Shrimp miR-965 transfers tumoricidal mitochondria

Animal treatment

All mice were in C57BL/6 (RRID: 2,159,769) background and purchased from Charles River. All experiments using animals were approved and performed by the Ewha Womans University Animal Care Committee (Guide for the care and use of laboratory animals; IRB number: ESM14-0260). Forty female mice were each given weekly 1 mg doses of 7,12-Dimethylbenzathracene (DMBA) in 0.2 ml of sesame oil by oral gavage for six weeks and were implanted with 30 mg pellets of compressed medroxyprogesterone acetate (MPA) subcutaneously beginning at 5 weeks of age. Doxorubicin was administered weekly by intraperitoneal injection (5 mg/kg body weight, 300 μl injection volume) for four weeks. Mice were then maintained for an additional week.

Cell culture

Human breast cancer cell lines, MDA-MB-453 (ATCC; RRID: CVCL_0418), was maintained in Dulbecco’s MEM (11,885, Gibco, USA) with 10% fetal bovine serum (16,000,044, Gibco, USA) at 37 °C under an atmosphere of 95% O2 and 5% CO2. Cells were exposed to miRNA negative control (MMIR-000-PA-1, System Biosciences), synthetic miR-965 or its antagomir (Bioneer, South Korea) using a kit from System Biosciences (EXFT10A-1, System Biosciences) for 5 h.

Optical tomographic microscope

Green light (λ = 520 nm, exposure 0.2 mw/mm2) from a laser diode was split into cells and reference beam at Nanolive (3D cell explorer, Switzerland). Cells were illuminated with a laser beam inclined at 45° which rotated around the sample 360°. Holographic images were recorded on a digital camera by combining the beam that had passed through the cells with the reference beam. 3D cell images were recorded up to 30 μm depth of reconstruction. Mitochondria were visualized with MitoTracker (M7514, Thermo Fisher Scientific, USA) at 490 nm.

Experimental procedures using microfluidic device

Cotton candy sheets were sealed with polydimethylsiloxane (PDMS) to construct microfluidic mold. After hardening, cotton candy fibers were removed by perfusing with water to make microfluidic mold of approximately 950 nm. Each microfluidic device was connected by polythene tubing (PE10, Braintree scientific, USA) with an inner diameter of 0.28 mm. Fluid flow was controlled by individual peristaltic pump (3,200,243, Dolomite, UK). Isolated mitochondria were introduced to microfluidic devices at a flow rate of 10–30 μm/s. After passing through the microfluidic channels, mitochondria were transported into cells.

Analysis of RNA using real-time PCR

RNA levels of ENDOG (Hs00172770_m1) were determined using primer/probe set from Life Technology. Real-time PCR was performed with TaqMan universal PCR Master Mix on an ABI Real time PCR System 7000 (Applied Biosystems, USA). PCR conditions were 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. For each experimental sample, the relative abundance value was normalized to the value derived from ACTB (Hs03023943_g1, Life Technology) as housekeeping control gene. Relative mRNA levels were quantified using the comparative 2−ΔΔCT method. The extracted miRNA were determined using microarray service (Macrogen, South Korea).

ELISA for detection of apoptosis or caspase-3 activity

Apoptosis was determined using ssDNA ELISA Kit (APT225, Sigma-Aldrich, USA). Briefly, cell plates were fixed and incubated with ABTS solution for 30 min to allow binding to HRP at 37 °C. To denature DNA, cell plates were incubated for 20 min at 75 °C. After cooling at 4 °C for 5 min, plates were blocked in 5% skim milk (70,166, Sigma-Aldrich) in PBS at 37 °C for 1 h. Cells were incubated with antisera mixture for 30 min and washed with PBS. After treatment with stop solution, absorbance was measured at 405 nm. Caspase-3 activity was determined using Cleaved Caspase-3 ELISA kit (ab220655, abcam, USA). Cells were fixed and incubated with antibody cocktail at 37 °C for 1 h, then washed and incubated with TMB solution for 30 min. After treatment with stop solution, absorbance was measured at 450 nm.

Western blot

Cells were homogenized and centrifuged at 5,000 g for 20 min. Protein content of the supernatant was diluted, boiled with sample loading dye, and 100 mg were loaded in SDS-PAGE (4561033EDU, Bio-Rad). After blotting, membranes were blocked in 5% skim milk (70,166, Sigma-Aldrich) in PBS containing 0.1% Tween-20 (P1379, Sigma-Aldrich). Membranes were incubated with antisera directed against cytochrome C (1:1000; #11,940, RRID: AB_2637071, Cell signaling technology, USA), Endo G (1:1000; #4969, RRID: AB_2098768, Cell signaling technology, USA), Mcl-1 (1:1000; ab28147, RRID: AB_776246, abcam, USA), or β-actin (1:1000; sc-47778, RRID: AB_626632, Santacruz Biotechnology, USA), then with secondary antibodies (mouse-specific HRP-conjugated antibody or rabbit-specific HRP-conjugated antibody). Bands were visualized using ECL (32,106, Thermo Scientific) detection kit and quantified by densitometry.

Mitochondrial isolation and transfer

Cells were harvested from culture dishes with homogenization buffer (20 mM HEPES–KOH, 220 mM mannitol, and 70 mM sucrose) containing a protease inhibitor mixture (Sigma-Aldrich) and centrifuged at 2300 × g for 5 min. The cell pellet was resuspended with homogenization buffer and incubated on ice for 5 min at 4 °C. Cells were ruptured by 10 strokes using a 27-gauge needle. The homogenate was centrifuged at 5800 × g for 5 min, and mitochondria were harvested. The amount of isolated mitochondria was expressed as protein concentration by using the Bio-Rad protein assay kit (Bio-Rad, Richmond, USA). Mitochondrial transfer was conducted by co-incubating isolated mitochondria with cells (1 × 105 cells/well of a 6-well plate) at 37 °C under 5% CO2 for up to 10 h. For in vivo experiments, mice were administered weekly with 1 × 104 isolated mitochondria per gram of body weight via the tail vein.

Transmission electron microscopy

We fixed the tissues of Marsupenaeus japonicas or mold of cotton candy-microfluidic with 3% buffered glutaraldehyde (G5882, Sigma-Aldrich) for 2 h and processed into resin (02,334, Polysciences, German). After embedding, the resin block was thin-sectioned by ultramicrotomy. Sections of 50–70 nm thickness were collected on metal mesh and stained with electron dense particles before imaging of ultrastructures, using the transmission electron microscope (H-7650, Hitachi-Science & Technology, Japan).

CRISPR-mediated gene deletion

Clustered regularly interspaced short palindromic repeats (CRISPR) transfection of ENDOG in MDA-MB-453 was performed using a kit from Santa Cruz (sc-395739, Santacruz Biotechnology, USA). Briefly, in six-well culture plates, 106 cells were plated and exposed to the ENDOG plasmid (sc-403263, Santacruz Biotechnology, USA) or negative control-CRISPR plasmid (sc-418922, Santacruz Biotechnology, USA) solution for 8 h at 37 ℃ in a CO2 incubator. Then, media was changed to Dulbecco’s MEM with 10% fetal bovine serum and incubated for another 18 h. The ENDOG expression was determined using RT-PCR.

Echocardiographic assessment

For echocardiography, Vevo 2100 was used at Cardiovascular Research Center in Seoul. Mice were anesthetized with 2% isoflurane and maintained with 1.5% isoflurane followed by application of depilatory cream to the chest and wiped clean to remove all hair in the area of interest. The scanning probe (20 MHz) was used to obtain 2D images of the parasternal long axis. These 2D images were converted to M-mode.

Statistical analysis

Values were means ± SE. The significance of differences was determined by a two-way analysis of variance (ANOVA), or a one way ANOVA followed by a Bonferroni post-hoc analysis where appropriate. Differences were considered significant when P < 0.05.

留言 (0)

沒有登入
gif