Breast cancer cell lines MDA-MB-231 and MDA-MB-453 and normal mammary epithelial cells MCF-10A, 184B5 cells were purchased from Chinese Academy of Sciences Cell Bank (Shanghai, China).
MDA-MB-231, MDA-MB-453 were cultured in Leibovitz's L-15 medium (Gibco) supplemented with 100 units/mL penicillin streptomycin, 10% FBS (Gibco) and kept at 37 oC in humidified 100% atmosphere. 184B5 cells were cultured in DMEM (Gibco) supplemented with 10% FBS and antibiotics and kept at 37 oC, 5% CO2. MCF10A cells were cultured in MEGM (Lonza) supplemented with 100 ng/mL cholera toxin, supplements and growth factors (Lonza) (BPE, hEGF, Insulin, Hydrocortisone, GA-1000), and kept at 37 oC, 5% CO2.
Generation of 3WJ-EGFRapt-siXBP1 NPs
Multifunctional pRNA-EGFRapt-siXBP1NPs were constructed by a bottom-up self-assembly approach. Briefly, NPs were assembled by mixing the equal molar amounts of RNA fragments and gradually annealing from 95 to 4 ℃ in 1×RNA annealing buffer (TMS buffer) on a PCR machine.
The therapeutic pRNA-EGFRapt-siXBP1 is composed of four strands. Lowercase letters indicate 2'-F modified nucleotides, and other sequences are adopted as previously described.
Strand 1: 5'-uucuuucGAucucuGGcAGuu-3’;
Strand 2: 5'-cuGccAGAGAucGAAAGAAuuuuGccAuGuGuAuGuGGG-3’;
Strand 3: 5'-ccc AcA uAc uuu Guu GAu ccG ccu uAG uAA
cGu Gcu uuG AuG ucG Auu cGA cAG GAG Gc-3' (underlined sequence is EGFR aptamer);
Strand 4: 5'-GGAucAAucAuGGcAA(Cy5)-3’.
The control pRNA-EGFRapt-siScramble is composed of strands with siScramble sequence instead of siXBP1 sequence. Lowercase letters indicate 2'-F modified nucleotides. The siScramble sequence is as follows:
Characterization of pRNA-EGFRapt-siXBP1 NPs
The size and zeta potential of our NPs were determined by DLS. To determine the size of NPs, NPs were filtered by 0.22 μm filter and then 100 μL NPs were (20 μM) diluted into TES buffer. To determine the zeta potential, the above NPs were further diluted to 500 μL TES buffer. The Tm value was detected by a SYBR green assay, as previously described. Briefly, the NPs were assembled in 1×TMS buffer in the presence of 1×SYBR Green II dye (Invitrogen) with final nanoparticle concentration of 250 nM. The samples were heated to 95 °C for 5 min and then slowly cooled down to room temperature at a rate of 0.11°C/s using the Roche Lightcycler 96 real-time PCR machine. The melting temperature was obtained from at least three independent measurements. As for the stability assay, the 2'F modified NPs or unmodified 3WJ control NPs were exposed to different concentrations of RNase A (0, 10, 100 µg/mL), or 10% FBS supplemented DMEM medium at 37 oC, respectively. The assembly and stability of our NPs was examined through 8% native PAGE gel electrophoresis.
In vitro cell binding and uptake
Flow cytometry and confocal microscope were used to evaluate the cellular binding and uptake of NPs. To do this, MDA-MB-231 cells were grown on slides overnight in L15 medium and incubated with 50 nM Cy5-conjugated NPs for 24 hrs at 37 oC. After washing three times with pre-cooling PBS, cells were fixed and stained with cytomembrane dye Alexa488-wheat germ agglutinin (Thermo Fisher) and DAPI (Sigma). The slides were imaging using a Nikon A1 Confocal Microscope System. For flow cytometry analyses, 50 nM Cy5 labeled NPs were incubated with MDA-MB-231 cells for 1, 3, 6, 12 and 24 hr(s). After that, the cells were washed and analyzed by a Cytoflex flow cytometer (Beckman).
Specific binding in mixture systemin vitro
To evaluate the specific binding of NPs in vitro, Dio pre-staining MDA-MB-231 cells were mixed with the same amount of normal mammary epithelial cells (MCF10A and 184B5) or control tumor cells (MDA-MB-453) with slightly rotating. Following this, 50 nM Cy5-conjugated NPs were added to the mixture and further incubated at 37 oC for 2 hrs. After that, the mixture was collected and analyzed by flow cytometer. The binding efficiency was calculated using the following formula:
Binding efficiency = ratio of Cy5 positive MDA-MB-231 cells/ratio of total MDA-MB-231 cells in mixture ×100%. The NPs binding efficiency was also represented by the value of MFI of Cy5 positive cells.
Cell viability, cell cycle and apoptosis assays
MDA-MB-231 cells were treated with 50 nM pRNA-EGFRapt-siXBP1 NPs or pRNA-EGFRapt-siScramble control. The cells viability, cell cycle and apoptosis were analyzed after three days culture. Cell viability was measured using the CCK8 assay (Dojindo), following the manufacturers protocol. In a dox sensitivity experiment, MDA-MB-231 cells were treated with dox (2, 0.5 and 0.25 µg/mL, respectively) alone, or with 50 nM NPs for 72 hrs. Then the cell viability was measured using the same method. To analyze cell cycles, cells were incubated with NPs for 72 hrs. After that, cell pellets were collected, permeabilized with 70% (v/v) ethanol, and re-suspended in 1 mL of PBS containing 1 mg/mL RNase and 50 mg/mL propidium iodide (PI). They were then incubated in the dark for 30 min at room temperature and analyzed by Cytoflex Flow Cytometer (Beckman). The cell cycle distribution was evaluated on DNA plots using a MODFIT software. To test apoptosis, annexin V and 7AAD staining (Southern biotech) was performed by flow cytometry.
Colony formation assay
The soft agar colony formation assay was performed as previously described with slight modifications. 1×103 pRNA-EGFRapt-siXBP1 NPs treated breast cancer cells were transferred to 0.8 % agarose with the same volume ratio (1:1) in 10% growth medium to make a final concentration of 0.4 % agarose. The cell mixture was plated on top of 0.8 % bottom layer of agar in the 10% growth medium in 6-well plates. Cells were fed every 5 days for 20 days with 10% growth medium containing 0.4 % agarose. For the dox sensitivity experiment, the breast cancer cells were firstly treated with 0.01 µg/mL dox alone, or dox plus prepared NPs for 48 hrs. Once completed the treated breast cancer cells were mixed with 0.4% agarose and seeded to the top of a solidified layer for further incubation. The experiment was repeated in triplicate and the statistical significance was calculated using Student's t test.
Total RNA was extracted from cultured cells or tumor tissues using a RNeasy mini kit (Qiagen), and reverse-transcription was performed by using HiScript II QRT SuperMix for qPCR (Vazyme Bioteche). Real-time PCR was performed as described previously.
Cells were lysed using RIPA buffer (CST) supplemented with cocktail protease inhibitors (Roche) and PMSF. Minced tumor tissues were homogenized in liquid nitrogen in RIPA buffer. 30 µg of total protein were separated by SDS-PAGE and the separated proteins were transferred to PVDF membranes (Millipore) for western blotting analysis using anti-XBP1 (Abcam, ab37152); anti-β-actin (CST).
Orthotopic Xenograft Breast Tumor Mouse Model
Six-week-old female athymic nude mice were purchased from Beijing Vital River Laboratory Animal Technology, and all animal procedures were performed under IACUC-approved protocols at the Zhejiang Chinese Medical University. Athymic nude mice were orthotopically injected with 1 × 107 MDA-MB-231 cells mixed with Matrigel into the fat pads of the fourth pair of mammary glands. When the tumor sizes reached 100-150 mm3, the mice were randomly divided into five groups (six/group) and intravenously injected with pRNAs (3.3 nmol/mice) twice a week for 3 weeks. For dox treatments, dox (Sigma) (3 mg/kg) was intravenously injected into mice twice a week for 3 weeks and 1 day before pRNA injection. When the tumor sizes reached 1000 mm3 (that is 25 days post-injection in this study), mice were sacrificed, and the tumors were collected, then lysed for extraction of total proteins and RNAs, or fixed in 10% neutral buffered formalin, respectively, for further Western blot, real-time PCR and IHC studies, etc. The tumor volume was measured, and tumor size was calculated using the formula: volume = 0.5 × (width)2 × (length).
To examine the biodistribution of NPs within the tumor issues, O.C.T.-embedded frozen sections (5 µm) were examined by confocal microscopy. To analyze tumor cell blood vessels, tumor tissue samples were collected and fixed in 10% neutral buffered formalin, followed by dehydration via a gradient series of ethanol (75%, 85%, 95% and 100%) and embedded in paraffin following routine methods. For CD31 immunostaining, slides were blocked with 3% H2O2-methanol for 15 min at room temperature, treated with mouse anti-CD31 antibody (Abcam, ab28364) overnight at 4 °C, with horseradish peroxidase (HRP)-conjugated secondary antibody, and with 3, 3'-diaminobenzidine (DAB). The tissue sections were counterstained with hematoxylin for nucleus visualization. The slides were analyzed on an LSM 510 Meta confocal microscope.