Synthesis RNAs
The full mRNA of RWPE-1 cells (human regular prostate epithelial cell) was extracted by Trizol methodology and reverse transcribed into cDNA, and all of the steps and PCR setup had been carried out in keeping with the information of Quick-Quant RT Equipment (TianGen, China) to acquire cDNA. Then, the cDNA was used as a template for the PCR to synthesize goal RNAs. The sequence of the PTENP1 gene (GeneID:11,191) was sequentially divided into 4 segments as templates to synthesize 4 RNAs (RNA1, RNA2, RNA3, RNA4) by PCR methodology, and the primers had been summarized within the supplementary supplies (Desk S1). Then, the RNAs had been gel purified with the Wizard SV Gel and PCR Clear-Up System (Promega Company, USA), and saved at -80 ℃.
Building, transformation, amplification and extraction of the plasmid
4 RNAs had been inserted into pGEM-T Straightforward Vector System I (Promega, USA) to synthesize plasmids, respectively. Then, 2 µg plasmid was blended with 100 µL of competent Escherichia coli and an ice bathtub was carried out for 30 min. It was positioned in a 42 ℃ water bathtub with thermal shock for 90 s and instantly positioned in an ice bathtub for two min. And 900 µL Luria-Bertani (LB) liquid medium was added, which was ready by including 10 g tryptone (OXOID, UK), 10 g NaCl (Solarbio, China), 5 g yeast extract (OXOID, UK), deionized water to 1000 mL, after which autoclaved and added 10 mg ampicillin. Then the combination was stirred at 225 rpm at 37 ℃ for 1 h, after which the bacterial resolution was planted on an agar plate containing 100 µg/mL ampicillin, which was then inverted and stored at 37 ℃ in a single day. After colonization, bacterial colonies of roughly 1 mm had been chosen with a sterile ring in 25 mL LB liquid medium containing 100 µg/mL ampicillin and shaken at 180 rpm at 37 ℃ for 16 h. A part of the bacterial resolution obtained was used for sequencing to confirm the sequence. The remaining was centrifuged at 4 ℃ at 6000 rpm for 10 min. After discarding the supernatant, the QIAGEN Plasmid Midi Equipment (QIAGEN, Germany) was used to extract the plasmids within the bacterial resolution.
Linearization, purification and restoration of the plasmid
A 50 µL plasmid linearization system was used for plasmid linearization. Particularly, 5 µL NcoI-HF (New England Biolabs, USA), 5 µL CutSmart Buffer (New England Biolabs, USA) and 10 µg plasmid had been added into the enzyme-free EP tube, and enzyme-free water was added to the system to 50 µL. After full mixing, the linearized plasmid was obtained after incubation at 37 ℃ for six h. The linearized plasmid was gel purified with the Wizard SV Gel and PCR Clear-Up System (Promega Company, USA), and saved at -20 ℃.
Synthesis of RNA by in vitro transcription
Linearized DNA was synthesized into RNA by means of in vitro transcription. Particularly, 10 µL of 5× transcription buffer (Thermo Fisher Scientific), 10 mM of ATP/GTP/CTP/UTP (Thermo Fisher Scientific) 1 µL every, 1 µg of linearized DNA, 1.25 µL RNAase inhibitor (Thermo Fisher Scientific), 1.5 µL T7 polymerase (Thermo Fisher Scientific), DEPC water to 50 µL had been added into the enzyme-free EP tube; then, the system was incubated at 37 ℃ for two h, and RNA was transcribed after which saved at -20 ℃ for later use.
Building of BP-PEI@RNAs
BP nanosheets had been ready by the liquid exfoliation methodology described in a earlier research [19], centrifuged at 4 ℃ (16,000 rpm, 60 min), and washed thrice with enzyme-free water to acquire a well-dispersed BP aqueous dispersion resolution in a focus of 1 µg/ml. Polyetherimide (PEI) (2.5k, 10 mg/mL, CAS: 9002-98-6, Sigma-Aldrich) was then added to the answer at a quantity ratio of 1:1.5 for BP: PEI, after which the combination was sonicated within the ice water bathtub for 3–4 h to acquire BP-PEI. BP-PEI (1 µg/ml) was resuspended and incubated with RNA (0.1 µg/ml) (BP to RNA mass ratio of 1:0.4) at 4 °C for 10 min with steady rotational mixing to acquire BP-PEI@RNAs platform.
Characterization of BP@RNA
The diameter and floor traits of BP nanosheets, BP-PEI and BP-PEI@RNAs had been characterised by transmission electron microscope (TEM, JEOL JEM-2100 F, Japan) and atomic pressure microscope (AFM, Dimension Icon, Germany). Raman spectroscopy evaluation of them was carried out utilizing a Raman spectrometer (Horiba scientific LabRAM HR evolution). The hydrated particle measurement and zeta potential had been analyzed by nanoparticle measurement analyzer (Zetasizer Nano ZS90, Malvern, UK).
Entrapment effectivity (EE%)
RNA EE% in BP-PEI was decided as beforehand described [20]. Briefly, RNA-loaded BP-PEI was centrifuged at 40,000 ×g for 20 min, after which the quantity of uncaptured RNA within the supernatant was measured utilizing RiboGreen Assay. EE% was calculated by the next formulation:
$$eqalign{{rm{RNAEE}}% & {rm{ = }} cr {{{rm{ Quantity of complete RNA – Quantity of uncaptured RNA }}} over {{rm{ Quantity of complete RNA }}}} instances {rm{100}} cr}$$
Analysis of RNA stability
The power of the BP-PEI@RNA complicated to guard RNA in opposition to RNase A digestion was investigated by agarose gel electrophoresis. Briefly, the optimum BP-PEI@RNA1 formulation (BP-PEI: RNA = 1:0.4) containing 1 µg RNA was incubated with RNase (0.01 µg/mL) (Sigma, USA) in PBS (pH 7.4) for 0, 15, 30 and 60 min at 37 °C. RNA was transferred from BP-PEI by incubating the samples with 1 µL of 1 mg/ mL sodium dodecyl sulfate (SDS) resolution (25 °C, 5 min). The identical quantity of RNase-treated free RNA was used as a management. The extracted RNA was subjected to 1% agarose gel electrophoresis at 120 V for 10 min. The ensuing gels had been stained with SYBR Inexperienced (Invitrogen, USA) and visualized on an imaging system.
RNA launch profile
RNA launch of the BP-PEI@RNA complicated was assessed at numerous pH circumstances (3, 5, 7, 9, and 11) and at numerous protein concentrations (2.5%, 5%, 7.5%, and 10% FBS). For options with totally different pH values, the pH was adjusted with HCl or NaOH utilizing a pH meter (Biofrontier Know-how). For options with totally different protein concentrations, a sure quantity of FBS was added to RPMI 1640 medium. The BP-PEI@RNA resolution was incubated at 37 ℃ and 100 rpm in a shaker. At every time level (1, 2, 4, 6, 12, 24, 48 and 96 h), the supernatant was collected by centrifugation at 15,000 rpm for 30 min, and the quantity launched was decided.
Fluorescent labeling of RNA
Within the technique of in vitro transcribed RNA preparation, 6-FAM or cy3 labeled ribonucleotides had been used as fluorescent substrates as an alternative of non-fluorescent labeled ribonucleotides, and RNA was synthesized in keeping with the above course of “Synthesis of RNA by in vitro Transcription” to acquire 6-FAM or Cy3 labeled RNA.
Uptake and subcellular distribution of BP@RNA
All PC3 cells had been cultured in RPMI 1640 medium (Gibco BRL Life Applied sciences Inc., Waltham, MA, USA) containing 10% FBS (Gibco BRL Life Applied sciences Inc.) and 1% antibiotics (Gibco BRL Life Applied sciences Inc.) and incubated at 37 ◦C in a humidified incubator with 5% CO2. When PC3 cells reached 50-60% confluence, they had been uncovered to the 6-FAM- and Cy3-labeled BP-PEI@RNA (1 µg/mL BP; 0.4 µg/mL RNA; BP-PEI@RNA: The mass ratio of BP-PEI and RNA was 1:0.4), respectively. The mobile uptake of BP-PEI@RNA-6FAM in PC3 cells was noticed by CLSM at totally different time factors (1, 3, and 6 h put up publicity). The subcellular localization of PEI@RNA-Cy3 in PC3 cells was detected by co-staining with lysosomes and noticed by CLSM at totally different time factors (6, and 24 h put up publicity).
Reside/ lifeless staining
When the confluence reached 50-60%, PC3 cells had been uncovered to BP-PEI@RNA (1 µg/mL BP; 0.4 µg/mL RNA; The mass ratio of BP-PEI and RNA was 1:0.4), and the cells had been then stained with the Viability/Cytotoxicity Assay Equipment for Animal Reside & Useless Cells (Proteintech, China) at 24 h put up publicity. The fluorescence sign of the cells was noticed by CLSM.
Cell viability check
PC3 cells had been cultured in 96 properly plates, when the confluence reached 50-60%, these cells had been uncovered to the free RNAs (0.4 µg/mL) or BP-PEI@RNA (1 µg/mL) for twenty-four h, after which the cell viability was examined by Cell Counting Equipment-8 assay (MedChemExpress, USA).
Autophagy, apoptosis and cell cycle check
PC3 cells had been cultured in RPMI 1640 medium (supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin). When 50-60% confluence was reached, these cells had been uncovered to BP-PEI@RNA (1 µg/mL BP; 0.4 µg/mL RNA) for twenty-four h. PC3 cells had been harvested and stained with Lyso Tracker Inexperienced DND-26 (Cell Signaling Know-how, USA), Annexin V-FITC apoptosis assay equipment (Beyotime Biotechnology, China) and Cell Cycle Staining Equipment (Abbkine, China).
RT-qPCR
PC3 cells incubated with BP-PEI@RNA for twenty-four h had been harvested. The miRNA of PC3 cells was extracted in keeping with the information of FastQuant RT Equipment (With gDNase) (TianGen, China), after which reversely transcribed into cDNA in vitro. RT-qPCR was carried out utilizing Common SYBR Inexperienced qPCR Supermix (US EVERBRIGHT, China). U6 was used as an inner management. The expression of miRNAs was quantified by 2−ΔΔCT methodology. The primers had been summarized in Desk S2.
Western-blot
PC3 cells had been harvested and washed 3 instances with RPMI 1640 medium. Then, these cells had been lysed by RIPA lysis buffer (Pierce IP Lysis Buffer, USA) and the protease inhibitor cocktail was added to inhibit the degradation of complete proteins. Protein focus was detected by BCA protein assay equipment (Solarbio Science & Know-how Co., Ltd., Beijing, China). Equal proteins had been used for sodium dodecyl sulfate-polyacrylamide gel after which incubated with totally different major antibodies (anti-β-actin, anti-GAPDH, anti-PTEN, anti-P-PI3K, anti-PI3K, anti-P-AKT, anti-AKT, anti-LC3-I, anti-LC3-II, anti-p62, anti-cleaved caspase 3, anti-cleaved caspase 8, anti-cleaved caspase 9, anti-Bax and anti-Bcl-2) (Proteintech, China) at 4 ℃ in a single day after which incubated with secondary antibodies at room temperature for 1 h. Lastly, the expression of various proteins was visualized by BIO-RAD ChemiDoc XRS chemiluminescence system (Bio-Rad Inc., CA, USA).
Animal experiment
With the approval of the Institutional Animal Care and Use Committee of Yi Shengyuan Gene Know-how (Tianjin) Co., Ltd., male Balb/C nude mice on the age of 4 weeks had been bought from HFK Co. (Beijing, China). We constructed the PC3 subcutaneous tumor mannequin by inoculating the collected PC3 cells (2 × 106) with Matrigel in an equal quantity ratio into the left belly subcutis of every mouse. The therapeutic intervention was carried out 2 weeks after mannequin institution, and equal volumes of PBS (15 µl), BP (20 µg), RNA2 (12 µg) and BP (20 µg)-RNA2 (12 µg) had been handled through intratumoral injection at 4 days intervals for a complete of 4 instances, respectively. The entire course of of fabric preparation and injection adopted the sterile precept. The size and diameter of the tumor had been measured with vernier calipers after which the tumor development curve was calculated and plotted in keeping with the formulation. On the thirty fifth day, the mice had been sacrificed and the tumors had been harvested for weight measurement and histological examination.
Tunel staining
Tumor tissues had been sectioned and stained with CoraLit-594 TUNEL Assay Apoptosis Detection Equipment (Proteintech, China) for mobile apoptosis detection. All of the steps had been carried out beneath the steering of the equipment and the cells present process apoptosis had been noticed by CLSM.
Immunohistochemistry staining
Paraplast-fixed tumor tissues had been sectioned, deparaffinized, dehydrated, and antigen retrieved in citrate buffer (pH = 6.0). The sections had been then incubated with totally different major antibodies (anti-PTEN, anti-Ki67, anti-p62, anti-caspase 8, and anti-Bcl-2) (Proteintech, China) at 4 ℃ in a single day. The sections had been then incubated with goat anti-rabbit IgG H&L coupled to horseradish peroxidase (HRP; Abcam) for 30 min at room temperature, and protein expression was visualized by 3-3-diaminobenzidine (DAB) staining and noticed beneath an optic microscope.
Statistical evaluation
Statistical evaluation was carried out utilizing GraphPad Prism 10 software program and introduced as imply ± SD. Comparisons had been carried out utilizing an unbiased t-test or one-way ANOVA check and variations between teams had been thought of statistically vital at P < 0.05.
