Ultrasound and microbubble-mediated delivery of miR-424-5p has a therapeutic effect in preeclampsia

Ethical statement

Written informed consent form was acquired from patients or their families before collection and use of placental tissues in this study. All experiments have been allowed by the ethical committee of the Second Affiliated Hospital of Harbin Medical University and were carried out in strict accordance with the ethical principles for human subject research in Declaration of Helsinki.

Bioinformatics analysis

GSE143953 dataset was recruited from GEO database (http://www.ncbi.nlm.nih.gov/geo), which included the mRNA profile of placental tissues from 4 patients with PE and from 4 normal pregnant women. Volcano plot of gene expression profile was described, in which the differentially expressed genes were highlighted. Log|FC|> 2 and P < 0.05 were set as filter criteria.

Clinical samples

PE patients in early stage (n = 15) and normal pregnant women (n = 15) at the Second Affiliated Hospital of Harbin Medical University were enrolled in this study. PE patients recruited were diagnosed by at least two deputy chief physicians or chief physicians of the obstetrics and gynecology department. Include criteria were as follows: gestational age of 20 ~ 34 weeks; no specific medication during the pregnancy; the subjects were informed and agree to participate in this study; pregnant women underwent caesarean section. Pregnant women with one of the following conditions were excluded: multiple pregnancy; fetal chromosomal or structural abnormalities; chronic hypertension plus PE during pregnancy or pregnant woman with hypertension; pregnant woman with liver and kidney disease, cardiopathy or endocrine system disease. After caesarean section, placental tissues (1 cm3) were collected from maternal placentae within 10 min after delivery of placenta. The tissues were washed with normal saline, dried with sterile gauzes and preserved in liquid nitrogen. All recruited patients were subjected to blood pressure measurement for three times at different days, and the average value of these three independent data was regarded as the blood pressure. Urine of enrolled patients was collected in 24 h to measure the urine volume and the 24-h proteinuria.

Cell culture

Human trophoblast cells (HTR-8/Svneo and TEV-1) and human embryonic kidney cells (HEK293T) acquired from American Type Culture collection (ATCC, Manassas, Virginia, USA) were maintained in DMEM/F12 (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) and DMEM (Gibco, Grand Island, NY, USA) containing 10% FBS and 1% penicillin/streptomycin, respectively. Cell culture was carried out at 37℃ with 5% CO2.

Cell transfections

miR-424-5p mimic (50 nM), vectors with AOC1 overexpression (2 μg), and the corresponding negative controls (mimic NC and pcDNA3.1) were synthesized by Shanghai GenePharma Co.,Ltd (Shanghai, China) and transfected into cells as the instructions on Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). Following measurements were carried out 48 h after the transfections.

5-Ethynyl-2’-deoxyuridine (EdU)

An EdU labeling/detection kit (Ribobio, Guangzhou, China) was used to examine cell proliferation. HTR-8/Svneo and TEV-1 cells in logarithmic phase were pipetted into 96 well plates at a density of 105 cells/well. Culture medium was used to dilute EdU solution at 1000:1 to prepare culture solution containing 50 μM EdU. The HTR-8/Svneo and TEV-1 cells in each well were cultured with 100 μl EdU for 2 h. After the culture solution was discarded, the cells were washed with PBS once or twice for 5 min each time. Then, the cells in each well were fixed by 4% paraformaldehyde in 50 μl PBS for 30 min, cultured with 50 μl 2 mg/ml glycine for 5 min and washed in 100 μl PBS for 5 min. Afterwards, 100 μl 0.5% TritonX-100 was cultured with the cells for 10 min and then washed off by PBS for 5 min. Prepared 100 μl 1 × Apollo® solution was added onto the plate and cultured at room temperature away from light. Thirty minutes later, the 1 × Apollo® solution was removed and the cells were washed by 100 μl 0.5% TritonX-100 twice or three times for 10 min each time. Then, TritonX-100 solution was pipetted off and the cells were successively washed in 100 μl formaldehyde (once or twice, 5 min each time) and in PBS (once, 5 min). The cells were cultured with 100 μl 1 × Hoechst 33,342 solution in dark at room temperature for 30 min and washed by 100 μl PBS once to three times before being observed by a fluorescence microscope.

Flow cytometry

Density of HTR-8/Svneo and TEV-1 cells in each group was adjusted to 105 cells/ml, and 3 ml cell suspension was pipetted into a 10 ml centrifuge tube and centrifuged for 5 min at 500 rpm. After culture medium was removed, the cells were washed with PBS and centrifuged at 500 rpm for 5 min. Then, the supernatant was discarded and the cells were resuspended in 100 μl binding buffer. Following 5 μl Annexin V-FITC and 5 μl PI were added for culture for 5 min at room temperature in dark, the fluorescence intensity of FITC and PI was visualized by flow cytometry to measure cell apoptosis.

Scratch assay

Transfected HTR-8/Svneo and TEV-1 cells were seeded onto six well plates at a density of 1 × 106 cells/well in triplicate and then cultured at 37℃ with 5% CO2 for 24 h until monolayer cells covered the whole well. A sterile 200 μl pipette tip was used to scratch the monolayer cells. Afterwards, the cells were washed by PBS and further cultured for 48 h at 37℃ with 5% CO2. Gap of the scratch was observed and recorded by a microscope at the 0th h and the 48th h. Migration rate of trophoblast cells was measured based on the changes of the gap. Migration rate = (gap of the scratch at 0 h – gap of the scratch at 48 h)/gap of the scratch at 0 h.

Transwell invasion assay

Transfected HTR-8/Svneo and TEV-1 cells were resuspended in serum-free culture medium and the density of the cells was adjusted. Then, 100 μl cells (1 × 105 cells) were pipetted onto matrigel-coated transwell chamber (354,480, Corning, NY, USA). The basolateral chamber was covered with 600 μl culture medium containing 10% FBS. After cell culture for 24 h at 37℃, the transwell chamber was collected and the excess culture medium was removed. Non-invasive cells in the chamber were scraped off by wet cotton swabs. Cells in the chambers were fixed by 4% paraformaldehyde for 30 min, stained with Giemsa dye for 20 min and counted under a microscope.

ELISA

Levels of PLGF, human chorionic gonadotropin (β-hCG) and tumor necrosis factor-α (TNF-α) in supernatant of HTR-8/Svneo and TEV-1 cell culture medium were quantified as described in the instructions of ELISA kit (R&D Systems, Minneapolis, MN, USA).

RNA immunoprecipitation (RIP)

HTR-8/Svneo and TEV-1 cells were collected, washed with PBS twice and centrifuged at 1,500 rpm for 5 min. Afterwards, the cells were mixed with an equal volume of RIP lysis buffer. Magnetic beads were resuspended in 100 μl RIP Wash Buffer and incubated with 5 μg anti-Ago2 antibody (ab32381, 1:100, Abcam, Cambridge, MA, USA) at room temperature for 30 min. Beads in the negative control group were incubated with anti-IgG antibody. The centrifuge tubes containing the magnetic beads were placed on magnetic rack and the supernatant was removed, after which 500 μl RIP Wash Buffer was added and shaken by vortexing to remove the supernatant. The aforementioned procedures were repeated once. Then, 500 μl RIP Wash Buffer was added into the centrifuge tube. Afterwards, the tube was shaken by vortexing and placed on ice. The prepared tubes containing magnetic beads were placed on magnetic rack to remove the supernatant and then were added 900 μl RIP Immunoprecipitation Buffer. Prepared cell lysate was rapidly unfrozen and centrifuged at 14,000 rpm and 4℃ for 10 min. The supernatant of the cell lysate (100 μl) was added into bead-antibody complexes and cultured at 4℃ overnight. After transient centrifugation, the centrifuge tube was placed on magnetic rack to discard the supernatant. RIP Wash Buffer (500 μl) was added into the tube, shaken by vortexing and placed on magnetic rack to remove the supernatant, which was repeated for 6 times. Proteinase K Buffer (150 μl) was added into each tube to resuspend the bead-antibody complexes. Then, the mixture was cultured for 30 min at 55℃, after which the tube was placed on magnetic rack to pipette the supernatant. qRT-PCR was used to detect the expression of genes after RNA extraction.

Dual luciferase reporter assay

Online software TargetScan (http://www.targetscan.org/vert_72/) was used to predict the binding sites of miR-424-5p in the 3’UTR of AOC1. Based on the results, wild and mutated sequences of the binding sites were separately synthesized and termed wt-AOC1 and mut-AOC1. Then, the sequences were inserted into luciferase reporter vectors (pGL3-Promoter, Promega, Madison, WI, USA) and cotransfected with miR-424-5p mimic (50 nM) or its negative control into HEK293T cells. The vectors containing wt-AOC1 or mut-AOC1 were also transfected with pRL-TK as the internal reference. After transfection, dual luciferase reporter gene detection kit (Promega, Madison, WI, USA) was used to assess firefly and renilla luciferase activities with renilla luciferase activity as the internal reference. Relative luciferase activity = firely luciferase activity/renilla luciferase activity.

Microbubble preparation and cell treatment

Distearoyl phosphatidylcholine (5 mg), dipalmitonyl phosphatidyl ethanolamine (2 mg), plasmid (EGFP-miR-424-5p, 2 mg, Genepharma), Span 60 (1 mg) and glycerinum (50 μl) were added into a 1.5 ml tube and dissolved in PBS to make a final volume to 0.5 ml. Afterwards, the mixture were cultured at 37℃ for 30 min and then the tube was supplemented with perfluoropropane to replace air. The tube was shaken for 60 s and added 0.5 ml PBS before being placed for 5 min. Following disinfection, the tube was preserved at -80℃. When trophoblast cells HTR-8/Svneo and TEV-1 were treated with microbubbles, the mixture was exposed to ultrasound at 300 kHz and 0.5 W/cm2 for 30 s.

MTT

HTR-8/Svneo and TEV-1 cells in logarithmic phase were digested by trypsin before being seeded onto 96 well plates (1000 cells/well, in triplicate). After being cultured at saturated humidity and 37℃ with 5% CO2 for 24 h, the cells were further cultured with 200 μl (1%, 5%, 10% or 20%) perfluoropropane microbubbles at the same condition. After cell culture, the supernatant was discarded and 10 μl DMSO-dissolved MTT (5 mg/ml, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) was mixed and cultured with the cells for 4 h at 37℃. Afterwards, the absorbance was detected at 570 nm (OD570). The absorbance was positively related to the number of living cells.

qRT-PCR

RNA was isolated from placental tissues and trophoblast cells (HTR-8/Svneo and TEV-1) using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA), and complementary DNA was obtained from RNA using reverse transcription kit (TaKaRa, Tokyo, Japan). RT-PCR amplifications were conducted based on the instructions of SYBR Green Mix kit (Roche Diagnostics, Indianapolis, IN) and the expression of genes of interest was determined on LightCycler 480 instrument (Roche, Indianapolis, IN, USA) by using U6 and GAPDH as the internal references of miRNA and mRNA, respectively. Thermal cycle parameters of RT-PCR amplifications were as follows: 95℃ for 10 s; 45 cycles of 95℃ for 5 s, 60℃ for 10 s and 72℃ for 10 s; extension at 72℃ for 5 min. Each PCR was performed in triplicates. Analysis of gene expression was conducted by 2−ΔΔCt method. ΔΔCt = (Ct target gene – Ct internal reference)experimental group – (Ct target gene – Ct internal reference)control group. Primer sequences of target genes and their internal references are exhibited in Table 1.

Table 1 Primer sequences of target genes and their internal referencesWestern blotting

Placental tissues and trophoblast cells (HTR-8/Svneo and TEV-1) were lysed by RIPA lysis buffer (Beyotime, Shanghai, China) for protein extraction and the protein concentration was quantified by BCA kit (Beyotime, Shanghai, China). An equal volume of protein was mixed with loading buffer (Beyotime, Shanghai, China) and denatured through boiling water bath for 3 min. Then, electrophoresis was initiated at 80 V for 30 min and then switched to120 V for 1 ~ 2 h to separate the protein. Membrane transferring was carried out at 300 mA for 60 min through ice bath. Afterwards, the membrane was rinsed for 1 ~ 2 min and blocked at room temperature for 60 min or at 4℃ overnight. Primary antibodies against GAPDH (ab8245, 1:2000), AOC1 (ab231558, 1:1000), β-catenin (ab32572, 1:5000) and c-Myc (ab32072, 1:1000) (Abcam, Cambridge, MA, USA) were incubated with the membranes at room temperature for 1 h, respectively. Thereafter, the membranes were washed 3 × 10 min before being incubated with secondary antibody at room temperature for 1 h. Following washes 3 × 10 min, the membranes were subjected to color development. Chemiluminescence imaging system (Gel Doc XR, Bio-rad) was applied to visualize and quantify the protein bands.

Animal experiments

Eight-week-old female C57BL/6 mice (25–30 g, Shanghai SLAC laboratory animal center, Shanghai, China) were reared under standard conditions (25 ± 5℃, 60–80% humidity, 12 h dark/light cycles, and standard diet) for one week of acclimation. Vaginal smears were daily obtained from female mice until the smears showed that they were oestrous. A PE mouse model was induced by subcutaneous injection of L-NAME [20].

Female mice were mated with male mice at a ratio of 2:1, and vaginal plug formation was detected on the next and defined as gestational day (GD) 0.5. The pregnant mice were divided into control, PE, and ultrasound microbubble groups (n = 6/group). During GD 7.5–18.5, the mice in PE and ultrasound microbubble groups were subcutaneously injected with 60 mg/kg L-NAME (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) every day, and mice in the control group were injected with an equal volume of normal saline. On GD 10.5, the mice in ultrasound microbubble group were injected with 100 μl microbubbles and irradiated by ultrasound at 1 MHz and 2 W/cm2 for 5 min with intervals of 10 s (on 10 s, off 10 s). The systolic pressure of the mice was measured on GD 6.5 and 18.5, and 24-h proteinuria was assayed using a BCA kit on GD 18.5. The mice were euthanatized on GD 18.5, and the placental tissues were collected for histological staining and gene expression detection.

Hematoxylin and eosin (H&E) staining

The collected placental tissues were fixed in 4% paraformaldehyde solution and sliced into 4-μm paraffin sections. The sections were dewaxed in xylene, hydrated in alcohol gradient, stained with hematoxylin for 5 min, and immersed in 1% hydrochloric acid alcohol for 30 s. Following 2-min eosin staining, the sections were dehydrated, permeabilized, sealed, and observed by a microscopy.

Statistical analysis

All data were analyzed by GraphPad prism 7 and summarized as mean ± standard deviation (SD). Normally distributed data between two groups were compared by t-test, and those among multiple groups were compared by one-way analysis of variance with Tukey’s multiple comparisons test. P value less than 0.05 was considered to have statistical significance.

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