Accepted Manuscript miRNA oligonucleotide and sponge for miRNA-21 inhibition mediated by PEIPLL in breast cancer therapy Shiqian Gao, Huayu Tian, Ye Guo, Yuce Li, Zhaopei Guo, Xiaojuan Zhu, Xuesi Chen PII: DOI: Reference:

S1742-7061(15)30018-0 http://dx.doi.org/10.1016/j.actbio.2015.07.020 ACTBIO 3788

To appear in:

Acta Biomaterialia

Received Date: Revised Date: Accepted Date:

19 March 2015 27 June 2015 9 July 2015

Please cite this article as: Gao, S., Tian, H., Guo, Y., Li, Y., Guo, Z., Zhu, X., Chen, X., miRNA oligonucleotide and sponge for miRNA-21 inhibition mediated by PEI-PLL in breast cancer therapy, Acta Biomaterialia (2015), doi: http://dx.doi.org/10.1016/j.actbio.2015.07.020

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miRNA oligonucleotide and sponge for miRNA-21 inhibition mediated by PEI-PLL in breast cancer therapy Shiqian Gaoa,c, Huayu Tiana, Ye Guob, Yuce Lia, Zhaopei Guoa, Xiaojuan Zhub,* , Xuesi Chena, * a

Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of

Sciences, Changchun 130022, China b

School of Life Science, Northeast Normal University, Changchun 130024, China

c

Graduate School of Chinese Academy of Sciences, Beijing 100039, China

ABSTRACT

MicroRNA-21 (miR-21) inhibition is a promising biological strategy for breast cancer therapy. However its application is limited by the lack of efficient miRNA inhibitor delivery systems. As a cationic polymer transfection material for nucleic acids, the poly (L-lysine)-modified polyethylenimine

(PEI-PLL)

copolymer

combines

the

high

transfection

efficiency

of

polyethylenimine (PEI) and the good biodegradability of polyllysine (PLL). In this work, PEI-PLL was successfully synthesized and confirmed to transfect plasmid and oligonucleotide more effectively than PEI in MCF-7 cells (human breast cancer cells). In this regard, two kinds of miR-21 inhibitors, miR-21 sponge plasmid DNA (Sponge) and anti-miR-21 oligonucleotide (AMO), were transported into MCF-7 cells by PEI-PLL respectively. The miR-21 expression and the cellular physiology were determined post

transfection. Compared with

the negative control,

PEI-PLL/Sponge or PEI-PLL/AMO groups exhibited lower miR-21 expression and cell viability. The anti-tumor mechanism of PEI-PLL/miR-21 inhibitors was further studied by cell cycle and western blot analyses. The results indicated that the miR-21 inhibition could induce the cell cycle arrest in G1 phase, upregulate the expression of Programmed Cell Death Protein 4(PDCD4) and thus active the caspase-3 apoptosis pathway. Interestingly, the PEI-PLL/Sponge and PEI-PLL/AMO also sensitized the MCF-7 cells to anti-tumor drugs, doxorubicin (DOX) and cisplatin (CDDP). These results demonstrated that PEI-PLL/Sponge and PEI-PLL/AMO complexes would be two novel and promising gene delivery systems for breast cancer gene therapy based on miR-21 inhibition. Keywords:Breast cancer therapy, Polyethylenimine, Polylysine, MiRNA-21 sponge, MiRNA-21 AMO 1. Introduction 1

Breast cancer (BC) is one of the most common cancers in women, accounting for 25% of the all cases [1]. Traditional treatments for BC include surgery, radiation therapy and chemotherapy. However, the surgery or radiation therapy has side effects which did harm to the health of the patients, and the recent studies have reported that chemotherapy could contribute to tumor recurrence [2]. Gene therapy is a nascent but one of the most promising treatments for BC in recent years [3]. Among the therapeutic genes, microRNAs (miRNAs) are highly conserved small non-coding RNAs which effectively regulate over 30% of protein-coding genes by imperfect pairing with the target mRNAs [4]. Each miRNA can adjust the expression of hundreds of genes which contribute to several biologic processes including proliferation, apoptosis, differentiation, and moreover act as oncogene or tumor suppressor during tumor formation [4, 5]. MiRNA-21 (miR-21) is a typical overexpressed miRNA in BC cells and solid tumor [6]. Previous reports showed that miR-21 endowed the oncogenic activity including high proliferation, low apoptosis, and high invasion ability to the BC cells [7, 8]. The down-regulation of miR-21 expression has been proven to be a rational therapeutic approach for BC through regulating the target genes expression which related to the cell cycle, proliferation or apoptosis pathways, and subsequently led to a synergistic anticancer effect with other chemical drugs [6, 9]. Traditional strategy to achieve function-loss of miRNA utilized anti miRNA oligonucleotide (AMO) which is complementary to the mature miRNA strand. However, the poor pharmacokinetic profile of oligonucleotide restricted its systemic applications [10]. MiRNA "sponge" is a sequenced plasmid DNA that encoding several copies of the complement of the target miRNA. It is a novel technique for miRNA down-regulation [11]. After transfection into the cells, the miRNA sponge transcribes into the encoding sequence which will absorb the target miRNAs through the multiple binding sites just like a sponge. Then the compounds will saturate the miRNA-induced silencing complexes (miRISC) and further regulate the activity of the target miRNA [12]. Although the chemical structure of miRNA sponge is more stable than AMO, the naked oligonucleotides or plasmids exposed to cell lysates and serum nucleases would still be rapidly degraded [13]. Moreover, the negative charge of the oligonucleotides or plasmids also excluded their interaction with the cell membrane, resulting in poor cellular uptake [14]. To overcome these barriers, many delivery strategies have been developed, such as the chemical modification of AMO and the application of carriers [10]. Polyethylenimine (PEI) is one of the most widely utilized and studied synthetic material for DNA or siRNA delivery [15-17]. As a non-viral delivery system, the cationic PEI could condense the negatively charged nucleic acids by electrostatic interaction [3]. However, the non-degradability restricted the application of PEI in vivo [18, 19]. To improve these capacities of PEI, a poly(L-lysine)-modified polyethylenimine (PEI-PLL) 2

copolymer had been established and utilized as an efficient gene carrier to transfect therapeutic plasmid DNA into the human cervical cancer cells and induce the tumor apoptosis both in vitro and in vivo in our previous work [20]. In this work, the PEI-PLL copolymer was synthesized based on the previous work [20]. The ability of PEI-PLL to complex with AMO and Sponge to form nanoparticles was confirmed by testing the particle size, zeta potentials and nucleic acids binding ability of the complexes. The best transfection ratio of PEI-PLL to the oligonucleotide and plasmid were determined by the cytotoxicity, in vitro transfection efficiency, and cell internalization measurement. The miR-21 AMO or Sponge was transfected into MCF-7 cells (human breast cancer cells) by PEI-PLL in this ratio and the bioactivities of the transfected MCF-7 cells were evaluated through the assay of cell proliferation, and chemical drug sensitivity (Scheme 1). Moreover, the cell cycle distribution and the expression profile of genes downstream to miR-21 were evaluated to reveal the underlying anticancer mechanism.

Scheme 1. Schematic illustration of formation, cellular uptake, intracellular fate and biological effects of the PEI-PLL/Sponge and PEI-PLL/AMO complex particles. 2. Materials and methods 2.1. Materials

Branched PEI (Mw = 25 kDa) was purchased from Aldrich (St. Louis, MO, USA). Benzyloxycarbonyl-L-lysine was prepared as reported by Daly [21, 22]. Trifluoroacetic acid was 3

purchased from GL Biochem Ltd. (Shanghai, China). Hydrobromic acid in acetic acid 33% (v/v) was purchased from ACROS. Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Japan). Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, USA). Cell lysate and the luciferase reporter gene assay kit were purchased from Promega (Mannheim, Germany). The BCA protein assay kit was purchased from Pierce (Rockford, IL, USA). Primary antibody anti-PARP1 was purchased from Ebioscience (USA), anti-cleaved PARP1 from Abcam (England), anti-PDCD-4 and anti-caspase-3 from Cell Signaling Technology, anti-α-tublin from Sigma (USA). Secondary antibody HRP-conjugated rabbit anti-mouse IgG and goat anti-rabbit IgG were also purchased from Sigma (USA). Anti-hsa-miR-21 oligonucleotide (AMO), AMO negative control (AMO-NC), and FAM labelled AMO negative control (AMO-FAM) were synthesized by GenePharma (China). Three copies of anti-miRNA-21 oligonucleotide sequence and negative control sequence were cloned into miRNA Sponge vector (C08004, GenePharma, China), resulting in miR-21 inhibition plasmid (Sponge) and its negative control plasmid (Sponge-NC). The plasmid encoding firefly luciferase (pGL3) was purchased from Promega (Mannheim, Germany). 2.2. Synthesis and characterization of PEI-PLL

ε-benzyloxycarbonyl-L-lysine N-carboxyanhydride (Lys(Z)-NCA) was synthesized as reported by Daly [26, 27]. PEI-PLL was obtained by the ring-opening polymerization reaction of Lys(Z)-NCA by PEI in anhydrous chloroform solution described in our previously work[20]. Briefly, PEI (700 mg) and Lys(Z)-NCA (700 mg) were respectively dissolved in anhydrous chloroform (50 ml). After thorough resolution, the Lys(Z)-NCA solution was added to the PEI solution and the mixture was stirred up for 72 h at 30°C. Then the products were concentrated and precipitated by excess diethyl ether and the obtained PEI-PLys(Z) was filtrated, followed by drying under vacuum at room temperature for 24 h. For deprotection of benzyloxycarbonyl groups, PEI-PLys(Z) (1.0 g) was dissolved in trifluoroacetic acid (10 mL) at room temperature followed by mixing with hydrobromic acid in 33% acetic acid (v/v) (3 mL). The mixture was added dropwise to an excess of ethyl ether 2 h later. The yellow solid was dried under vacuum at room temperature overnight and then dissolved in MilliQ water and dialyzed against MilliQ water before lyophilization (molecular weight cutoff (MWCO) = 7000 Da, Shanghai Green Bird Science & Technology Development Co., Ltd., Shanghai, China). 1H NMR was performed on a Bruker AV 400 NMR spectrometer (Bruker, Ettlingen, Germany) in CF3COOD at room temperature. 2.3. Polymer/nucleic acids complexes characterization 4

The binding efficiency of the carriers with nucleic acids (Sponge or AMO) was studied through gel retardation. 10 μL polymer/Sponge and polymer/AMO with different weight ratios were prepared. After 20 min incubation at room temperature, the complex solutions were analyzed by 1% agarose gel electrophoresis. Sponge or AMO bands were visualized by a UV illuminator using a UVP EC3 bioimaging system (Upland CA, USA). The zeta potentials, particle sizes and the polydispersity index (PDI) of the PEI-PLL/AMO, PEI/AMO, PEI-PLL/Sponge and PEI/Sponge complexes at the different weight ratios were determined by a Zeta Potential/BI-90Plus Particle Size Analyzer (Brookhaven, USA) after 20 min of incubation at room temperature. Atomic force microscopy (AFM) imaging of the complexes was performed by 5500 AFM (Agilent Technologies, Chandler, AZ). 2.4. Cell culture

MCF-7 and HeLa (Human cervical cancer cells) cells were purchased from Shanghai Cell Bank of the Chinese Academy of Sciences, and 293T cells (human embryonic kidney cells) was purchased from Invitrogen Corporation (USA). The cells were cultured in DMEM supplied with 10% (v/v) FBS at 37°C in 5% CO2 (v/v) (Thermo Forma, USA). 2.5. Cell viability

Cell viability was determined by CCK-8. MCF-7 cells were seeded into 96-well plates at 1.0 ×104 cells well-1 in DMEM (200 μL) containing 10% FBS for 24 h and then incubated with the polymer/plasmid or polymer/oligonucleotide complex particles at the different concentrations for another 48 h. CCK-8 solution (20 μL/well) was added 4 h before measurement. The absorbance at 450 nm of each well was recorded by an ELISA micro plate reader (Bio-Rad Laboratories, Hercules, CA, USA). The cell viability (%) was calculated as follows: Cell viability (%) = (Asample/Acontrol) × 100 Asample and Acontrol are the absorbance of the cells treated by the complexes and the untreated cells. Each experiment was repeated in triplicate. 2.6. Transfection efficiency

The transfection efficiency of PEI-PLL particles in vitro was evaluated in MCF-7 cells compared to PEI ones. The cells were seeded into 96-well plates at the density of 1.0×104 per well 5

24 h before transfection in DMEM (200 μL) containing 10% FBS. The pGL3 plasmid (0.2 μg) was added to the polymer solutions at different weight ratios. The complex particles solutions were mixed and incubated for 15 min at room temperature and added to cells. The medium was replaced by fresh complete medium 6 h later and maintained for another 48 h at 37°C. The luciferase (LUC) expression was determined by a Promega Luciferase Assay system and normalized by the total protein contents of the cells evaluated by the Micro BCA Protein Assay Kit (Pierce). 2.7. Cellular internalization

For cellular uptake analysis, MCF-7 cells (2.0×105 per well) were seeded into 6-well plates for 24 h and then transfected with PEI-PLL/AMO-FAM or PEI/AMO-FAM complex particles. The cells were harvested by trypsin at 0, 4, 8, and 24 h post transfection and washed with phosphate buffered saline (PBS) once. The internalization efficiency was determined by a flow cytometer (FACS Caliber, Becton-Dickinson, San Jose, CA, USA). 2.8. Quantification of mature miR-21 expression

To measure the expression of mature miR-21, the cells in 6-well plates were washed with PBS twice and total RNA was extracted with a TRIzol reagent (Invitrogen) following the instruction. After appropriate drying, the RNA was resolved by diethylpyrocarbonate-treated water and reversely transcribed into cDNA by using a Hairpin-itTM miRNA reverse transcription kits (GenePharma, Shanghai, China). The qRT-PCR was carried out using hairpin-it™ miRNAs qPCR quantitation kits (GenePharma, Shanghai, China) according to the protocol. The relative expression of mature miR-21 was evaluated by the ΔΔCT method and normalized to the expression of U6 RNA, which was the endogenous control in the corresponding groups [23]. Same treatments were carried out for the measurements of the miR-21 silence efficiency of the polymer mediated miR-21 inhibitors 48 h post transfection. All the measurements were performed in triplicates. 2.9. Cell cycle assay

MCF-7 cells (2.0×105 per well) were seeded into 6-well plates in 2 ml of the culture medium and allowed to adhere for 24 h. The cells were treated with PBS, or transfected with miR-21 inhibitors (AMO, Sponge) or miR-21 inhibitor negative controls (AMO-NC, Sponge-NC) by PEI-PLL carriers for 48 h. The cells were then trypsinized and fixed in cold 70% ethanol overnight, followed by staining by propidium iodide and analyzed with a flow cytometer immediately (FACS Caliber, Becton-Dickinson, San Jose, CA, USA). 6

2.10. Western blots

Cells were seeded into 6-well plate and transfected with two kinds of miR-21 inhibitors or negative controls for 48 h. The total cellular protein was extracted with RIPA buffer containing a protease inhibitor. The supernatant was removed and collected after centrifuging at 12, 000 rpm for 15 min. The protein concentration was detected by a BCA protein assay kit (Thermo Scientific, Rockford, USA). Approximately 40 mg protein was loaded into 12% polyacrylamide SDS-PAGE gel and transferred to PVDF membranes. The membranes were blocked with 5% BSA solution for 1 h, and immunoblotted overnight at 4°C with primary antibodies (anti-PARP1, anti-cleaved PARP1, anti-PDCD-4, anti-caspase-3, and anti-β-actin) in tris-buffered saline (TBS) overnight. After three time washes with TBST (TBS with 0.1% tween), the membrane was incubated with secondary antibodies (HRP-conjugated goat anti-rabbit IgG or rabbit anti-mouse IgG) at room temperature for 1 h. After another three washes with TBST, the signal was detected by ECL kit (GE, London, UK). 2.11. Statistics

The data were recorded as mean standard deviation (SD) of triplicate groups (n = 3). The data differences were statistically determined by one way analysis of variance (ANOVA). A P-value < 0.05 was considered to indicate significant differences. 3. Results

3.1. Characterization of PEI-PLL

The chemical structure and the 1H NMR spectrum (Fig. 1) of PEI-PLL copolymer were resembled as the previous study [20]. The characteristic signal of l-lysine residue at 1 – 2 ppm (–CH–CH2CH2CH2–), 2.7 ppm (–CH2–NH2) and, 4.0 ppm (–CH–) and PEI at 2.8 – 3.5 ppm (–CH2CH2NH–) confirmed the chemical structure of PEI-PLL. The molar ratio of PEI and l-lysine residue calculated by 1H NMR was 1:90, close to the feeding ratio of PEI and Lys(Z)-NCA, which was determined in our previous work for the best transfection activity in MCF-7 cells. The structure of PEI-PLL was further confirmed by FT-IR absorption spectrum (Fig. 2). The typical C=O stretching absorption at 1676 cm-1 and CON-H stretching absorption at 2061 cm-1 demonstrated the chemical structures of PLL moieties of PEI-PLL. 7

Fig. 1. 1H NMR spectra of PEI-PLL chemical structure.

Fig. 2. FT-IR spectrum of PEI-PLL copolymer. 3.2. Cytotoxicity of the polymers and polymer/nucleic acids complexes

High dose of cationic polymers will induce cytotoxicity and restrict their application in transfection, so CCK-8 assay was firstly utilized to select the appropriate concentration of the polymers. As shown in Fig. 3A, both PEI and PEI-PLL showed the dose dependent increase of cytotoxicity. However, PEI-PLL displayed relative lower toxicity than PEI to MCF-7 cells. Plasmid and oligonucleotide are different kinds of nucleic acids, so the toxicity of polymer/Sponge-NC plasmid particles (Fig. 3B) or polymer/AMO-NC oligonucleotide particles (Fig. 3C) at different weight ratios was measured in the same plasmid or oligonucleotide concentration (0.2 mg/L) respectively. For PEI-PLL copolymer solution, the ratios of 10/1 and 5/1, showed significant less toxicity than the ratio of 15/1 or the other higher ratios to the cells, and were selected to conduct the further gene transfection experiments. As a positive control, two low toxicity ratios (5/1 and 2.5/1) were utilized for PEI experiments.

8

3.3. In vitro gene transfection activities

Luciferase assay was used to evaluate the plasmid transfection efficiencies of PEI and PEI-PLL to MCF-7 cells. PEI-PLL (10/1, 5/1) and PEI (5/1, 2.5/1) at lower toxicity ratios were utilized to determine their transfection efficiencies. PEI-PLL at the ratio of 10/1 showed much higher transfection ability than the ratio of 5/1 of PEI-PLL or the other ratios of PEI (Fig. 3D). So 10/1 was selected as the best transfection ratio for PEI-PLL to conduct the rest transfection experiments. Though the higher weight ratio (15/1) could induce higher transfection efficiency (data not shown), this ratio was abandoned because of its higher cell toxicity as illustrated in Fig. 3B.

Fig. 3. (A) Viability of MCF-7 cells exposed to various polymer concentrations for PEI and PEI-PLL. (B) Viability of MCF-7 cells exposed to various weight ratios of polymer and Sponge-NC plasmid complexes. (C) Viability of MCF-7 cells exposed to various weight ratios of polymer and AMO-NC oligonucleotide complexes. (D) Transfection efficiencies of PEI/LUC-pDNA and PEI-PLL/LUC-pDNA in MCF-7 cells (*p < 0.05).Data represent mean ± standard deviation (n = 3). 3.4. Cellular internalization activities.

PEI-PLL with the best ratio of 10/1 had been proven to be a better plasmid carrier than PEI. To evaluate whether this ratio had a good cellular internalization activity for oligonucleotide, a kind of 9

FAM labeled oligonucleotide (AMO-FAM) was utilized to transfect into MCF-7 cells. Cellular internalization activity was measured at different time point(2 h, 4 h, 8 h and 24 h)after transfection by flow cytometric analysis. As shown in Fig. 4, the FAM fluorescence signal in cells transfected with the PEI-PLL/AMO-FAM complexe particles (10/1) was much higher than that of the PEI/AMO-FAM ones (5/1) at the time points of 2 h, 4 h and 8 h post transfection. The fluorescence signal of the cells started to fade after 8 h, and there was no significant difference between the uptake activities of the both polymers at 24 h. This was probably due to the quenching of the FAM fluorescent group after 24 h. Based on the above results, PEI-PLL copolymers showed higher oligonucleotide transportation ability than PEI.

Fig. 4. (A) Cellular uptake analysis of PEI/AMO-FAM complexes and PEI-PLL/AMO-FAM complexes at different time point post transfection by flow cytometry in MCF-7 cells: PBS blank, gray; PEI, red; PEI-PLL, blue. (B) Mean fluorescence intensity analysis of FAM by flow cytometry. 10

3.5. Characterization of polymer/nucleic acids complexes

Characteristics such as particle size and zeta potentials both affect the transport efficiency and toxicity of the polymer/nucleic acid complex particles. So the size and zeta potentials were studied for the complex particles of PEI-PLL and PEI in respective best transfection ratios. As shown in Fig. 5A, both PEI-PLL/Sponge-NC plasmid and PEI-PLL/AMO-NC oligonucleotide complex particles had a relatively lower but still positive surface charge compared with the complex particles of PEI. Previous studies have shown that cells can take up the nanoparticles ranging from fifty to several hundred nanometers [24]. From the results in Fig. 5B, it was found that the complexes of PEI-PLL/Sponge-NC plasmid (PDI 0.141) and PEI/Sponge-NC plasmid (PDI 0.091) had a resemble particle size (~ 140 nm). For AMO-NC oligonucleotide, there was also no significant difference between PEI (PDI 0.131) and PEI-PLL (PDI 0.112) complexes in particle size (~ 300 nm). Above results suggested that both PEI-PLL and PEI could condense plasmid or oligonucleotide and formed the positive charged particles ranging from 100 nm to 300 nm. This characteristic would enhance the ability of cells to take up the nanoparticles. The particle sizes of the complexes were further confirmed by AFM imaging as shown in Fig. S1. To confirm the ability of PEI-PLL to condense plasmid and oligonucleotide, the gel retardation assay was utilized at various weight ratios, and PEI was used as the control. As shown in Fig. 6, both PEI and PEI-PLL could totally complex with plasmid to form particles when the weight ratios were higher than 1/1. However, PEI-PLL could complete oligonucleotide retardation at the ratio of 1/1 which is higher than the ratio of PEI (0.5/1).

Fig. 5. Zeta potential (A) and particle size (B) of PEI/Sponge-NC plasmid complexes, PEI-PLL/ Sponge-NC plasmid complexes, PEI/AMO-NC oligonucleotide complexes, PEI-PLL/AMO-NC oligonucleotide complexes in each best transfection ratio (*p < 0.05). Data represent mean ± standard deviation (n = 3).

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Fig. 6. Gel retardation assay for complexes of (A)PEI/Sponge-NC, (B)PEI/AMO-NC, (C) PEI-PLL/Sponge-NC and(D)PEI-PLL/AMO-NC at various polymer/nucleic acid weight ratios. 3.6. MiR-21 expression in vitro.

In this study, miR-21 expression was profiled in breast cancer cell line MCF-7, cervical cancer cell line HeLa and normal human embryonic kidney 293T cells by qRT-PCR (Fig. 7A). The higher expression levels of miR-21 in both MCF-7 (106 folds) and HeLa (70 folds) cancer cells were detected than 293T normal cells, and this was in correlation with the previous studies [7]. PEI-PLL could effectively transported plasmid and oligonucleotide into MCF-7 in vitro and miR-21 was overexpressed in MCF-7. Thus, a plasmid expression vector which including several miR-21 binding sites (Sponge) was constructed and the anti-miR-21 oligonucleotide (AMO) was synthesized to inhibit the expression of miR-21 in MCF-7. PEI-PLL was used to deliver Sponge and AMO into MCF-7 cells and the miR-21 expression was detected at 48 h post transfection by qRT-PCR. As illustrated in Fig. 7B, both PEI-PLL/Sponge and PEI-PLL/AMO complex particles could significantly reduce miR-21 expression level by ~80% in comparison with the negative group (P < 0.001).

12

Fig. 7. (A) MiR-21 expression level in 293T, HeLa and MCF-7 cell lines by qRT-PCR. (B) MiR-21 expression level in MCF-7 cells 48 h after transfection with PEI-PLL mediated Sponge, Sponge-NC, AMO or AMO-NC by qRT-PCR (***p < 0.001). Data represent mean ± standard deviation (n = 3). 3.7. Effects of AMO or Sponge complexes with PEI-PLL on cell viability

To evaluate whether the down regulation of miR-21 induced by PEI-PLL/AMO complex particles would correlate with any changes in cell viability, the CCK-8 assay was utilized for the cell viability 48 h after the transfection with different concentration of AMO or AMO-NC. As shown in Fig. 8A, 200 mM of AMO showed the most significant reduction effect on cell viability when compared with the negative group AMO-NC. In this consideration, 200 mM was selected as the final concentration of AMO to conduct the rest experiments. The viability of the cells transfected with AMO or Sponge complexes with PEI-PLL at different time point was also detected. As shown in Fig. 8B, a significant decrease in the percentage of viable cells was observed when cells were transfected with miR-21 Sponge (51.5 ± 6.5%) or AMO (47.7 ± 2.4%) at 48 h. PEI-PLL/Sponge-NC or PEI-PLL/AMO-NC also showed certain cytotoxicity, which was due to the low cell density and the toxicity of material itself. When the transfection time prolonged to 72 h, 96 h, and 120 h, with the increase of cell density, the cell viability of the Sponge-NC and AMO-NC transfection groups would also increase. Interestingly the cell survival percentage of Sponge or AMO transfection group was maintained at ~50%. The Sponge transfection group reached the lowest cell viability (36 ± 5%) at 120 h post transfection.

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Fig. 8. (A) Effect of different concentration (10 nM to 250 nM) of AMO or AMO-NC with PEI-PLL on cell viability (*p < 0.05); (B) Effect of Sponge, Sponge-NC, AMO or AMO-NC complex particles with PEI-PLL on cell viability at different time point (48 h, 72 h, 96 h, 120 h) post transfection by CCK-8 assay. Data represent mean ± standard deviation (n = 3). 3.8. Cell cycle analyze of miR-21 knocking down cells

Aiming to evaluate whether the reduction of cell viability would correlate with cell cycle arrest, the cell cycle assay was measured after the transfection with these two miR-21 inhibitors by PEI-PLL. As shown in Fig. 9, there was no significant cell cycle distribution difference between blank and PEI-PLL/Sponge-NC groups, however PEI-PLL/Sponge groups induced a small, but significant arrest in G1 phase (from 51.4 ± 1.9% to 63.5 ± 4.1%) in comparison with the negative control. PEI-PLL/AMO particles could also induce G1 phase arrest, but the variation (from 50.7 ± 1.1% to 58.8 ± 4.5%) was weaker than PEI-PLL/Sponge group. Above results suggested that the miR-21 down regulation after transfection with PEI-PLL/Sponge or PEI-PLL/AMO could induce cell cycle G1 arrest, and this arrest could explain the cell viability reduction which had shown in the previous results (Fig. 8).

Fig. 9. The cell cycle distribution after PEI-PLL mediated Sponge, Sponge-NC, AMO, or AMO-NC transfection in MCF-7 cells by flow cytometry. Data represent mean ± standard deviation (n = 3). 14

3.9. Western blot

Some studies have shown that miR-21 could target tumor suppressor gene PDCD4, which plays an important role in the caspase-3 apoptosis pathway, and promote its degradation [25, 26]. In this regard, to evaluate whether the down regulation of miR-21 induced by PEI-PLL/Sponge and PEI-PLL/AMO particles would correlate with PDCD4 or other caspase-3 pathway relate gene expression in MCF-7 cells, western blot was utilized after the transfection with these two miR-21 inhibitors. As shown in Fig. 10, both PEI-PLL/Sponge and PEI-PLL/AMO particles could enhance PDCD4 expression in comparison with respective NC groups. Then, the expressions of full-length caspase-3, PARP, and cleaved PARP, three key components of caspase-3 apoptosis pathway were detected after the same treatments. The results showed that the high expression of PDCD4 could attenuate the expression of full-length caspase-3. The lack of full-length caspase-3 could lead to downstream cleavage substrate PARP and our results confirmed the decrease expression of PARP and the increase expression of cleaved PARP after transfection with Sponge or AMO in comparison with the NC groups or the blank groups. In total, our results suggested that these two miR-21 inhibitors/PEI-PLL complex particles could active the caspase-3 apoptotic pathway by the down regulation expression of PDCD4.

Fig. 10. Western blot detection of endogenous control Tubulin, and several apoptosis related proteins: PDCD4, Caspase-3, PARP, and Cleaved PARP expression in MCF-7 cells after transfection with Sponge, Sponge-NC, AMO, or AMO-NC carried by PEI-PLL. 15

3.10. Chemical drug sensitivity

Recent studies reveal that miR-21 may modulate sensitivity of lung cancer cells to chemotherapy [27]. The CCK-8 assay was utilized to test if the PEI-PLL mediated miR-21 Sponge or AMO complex particles would have any synergistic anti-tumor effects with two FDA approved chemotherapy drugs, doxorubicin (DOX) and cisplatin (CDDP), in MCF-7 cells. As shown in Fig. 11A and Fig. 11B, after the transfection with Sponge or AMO, the cells have lower viability compared with the respective NC transfection groups or the blank groups at the concentration less than 0.675 μg/ml for DOX, but this synergistic antitumor viability effect would disappear when the drug concentration increase to 1.25 μg/ml. This might be attributed to the susceptibility of MCF-7 cell to DOX, and drug toxicity would hold a dominant position in comparison with the Sponge or AMO transfection at relatively high concentrations. However, after the calculation of the 50% inhibitory concentration (IC50) of DOX in MCF-7, it was found that the Sponge groups would decrease the IC50 from 0.565 μg/ml to 0.247 μg/ml as compared with the negative groups. For AMO transfection groups, the IC50 would also decrease from 0.585 μg/ml to 0.415 μg/ml. These illustrated that the miR-21 inhibition induced by PEI-PLL/Sponge or AMO complexes could increase DOX susceptibility of MCF-7 cells. For CDDP, the synergistic effect was not as significant as DOX. The IC50 of CDDP would reduce from 1.29 μg/ml to 1,067 μg/ml after the transfection with Sponge in comparison with Sponge-NC groups (Fig. 11C, D). For AMO transfection groups, the IC50 would reduce to 0.940 μg/ml from 1.051 μg/ml. However the synergistic anti cell viability was also more apparent in the lower concentration of CDDP (≤0.168 μg/ml for Sponge, ≤0.675 μg/ml for AMO). Taken together, these results illustrated that the cells transfecting with Sponge or AMO were more sensitive to chemotherapy drugs than negative controls.

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Fig. 11. Cell viability in MCF-7 cells after transfection with Sponge or Sponge-NC, either per se or in combination with different concentration of DOX (A) or CDDP (C). Cell viability in MCF-7 cells after transfection with AMO or AMO-NC, either per se or in combination with different concentration of DOX (B) or CDDP (D). Data represent mean ± standard deviation (n = 3). 4. Discussion

PEI is an efficient non-viral nucleic acid carrier which is widely used in the research of cancer gene therapy [28]. As another common used transfection material, Poly (L-lysine) (PLL) could not condense DNA as effective as PEI, but prevailed because of its low toxicity in vivo [19]. Previous study had shown PEI-PLL could induce cervical cancer cell apoptosis after the transfection of the rev-caspase-3 pDNA both in vitro and in vivo [20]. Our study focused on that whether PEI-PLL could effectively deliver two different kinds of nucleic acids, oligonucleotide and plasmid into the breast cancer cells and achieve the anticancer effects in vitro. PEI modified transfection agents have a serious limitation because of its toxicity on cell viability. In formation of gene carriers, the weight ratio of cationic polymer and oligonucleotide or plasmid plays a critical role in transfection efficiency and cytotoxicity [29]. High weight ratio will not only increase cell transfection efficiency but also reduce the cell viability. So the cell viability assay was firstly utilized to select the appropriate concentration range for transfection in MCF-7. In our study, the best transfection weight ratio between PEI-PLL and oligonucleotide or plasmid was determined 17

as 10/1 which had less cytotoxicity and more plasmid transfection (Fig. 3) and oligonucleotide uptake efficiency (Fig. 4) than PEI. Interestingly, the particle size of PEI-PLL/ plasmid or PEI-PLL/oligonucleotide complexes had no significantly difference between those of PEI at each best transfection ratio, but both PEI-PLL and PEI/oligonucleotide complex particles have relative bigger diameter size (~300 nm) than the polymer/plasmid complex particles (~140 nm) (Fig. 5B). This would be attributed to the unstable structure of polymer/oligonucleotide complexes [28]. Next the zeta potentials of the complexes formed by polymers and pDNA or oligonucleotide were measured at the same transfection ratio (Fig. 5A). According to previous reports, the positive charge of the nanoparticle would improve the interaction with the negatively charged cell membrane, but also offer more cytotoxicity [18]. Our result showed that both PEI-PLL and PEI formed the positive nanoparticles with pDNA or oligonucleotide. In addition, it was also found that PEI-PLL mediated pDNA or oligonucleotide had a relative lower zeta potential in comparison with those of PEI, this result exactly explained the less toxicity of PEI-PLL complexes in Fig. 3. Interestingly, the minus ratio for PEI-PLL (1/1) to complete oligonucleotide retardation is higher than that of PEI (0.5/1). This may be due to the modification of PLL and reduces the surface positive charge of PEI, while the negative charge of oligonucleotide is weaker than plasmid and prone to detach from the electrostatic complex. Thus it is necessary to increase the PEI-PLL proportion to complete oligonucleotide retardation. MiR-21 has been widely studied as an oncogene in cancer development [7]. Our results also illustrated that miR-21 had a higher expression level in breast cancer cells MCF-7 in comparison with cervical cancer cells HeLa or normal cells 293T. Thus we proposed a hypothesis that the down regulation of miR-21 would have therapeutic effect towards MCF-7 cancer cells. To verify this assumption, miR-21 Sponge and AMO, two kinds of miR-21 target inhibitors, were respectively transported into MCF-7 cells by PEI-PLL at the best transfection ratio 10/1. The results in Fig. 7B illustrated that both of these two complex particles effectively inhibited the miR-21 expression in vitro. In addition, the results in Fig. 8 illustrated that the down regulation of miR-21 significantly induced cell viability reduction. Interestingly, the miR-21 Sponge transfection groups had a stronger anti-tumor via bioactivity when the transfection time prolonged to 120 h in comparison with the AMO group. This may be attributed to the Sponge plasmid transfected into the cells by PEI-PLL would inhibit miR-21 expression for a long term. In contrast the AMO oligonucleotide has an unstable structure and is more likely to be degraded by intracellular nuclease [13]. This result suggested that the PEI-PLL mediated miR-21 Sponge groups would be more suitable for further experiment in vivo. The viability reduction of the PEI-PLL mediated miR-21 inhibitors could result from cell cycle arrest [25]. As shown in Fig. 9, there was a significant increase of cell population in G0-G1 phase after transfected with miR-21 Sponge or AMO compared with each negative transfection groups. 18

During G0-G1 phase, the cells synthesis DNA and prepare for the further mitotic division. The arrest of G0-G1 may interfere with normal cell division and lead to cell viability reduction. Notably, the AMO transfection groups had a relative slight effect in cell cycle arrest when compared with the Sponge transfection groups. This just explained the former results of the different bioactivities of these two miR-21 inhibitors in cell viability reduction (Fig. 8). It has been widely reported that the anticancer bioactivity of miR-21 are relevant to the repression of a serious of cancer suppressor genes, such as Bcl-2, PTEN, TPM1 and PDCD4 [25, 30]. MiR-21 could target mRNA of these suppressor genes and block their further translation. In our study, the evidence was provided that the down-regulation of miR-21 induced by PEI-PLL/Sponge or PEI-PLL/AMO could increase the expression level of tumor suppressor PDCD4 by western blot assay. PDCD4 is a frequently disrupted cancer suppressor in breast cancer cells and has been shown to affect the cell viability, cell cycle and apoptosis [25]. The high expression of PDCD4 could active multiple components in caspase-3 or p53 apoptosis pathways [26]. So the expression of several key factors of these pathways was determined. Interestingly, the caspase-3/PARP activation increased after the transfection of these two miR-21 inhibitors by PEI-PLL particles. The increase expression level of the tumor suppressor PDCD4 and the activation of caspase-3 apoptosis signal pathway not only explained the reduction observed in the viability of MCF-7 cells, but also explained the increase sensitivity of MCF-7 cells to chemical drugs. In this regard, several studies have proven that the knocking down of miR-21 increases the cytotoxic effect of anticancer drugs in vitro and in vivo [31]. Our results, combining the miR-21 inhibitors with doxorubicin or cisplatin, also revealed this synergistic anticancer viability effect. Both doxorubicin and cisplatin are approved by the FDA for the treatment of breast cancer cells. But recent reports showed that the classic anticancer chemotherapy drugs would elicit a side effect to induce the hyper proliferation of cancer stem cells and further increase the tumor recurrence after the chemotherapy [2]. Many studies suggested that the cancer stem cell (CSC) characteristics are relevant with the miR-21 expression level. The inhibition of miR-21 may contribute to the suppress CSC phenotype of breast cancer [32]. For this consideration, the synergistic effect of miR-21 inhibitors and chemical drugs may have deeper application prospects in anti-cancer researches. 5. Conclusions In this study, a cationic transfection polymer PEI-PLL was successfully synthesized. It combined the following characteristics: the high plasmid transfection efficiency, the high oligonucleotide uptake efficiency and the low cytotoxicity. The particle size and zeta potential data demonstrated the plasmid and oligonucleotide condensation capability of PEI-PLL. The MiR-21 silence assay further confirmed the ability of PEI-PLL to transport two kinds of miR-21 inhibitors, 19

miR-21 Sponge and AMO, into the MCF-7 cells. In addition, PEI-PLL mediated Sponge or AMO will induce the cell viability reduction, and this probably due to the cell cycle arrest in G1 phase. These two kinds of miR-21 inhibitor complex particles also regulate the expression of PDCD4 and thus activate caspase-3 apoptosis pathway. Interestingly, the PEI-PLL mediated Sponge transfection would have more remarkable anti-tumor cells viability when combined used with DOX rather than CDDP. Our results revealed the great potential of PEI-PLL as the carrier in a plasmid or oligonucleotide deliver system in breast cancer cells. PEI-PLL/Sponge and PEI-PLL/AMO complexes would be two novel and promising gene delivery systems for breast cancer gene therapy based on miR-21 inhibition. Acknowledgements This work was financially supported by the Ministry of Science and Technology of China (International cooperation and communication program 2011DFR51090), the Project of National High Tech R&D Program (2014AA020708), the National Natural Science Foundation of China (Grant Nos. 51222307, 21474104, 51233004, 51390484 and 51321062), Jilin province science and technology development program (20130521011JH), Youth Innovation Promotion Association, CAS.

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Graphical Abstract

miRNA oligonucleotide and sponge for miRNA-21 inhibition mediated by PEI-PLL in breast cancer therapy.

MicroRNA-21 (miR-21) inhibition is a promising biological strategy for breast cancer therapy. However its application is limited by the lack of effici...
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