Development of a Non‐Coding‐RNA‐based EMT/CSC Inhibitory Nanomedicine for In Vivo Treatment and Monitoring of HCC

Abstract The objective of this study is to improve the overall prognosis of patients with hepatocellular carcinoma (HCC); therefore, new therapeutic methods that can be used in vivo are urgently needed. In this study, the relationship between the quantities of microRNA (miR)‐125b‐5p in clinical specimens and clinicopathological parameters is analyzed. A folate‐conjugated nanocarrier is used to transfect miR‐125b‐5p in vivo and to observe the therapeutic effect on HCC. The inhibitory effect and mechanism of miR‐125b‐5p on hepatoma cells are also studied. Data from clinical specimens and in vitro experiments confirm that the miR‐125b‐5p quantity is negatively correlated with progression, and the target protein that regulates the epithelial–mesenchymal transition (EMT)/cancer stem cells (CSC) potential in HCC is STAT3. The miR‐125b‐5p/STAT3 axis inhibits the invasion, migration, and growth of HCC via inactivation of the wnt/β‐Catenin pathway. miR‐125b‐5p‐loaded nanomedicine effectively inhibits the EMT/CSC potential of hepatoma cells in vivo together with their magnetic resonance imaging (MRI) visualization characteristics. An HCC‐therapeutic and MRI‐visible nanomedicine platform that achieves noninvasive treatment effect monitoring and timely individualized treatment course adjustment is developed.


Real-time quantitative RT-PCR (qPCR)
Total RNA was extracted and purified using TRIzol reagents (Invitrogen, Carlsbad, CA) and RNeasy mini kit (Qiagen Inc, Valencia, CA) according to the manufacturer's instructions. The relative mRNA levels were determined by reverse transcription and qPCR with Sybr Green dye (Invitrogen) as described previously. [2] For miRNA analysis, cDNAs were synthesized using the PrimeScript RT reagent kit (Takara). Then, qPCRs were performed by using the Takara SYBR Premix Ex Taq II (Takara) and the 7500 Real-Time PCR System (Applied Biosystems). Quantitative real-time PCR (qRT-PCR) analysis of miR-125b-5p was performed by using a commercially purchased kit specifically designed for quantifying this miRNA

Western blotting analysis
Three groups of HCCLM3 and HUH7 were harvested 24 h after transfections respectively. Equal amounts of protein lysates (30 g) were separated by 10 %

SDS-PAGE for immunoblots with primary antibodies. A horseradish
peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin-G antibody was used as the secondary antibody. Signals were detected using enhanced chemiluminescence reagents (Amersham Biosciences). All the experiments were independently performed three times as the least. Information and dilution of antibodies used in this study were listed in Supplementary Table 1.

Plasmids and stable cell lines
The pre-miR-125b-1 was synthesized by Invitrogen and ligated into the EcoR I/ Bgl II sites of the pMSCV-neo vector. For establishment of miR-125b-5p overexpression stable cell lines, pMSCV-neo-miR-125b-1 plus with pVPack-GP and pVpack-VSV-G were co-transfected into 293T cells to package viral particles. The viruses were collected and infected HCCLM3 and HUH7 cells. 48 h after infection, the cells were selected with G418-containing (500 g/ml) medium for at least 10 days.
Then the expression of miR-125b-5p was detected by quantitative real-time PCR. The expression of miR-125b-5p was defined based on the threshold cycle (Ct), and relative expression levels were calculated as 2 -[(Ct of miR-125b-5p) -(Ct of U6)] after normalization with reference to expression of U6 small nuclear RNA. After validating the successful overexpressing miR-125b-5p, the cells were maintained in a low concentration of G418 (200 g/ml) containing medium and the expression of miR-125b-5p was assessed monthly.

Wound-healing assays
For wound-healing assays, transfected cells were plated in six-well plates, and cells were maintained in serum-free medium. When the HCCLM3 and HUH7 including miR-125b-5p group, negative control group and blank control group confluence reached 80-90%, a scratch was made using a 10-l pipette tip, after serum starved for 24 h. The wounded monolayer was washed with PBS and imaged 48 h after scratching using an Olympus camera system. Wound healing assays were conducted in triplicate.

Cell invasion assay
For invasion assays, three groups of HCCLM3 and HUH7 were resuspended in 200 l of serum-free DMEM medium and seeded onto Boyden chambers (Corning, NY, USA) coated with Matrigel. The chambers were then incubated in DMEM with 20% FBS at 37 °C in 5% CO 2 . After 24 h, the cells adhering to the chamber's lower surface were fixed, whereas cells remaining on the upper surface were removed. After staining in a dye solution containing 0.05% crystal violet, the cells from three randomly selected high power fields were counted under a microscope (Olympus, Tokyo, Japan). Invasion assays were conducted in triplicate.

Tube formation assay
The HUVEC cells commercially obtained from Vec Technologies (Rensselaer, NY), and the tube formation assay was carried out as described. [3] In brief, 96-well plates were coated with 50 l Matrigel. 2 × 10 4 HUVEC cells were added to each well and incubated respectively in medium preconditioned with three groups of HCCLM3 and HUH7 cells at 37°C for 6-8 h. Images were acquired under an inverted microscope. Angiogenesis potential was quantified by measuring the length of tube walls formed between discrete endothelial cells in each well relative to the control.
Tube formation assays were conducted in triplicate.

Flow cytometry
Three groups of HCCLM3 and HUH7 were digested using trypsin and resuspended at 1×10 6 cells per mL in DMEM that contained 2% FBS and pre-incubated at 37°C for 30 min with or without 100 M verapamil (Sigma-Aldrich, 1711202) to inhibit ABC transporters. Cells were subsequently incubated for 90 min at 37°C with 5 g/ml Hoechst 33342 (Sigma-Aldrich B2261). Then, cells were incubated on ice for 10 min and washed with ice-cold PBS before flow cytometry analysis. Data were analyzed using Summit 5.2 software (Beckman Coulter).

Sphere formation assay
Various cells (1000 cells per well) were plated onto a 24-well ultra-low attachment plate (Corning) in serum free DMEM/F12 medium supplemented with N2 plus media supplement (Invitrogen), 20 ng/ml epidermal growth factor (Invitrogen), 20 ng/ml basic fibroblast growth factor (Invitrogen) and 4 mg/ml heparin

Synthesis and characterization of delivery agents
The Folate-conjugated block copolymer, Fa-PEG-g-PEI, was synthesized according to the previously described method. [4] Briefly, HO-PEG-NH 2 [5] and folic acid (1 g) were dissolved in anhydrous DMSO (20 mL). N-hydroxysuccinimide (NHS, 0.9 g) and dicyclohexylcarbodiimide (DCC, 0.5 g) were added and the mixture was stirred overnight at room temperature. Under an argon atmosphere, the mixture was 14000 Da) in distilled water for 3 day and then lyophilized. Nontargeted mPEG-g-PEI was synthesized using the same approach.

Preparation of magnetic nanocarriers (Fa-PEG-g-PEI-SPION)
SPION was complexed with Fa-PEG-g-PEI by the ligand exchange method as reported. [6] In brief, Fa-PEG-g-PEI (200 mg) was dissolved in 2 mL of chloroform, and SPION (10 mg) was dispersed into the solution. The reaction mixture was stirred overnight at room temperature before precipitating into large amount of hexane. The precipitate was collected by centrifugation, washed twice with hexane and vacuum-dried. The product thus prepared was dispersed into double distilled water under sonication and filtered through a 220 nm membrane to remove large aggregates.

Nanomedicine formation.
miR-125b-5p or negative control-miR plasmid (4 mg) and an appropriate amount of nanocarrier were dissolved separately in 0.9% sodium chloride solution. The two solutions were mixed by vigorous pipetting and then kept at room temperature for 60 min to allow polyplex formation. The amount of delivery agent (Fa-PEG-g-PEI-SPION) to complex plasmid was based on the designed experimental N/P ratio (10), which was calculated as the number of nitrogen atoms in delivery agents over that of the phosphate groups in plasmids.

In vitro and vivo MRI
The HCCLM3 or HUH7 cells cultured in vitro were imaged using a clinical 1.5-T

Fluorescence study on in vitro cellular
To enable confocal laser scanning microscopic (CLSM) observation of the cells after incubation, the nanocarrier Fa-PEG-g-PEI-SPION and pDNA were labeled with Oregon Green 488 and POPO-3, respectively, according to the manufacturer's protocols. [7] HCCLM3 or HUH7 cells were seeded at a density of 5×10 5  calibration. [8,9] Prussian blue staining In addition, some slides of HCCLM3 or HUH7 tumors were pretreated with an antigen retrieval method by heating the slides in an autoclave in citrate buffer (10 mM, pH 6.0) for 90 seconds except those stained for STAT3 and -catenin. EDTA-Tris buffer (1 mM, pH 9.0) was used for pretreating before staining. After rinsing in TBS (pH7.6), the specimens were incubated for 2 h at 37°C with anti-STAT3 or anti--catenin antibody. Subsequently, all slides were incubated with Envision HRP antibody working fluid (Dako Company) for 30 minutes at 37°C, and then developed with DAB-H 2 O 2 solution (Dako Company). The cell nuclei were stained with Meyer's hemotoxylin.

Statistical analysis
The data are expressed as the means ± standard deviation (S.D.). Experimental data were analyzed by Student's t-tests for two groups and two-way analysis of variance for multiple groups. All statistical tests were performed using SPSS 16.0 software (SPSS, Inc., Chicago, IL), and statistical significance was set at P < 0.01.