000g. These were then analyzed for miR-122 level by qPCR. We plotted the C t values for each fraction and observed comparable levels of miR-122 in the equivalent amount of post exosomal and exosomal fractions. For this experiment n=3 and qPCR was carried out in triplicate. (B) Activity assays were then performed by adding cell equivalent amounts of Huh7 exosomes and post exosomal supernatant to reporter transfected HepG2 cells. Also the 10,000g pellet obtained during exosome isolation was also dissolved in medium and added to the reporter transfected HepG2 cells. We observed that miR-122 mediated repression increases in HepG2 cells only when Huh7 exosomes are added. n=3 and P values were calculated by unpaired t test   As a control the cells were also cultured separately for the same time durations and then mixed together. Cells harvested at each time point were fixed and analysed by FACS to detect the percentage of DsRed and GFP cells.
The relative growth rate was then plotted by dividing the percentage of GFP / Ds Red positive cells in the cocultured samples with that of the control samples. We found that the increased growth rate of co-cultured DsRed Huh7 cells observed in Figure

Immunofluorescence
For immunofluorescence, cells were grown on gelatin coated coverslips and were fixed with 4% paraformaldehyde for 30 mins, washed with 1 X PBS twice to remove the paraformaldehyde.
Cells were then blocked and permeabilized using 1XPBS containing 10% goat serum, 1% Bovine Serum Albumin (BSA), and 0.1% Tritin-X-100 for 30 mins at room temperature. Primary antibody incubation was done in 1XPBS with 1% BSA at 4⁰C overnight in a humid chamber.
The anti-mitosin antibody was used at a dilution of 1:100. Secondary antibody incubation was done in 1XPBS with 1% BSA for 1h at room temperature. Secondary anti-rabbit antibodies labeled either with Alexa Fluor® 488 dye (green) or Alexa Fluor® 594 dye (red) fluorochromes (Molecular Probes) were used at 1:500 dilutions. Coverslips were then mounted with Vectashield containing DAPI and observed under a fluorescence microscope. Images were captured with a Nikon Eclipse Ti microscope. All post capture analysis and processing were done using Nikon NIS-Element AR 3.1 software.

Western blotting
Cells were lysed in 1X Passive Lysis Buffer (PLB) (Promega) and quantified using Bradford reagent (Thermo Scientific). Samples were assayed as per standard protocol . Imaging of all western blots was performed using an UVP BioImager 600 system equipped with VisionWorks Life Science software (UVP) V6.80. Details of all antibodies used are given at the bottom.

Cell senescence assay
Cells were assayed for senescence using Senescence Cells Histochemical Staining Kit from Sigma as per manufacturers' protocol. Assayed cells were thoroughly washed with PBS and finally mounted on a slide with Vectashield containing DAPI (H-1200, Vector Laboratories) for observation with a motorized Nikon Eclipse Ti microscope equipped with 10X Plan Fluor 10X/0.30 objective. Images were captured using a Nikon Ri1 camera.

Invasion assays
Using sterile forceps 24 well cell culture inserts from Nunc (Catalog no. 140629) were removed from their packaging and put in to wells of a 24-well plate. Pre-cooled inserts were washed twice with cold DMEM and then 100µl of 1:6 diluted Matrigel (BD catalog no. 354248) was coated onto the centre of each insert using pre-cooled pipette tips. The matrigel was spread across the entire surface and the coated inserts were kept at 37°C to solidify. For Figure 6C, matrigel with reduced amounts of growth factors were used. (BD catalog no. 354230).
For cell seeding, 24h after transfection cells were collected by trypsinization and 4X10 4 of transfected HepG2 cells, along with the cell used for co-culture, were seeded on to the upper chamber of the cell culture insert in 200µl of culture medium. 300µl of medium was placed in the lower chamber and cells were cultured for 48 h. For Figure 6C, 1X10 5 of nontransfected Huh7 cells were seeded to form a layer on the reduced growth factor matrigel coated cell culture insert. 4x10 4 GFP transfected HepG2 cells were then seeded on top of that. After 24h, medium was aspirated from the lower chamber and replaced by 500µl of 4% Paraformaldehyde. This was kept for 30 mins. After 30 mins, the medium from the inner chamber was aspirated out and and the matrigel layer was removed by wiping with a moist cotton swab. The insert was taken and the membrane was cut out using a sharp blade. The membrane was mounted on DAPI such that cells on the outer surface were stained with DAPI. The number of DAPI and GFP/DsRed positive cells was counted under the fluorescence microscope. The percentage of GFP/DsRed cells from the total DAPI positive invaded cells were then determined.

Cell proliferation assay
5x10 4 cells were seeded in each well in a 24 well plate and incubated in a 37°C humidified 5% CO 2 incubator for 24 h. Cells were then washed with serum free media (DMEM) and then 1 ml of DMEM was added to each well to synchronize cells in a low growth state. After 24h media was replaced with serum containing media with the added exosomes. 24h afterwards 1µCi of 3 H thymidine was added to each well. Cells were incubated for 8 h in a 37°C humidified 5% CO 2 incubator.
To extract 3 H-thymidine labelled DNA, cells were washed with 1 ml of ice-cold PBS.
Then 1 ml of ice-cold 5% TCA was added and cells were incubated at 4°C for 30 mins. After 30 mins, TCA solution was aspirated out and cells were washed once with PBS. 0.5 ml of 0.5M NaOH/0.5% SDS solution was added at room temperature to the cells. The cell suspension was pipetted up and down to lyse the cells. The mixture was then added directly to scintillation vials containing scintillation fluid. The vials were counted in a scintillation counter for assessment of radionucleotide incorporation.

Animal handling and mouse primary hepatocyte isolation
Animals were obtained from the animal house of the institute and all experiments were performed according to the guidelines set by Institutional Animal Ethics committee following the were then washed (as per manufacturers' protocol) and stained with the Exo-FITC universal exosome stain provided in the kit. Stained exosomes were then visualized on a BD LSR Fortessa FACS machine. Affinity purified exosomes were also eluted from the beads (as per manufacturers' protocol) and experiments were done with the eluted vesicles ( Figure S6).

Transmission Electron microscopic imaging of Huh7 exosomes
Exosomes of Huh7 cells obtained after ultracentrifugation of cell culture supernatants (exosomes from ~6x10 6 cells resuspended in 1ml PBS) were resuspended in PBS and 10l spotted onto Formvarcoated grids (200 mesh). The exosomes were then directly visualized.
Grids were examined by a FEI Technai G 2 Spirit BioTWIN electron microscope at 100 kV.

Scanning electron microscopic imaging of Huh7 exosomes
Exosomes of Huh7 cells obtained after ultracentrifugation of cell culture supernatants were resuspended in PBS (exosomes from ~6x10 6 cells resuspended in 1ml PBS) and spotted onto sample slides. The sample slides were dried completely under Critical Point dryer (Quorum Technologies) after drying the samples were kept on the sample holder by using carbon tape.
Gold coating was done with the current 10 mA at 10 -6 -10 -8 mbar/Pa (Quorum Technologies, Model No. SC7620). SEM imaging was performed using TESCAN Vega II LSU (TESCAN Digital Microscopy Imaging). Imaging and measurements were done by Vega TC software.
Atomic force microscopic imaging of Huh7 exosomes 5ul of Huh7 exosomes obtained from Huh7 cells (exosomes from ~6x10 6 cells resuspended in 1ml PBS) and isolated by ultracentrifugation were resuspended in PBS and deposited onto freshly cleaved muscovite Ruby mica sheet (ASTM V1 Grade Ruby Mica from MICAFAB, Chennai) for 15-30 minutes , Mica sheets are negatively charged so that the molecule binds strongly to the mica surface. The sample was dried by using vacuum dryer for 15 minutes. The sample slides were then gently washed with 0.5 ml MilliQ water to remove molecules that were not firmly attached to the mica and dried as mentioned above. AAC mode AFM was performed using a Pico plus 5500 ILM AFM (Agilent Technologies USA) with a piezoscanner maximum range of 9um. Micro fabricated silicon cantilevers of 225 m in length with a nominal spring force constant of 21-98 N/m were used from Nano sensors, USA. Cantilever oscillation