The 19-base oligonucleotide probes were chemically synthesized and modified from Shanghai Shangon Bioengineering Co., Ltd. The ASON sequence was designed as 5′- AATTCTTAAATTGGGCTGG -3′, which was completely complementary to the HOTAIR fragment, and the ASONM（mismatched antisense oligonucleotides） sequence was 5′-AATACTTAGATTAGGCAGG-3′ (The underlined part is the substituted nucleosides). Two probes were modified with 2' methylation (2'-O-methyl) at both ends of the sequence and two bases at each end were phosphonothioate modified to improve its stability, NH2C6 is connected to 5’ end. Ultimately, the synthetic structure was 5’-NH2C6-ASON/ASONM-3'. 99mTc is obtained from the 99mTc radionuclide generator produced by China Atomic Energy Research Institute.
Synthesis and labeling of the probe
0.2mg ASON dissolved in 50μL buffer (2mol/l NaCl, 0.5mol/l NaHCO3, 2mmol/l EDTA), HYNIC (TriLink, US) dissolved in DMF solution (10mg/mL). Then mixed at a molar ratio of 25:1(HYNIC: ASON) and avoid light for 1 h. Then the mixture above were added with 60% methanol to the total volume of 500μL, using an ultrafilter tube (Sartorius, GER) 13000g to centrifuge for 10min (ensure that the volume after centrifugation was less than 50μL) to obtain HYNIC-ASON. Next, 100μL Tricine(100mg/mL), 20μL 99mTc(222MBq) as well as 4μL fresh SnCl2•2H2O (1mg/mL) were added to the above HYNIC-ASON in turn and reacted for 60min. After the reaction, using Sephadex G25(GE, US) to separate and purify. 15 tubes of eluents were collected with 5 drops per tube, then measured the radioactivity counts and nucleic acid concentration of each tube. Take the peak tube for the following up experiment.
Fresh human serum is provided by volunteers in our department. The institutional review committee of General Hospital of XXX approved the study and all the volunteers obtained informed consent. 99mTc-HYNIC-ASON was incubated in saline and fresh human serum at 37℃ and room temperature, respectively (the volume ratio of the probe to serum/saline was 1:1). The radiochemical purity was detected by thin layer paper chromatography (ITLC) at 0, 2, 4, 6, 8, 12 h.
Agarose gel electrophoresis
To identify the integrity of 99mTc-HYNIC-ASON and eliminate the degradation after labeling. 1% agarose gel was configured, followed by an unbonded ASON sample, 99mTc, 99mTc-HYNIC-ASON before and after purification. The voltage was 120V, electrophoresis for 20 minutes, then the band was observed under UV.
Cell culture and transfection
U87 glioma cells were purchased from the Chinese Academy of Sciences, and cultured in DMEM (Invitrogen, US) medium containing 15% fetal bovine serum（ABW,CHN） and 1% antibiotics in a CO2 incubator at 37 ℃ for 24 hours, then passaged when the cell density reached 90%.
For transfection (Lip-99mTc-HYNIC-ASON and Lip-99mTc-HYNIC-ASONM). Liquid A: 10μg purified 99mTc-HYNIC-ASON/ 99mTc-HYNIC-ASONM were added to 500μL DMEM without serum and antibiotics; Liquid B: 25μL Lipofectamine 2000 (Invitrogen, US) were added to 475μL DMEM, and Liquid A and B were placed at room temperature for 5min respectively, then mixed two of them for 20min. After added Lip-99mTc-HYNIC-ASON/ Lip-99mTc-HYNIC-ASONM to the cells and cultured in a 37℃ incubator for 6 hours, the medium was replaced by DMEM containing 15% fetal bovine serum for 24-48 hours to complete transfection.
U87 cells were inoculated in a 12-well plate at 5×105 density and cultured overnight in DMEM containing 15% FBS without antibiotics. The cells were divided into liposome transfected and non-transfected groups. In the transfection group, 200μL DMEM, 500ng 99mTc-HYNIC-ASON or 99mTc-HYNIC-ASONM (37kBq) and 3μL Lipofectamine 2000 were added into each well; 500ng 99mTc-HYNIC-ASON or 99mTc-HYNIC-ASONM (37kBq) in the non-transfection group. After cultured in a 37 ℃ incubator, each well of culture medium was collected at 0.5h,1h, 2h, 4h, and 6h, respectively, and washed three times with 100μL PBS collected into the same EP tube labeled Cout. Then the cell was collected with trypsin containing EDTA, 100μL PBS in each well was washed three times to EP tube labeled Cin. The radioactivity counts of Cin and Cout were measured by γ radioimmunoassay counter (Chinese Academy of Metrology), and the uptake rate of cells to the probe at each time was calculated. Calculation formula: cell uptake rate = Cin/ (Cin+Cout) × 100%.
Animal xenograft model
BALB/c nu/nu mice (female, weight ±SD, 206.0g, age 3~4wk) were fed in the Experimental Animal Center of XXX. Malignant glioma U87 cells (5×1011) were subcutaneously injected into the right fore axilla of each mouse. When the tumor reached 1.5-2.0cm, it was used in the follow-up experiment. All animal experiments have passed the ethical review of the Animal Experimental Center of XXX. and are conducted under the guidelines of the Animal Welfare Committee.
Twenty nude mice were randomly divided into 5 groups with 4 mice in each group. Lip-99mTc-HYNIC-ASON 1μg, 2.59MBq (100μL) was injected into the tail vein. Then the mice were killed by cervical dislocation at 1, 2, 3, 4 and 6 hours after 100μL of blood was taken from the ophthalmic vein. After that, tissues like the heart, liver, spleen, kidney, stomach, small intestine, bladder, muscle, bone, and tumor were removed and weighed, then radioactivity count was measured. The distribution results were recorded as the percentage of radioactivity per gram of tissue (% ID/g).
Images were performed by a SPECT scanner at 1h, 2h, 4h, 6h, and 8h respectively，after 4μg, 14.8MBq (150μL) Lip-99mTc-HYNIC-ASON/ Lip-99mTc-HYNIC-ASONM probe were injected into the tail vein. For blocking groups, 10μg liposome transfected unlabeled probes were injected 2 hours before Lip-99mTc-HYNIC-ASON injection. The collection counts were 100 KCounts and stored as a 64×64 matrix at 3.2 zooms, then T/M (Tumor/Muscle) and T/A (Tumor/Abdomen) ratio of regions of interest were calculated.
All data are processed by SPSS22.0 statistical software, variables are represented by χ̅ ±SD. T test was used for comparison between the two groups. P<0.05 is considered to be statistically significant.