Simultaneous Targeting of Multiple oncomiRs with Phosphorothioate or PNA-Based Anti-miRs in Lymphoma Cell Lines

Abstract

Purpose

MicroRNAs (miRNAs) are short (~ 22 nts) RNAs that regulate gene expression via binding to mRNA. MiRNAs promoting cancer are known as oncomiRs. Targeting oncomiRs is an emerging area of cancer therapy. OncomiR-21 and oncomiR-155 are highly upregulated in lymphoma cells, which are dependent on these oncomiRs for survival. Targeting specific miRNAs and determining their effect on cancer cell progression and metastasis have been the focus of various studies. Inhibiting a single miRNA can have a limited effect, as there may be other overexpressed miRNAs present that may promote tumor proliferation. Herein, we target miR-21 and miR-155 simultaneously using nanoparticles delivered two different classes of antimiRs: phosphorothioates (PS) and peptide nucleic acids (PNAs) and compared their efficacy in lymphoma cell lines.

Methods

Poly-Lactic-co-Glycolic acid (PLGA) nanoparticles (NPs) containing PS and PNA-based antimiR-21 and −155 were formulated, and comprehensive NP characterizations: morphology (scanning electron microscopy), size (differential light scattering), and surface charge (zeta potential) were performed. Cellular uptake analysis was performed using a confocal microscope and flow cytometry analysis. The oncomiR knockdown and the effect on downstream targets were confirmed by gene expression (real time-polymerase chain reaction) assay.

Results

We demonstrated that simultaneous targeting with NP delivered PS and PNA-based antimiRs resulted in significant knockdown of miR-21 and miR-155, as well as their downstream target genes followed by reduced cell viability ex vivo.

Conclusions

This project demonstrated that targeting miRNA-155 and miR-21 simultaneously using nanotechnology and a diverse class of antisense oligomers can be used as an effective approach for lymphoma therapy.

Introduction

MicroRNAs (miRNAs or miRs) are short endogenous non-coding RNAs that bind to complementary target messenger RNA (mRNA) and regulate protein translation by either degradation of the target mRNA or inhibition of mRNA translation [1]. miRNAs play a key role in physiological processes, cellular division, proliferation, and survival [2]. miRNA levels are dysregulated in several diseases like cancer and disorders such as cardiovascular, diabetes, immune and neurodegenerative disorders due to mutations, deletions, amplification, or downregulation of the enzymes involved in miRNA biogenesis [3]. The miRNAs involved in cancer pathogenesis are classified as oncogenic or tumor suppressive. In particular, the miRNAs that are upregulated in and promote cancer progression are called oncomiRs [4]. These oncomiRs can act to make the cancerous cells immortal and proliferative, promote sustained angiogenesis, resist cell death, evade immune surveillance, and promote tissue invasion and metastasis [5].

MiR-21 and miR-155 are well established oncomiRs that are dysregulated in various cancers. MiR-21 is upregulated in several cancers, including glioblastoma, lymphoma, breast cancer, colorectal cancer, pancreatic cancer, lung cancer, and ovarian cancer [6-9]. Increased miR-21 levels downregulate various targets, such as PDCD4 (programmed cell death protein 4), FOXO1 (forkhead box protein O1), SPRY2 (sprouty RTK signaling antagonist 2), PTEN (phosphatase and tensin homolog), RECK (reversion-inducing cysteine-rich protein with Kazal motifs), and TIMP3 (metalloproteinase inhibitor 3), which results in increased cell proliferation, metastasis, migration, and reduced apoptosis of cancer cells [10]. Similarly, miR-155 is upregulated in breast, colon, and lung cancer as well as various hematological malignancies [11]. The elevated miR-155 level is responsible for cell proliferation, tumor growth, and inhibition of apoptosis by reducing the expression of targets like BACH1 (BTB Domain and CNC Homolog 1), FOXO3 (forkhead box O3), SHIP1 (SH2 containing inositol phosphatase), and SOCS (suppressor of cytokine signaling protein) [12]. In two independent studies, the direct relationship of miR-21 and miR-155 with lymphoma has been established [13, 14]. MiR-21 inhibits the expression of tumor suppressor proteins, PTEN and FOXO1 by activating the PI3K–AKT–pathway and causes chemoresistance [15-17]. MiR-155 inhibits the expression of tumor suppressor proteins like SHIP, FOXO3, and SOCS [12].

These miRNAs can be targeted using synthetic antisense oligonucleotides (ASOs). ASOs bind sequence specifically to the target miRNAs by Watson–Crick base pairing. Since the miRNAs are bound to the ASOs, the miRNAs cannot act on the target mRNAs and affect protein expression [18]. The initial attempts to deliver ASOs systemically or locally were not successful. Also, large dose requirements and instability of the ASOs were the other hurdles [19]. Various chemical modifications have been incorporated into the ASO design to protect against endo and exonuclease-based degradation and to increase the binding affinity of the ASOs to their targets [20]. Encapsulating the antimiRs in polymers also protects the antimiRs against enzymatic degradation [21].

In this research work, we compared the anti-tumor efficacy of two different classes of nucleic acid analogs: phosphorothioates (PS) and peptide nucleic acids (PNAs) that can simultaneously target miR-21 and miR-155. In the PS modification, the phosphate group's non-bridging oxygen atom is substituted with a sulfur atom, which improves ASO stability [22-24]. On binding with the target miRNA, a PS modified ASO recruits RNase H1 enzyme and cleaves the target miRNA. The negative charge of the PS favors binding to serum proteins, thereby increasing the in vivo circulation time [25, 26]. Whereas, the PNA ASO consists of a neutral backbone of N-2 aminoethyl glycine units to which the nucleobases are attached by methyl carbonyl linker [27]. Like the PS ASO, PNA ASOs are also stable against enzymatic degradation [28]. PNA ASOs exert their antisense effect by steric hindrance. PNA ASOs have a neutral charge, which favors the binding of PNA to negatively charged DNA or RNA with ease. However, this neutral charge also limits the cellular uptake of the PNA ASO molecules [29, 30]. Recently, many delivery platforms, such as polymeric nanoparticles, lipid conjugates, and lipid nanoparticles, have been explored to encapsulate the ASOs to improve cellular uptake [31].

Here, we used PLGA (polylactic-co- glycolic acid)-based nanoparticle formulations to deliver both PS- and PNA-based antimiR-21 and 155. PLGA is a biodegradable and FDA-approved polymer that has been used to deliver several small drug molecules and macromolecules [32, 33]. We performed a comprehensive characterization of formulated Poly-Lactic-co-Glycolic acid (PLGA) nanoparticles (NP)s and established that targeting both miR-21 and miR-155 by two different classes of antimiRs decreases lymphoma cell viability. Overall, we present a novel platform where PS or PNA based antimiR ASOs can be employed to target both miRNAs together, and resulting in decreased cell viability in cell culture studies.

Materials and Methods

PNA Synthesis

Boc monomers (Adenine, Guanine, Cytosine, Thymine) were purchased from ASM chemicals (Hannover, Germany). All the monomers were vacuum dried before using for the synthesis. Lysine loaded resin was synthesized as per protocol cited in literature [34]. Around 100 mg lysine loaded resin was soaked in dichloromethane (DCM) for 5 h. The DCM was drained from the reaction vessel and the synthesis was started by deprotecting the resin using trifluoroacetic acid (TFA):m-cresol (95:5) three times for 5 min. For the coupling reaction, the monomer was dissolved in a mixture of N-methyl-2-pyrrolidone (NMP), N, N-diisopropylethylamine (DIEA), and O-Benzotriazole-N, N, N’, N’-tetramethyl-uronium-hexafluoro-phosphate (HBTU). The monomer solution was added to the reaction vessel and the reaction was continued for 2 h. Kaiser test was used to confirm the success of the coupling step. The unreacted amino groups were capped using a mixture of NMP:pyridine:acetic anhydride. The resin was washed with DCM 8 times followed by deprotection using TFA. The above consecutive steps were repeated until the last monomer was added. All the PNA sequences contain one lysine group at 3' and 5' end. The PNA was cleaved from the resin using cleavage cocktail (m-cresol, thioanisole, TMFSA, and TFA at 1:1:2:6) for 1.5 h. The cocktail solution was collected from the vessel and the PNA was precipitated using diethyl ether. The PNA was centrifuged at 3,500 rpm for 5 min at 4°C. The PNA was washed with diethyl ether for two additional times. The diethyl ether supernatant was decanted and the PNA sediment was vacuum dried overnight. The PNA pellet was reconstituted using water: acetonitrile (ACN) and purified using reverse phase high performance liquid chromatography (HPLC). The absorbance of the PNA solution was measured at 260 nm using Nanodrop One (Thermofisher Scientific, City, MA, USA). Extinction coefficient of individual monomers (13,700 M⁻¹ cm⁻¹ (A), 6,600 M⁻¹ cm⁻¹ (C), 11,700 M⁻¹ cm⁻¹ (G), and 8,600 M⁻¹ cm⁻¹ (T)) of the sequences was used to calculate PNA concentration.

Formulation of PLGA Nanoparticles

The NPs were formulated by double emulsion solvent evaporation technique. Nuclear localization signal (NLS) peptide was purchased from New England Peptide (Gardner, MA, USA). NLS was used to improve the loading of PS in acid terminated PLGA polymer. The ratio of PS:NLS used was 1:5. About 40 mg PLGA polymer was dissolved in 0.5 ml DCM. Forty nanomoles PS was added to 200 μl NLS and mixed. This encapsulant was added dropwise to a PLGA DCM polymer solution with continuous stirring followed by probe sonication (10 s × 3 cycles) to form the primary oil in water (o/w) emulsion. In case of PNA, the 40 nmol of PNA was added to an ester terminated PLGA DCM solution dropwise with continuous stirring and then probe sonicated (10 s × 3 cycles) to form primary o/w emulsion. This o/w emulsion was added dropwise to 1 ml 5% w/v PVA solution followed by probe sonication (10 s × 3 cycles) to form w/o/w double emulsion. This double emulsion was added to 10 ml 0.3% w/v PVA solution and left overnight for the DCM to evaporate. The NPs were washed with cold water by centrifuging at 9,500 rpm for 10 min at 4°C to remove the excess PVA. The NP were redispersed in 5 mg/ml trehalose solution and then freeze dried.

Dynamic Light Scattering (DLS)

The hydrodynamic diameter and polydispersity index (PDI) of the NPs were evaluated using Zetasizer Nano ZS (Westborough, MA, USA) based on DLS technique. Zeta potential of the NPs was also determined by Zetasizer Nano ZS (Westborough, MA, USA). To prepare the samples for the measurements, NPs (n = 3) were dispersed in 1,000 μl water by vortexing briefly, followed sonication for 5 mins.

Scanning Electron Microscopy

Dry NPs were gently smeared on carbon tape adhered on a stub and sputter coated with palladium for 2 min. The images were captured under high vacuum at 2 kV accelerating voltage. The dry size of the NPs was calculated using ImageJ software (Bethesda, MD, USA).

Release Profile

0.3 ml phosphate-buffered solution (PBS) was added to 2 mg NPs and agitated at 300 rpm at 37°C. Samples were withdrawn at 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72, 96, and 120 h by centrifuging the NPs at 15,000 rpm for 10 min at 4°C. The NP pellet was redispersed in 0.3 ml fresh PBS and agitated at 300 rpm at 37°C. NP samples from three different batches were used for the release study. The absorbance of the supernatant was measured at 260 nm wavelength using Nanodrop One (ThermoFisher, Waltham, MA, USA).

Loading Study

To 2 mg of NPs, 0.2 ml DCM was added and the NPs were agitated at 1000 rpm for 4 h. Next, 0.2 ml 1X TE buffer was added and the NPs were further agitated for 2 h. The tube was centrifuged at 15,000 rpm for 10 min at 4°C. The supernatant was carefully withdrawn from the tube without disturbing the pellet. The absorbance of the supernatant was measured at 260 nm wavelength using Nanodrop One (ThermoFisher, Waltham, MA, USA).

Confocal Microscopy

Approximately 50,000 HeLa cells were seeded overnight on a coverslip in a 24 well plate. The cells were treated with 1 mg/0.5 ml NPs for 24 h. In case of simultaneous treatment, 1 mg PS-21 FITC NPs and 1 mg PS-155 TAMRA NPs were added. The cells were then washed with PBS twice to remove the excessed NPs. The cells were fixed with 4% formaldehyde for 10 min, followed by washing with PBS. The cells were then permeabilized with 0.1% Triton X for 10 min and then washed with PBS. The cells were stained using DAPI in mounting medium and left to dry overnight. The slides were imaged using 60 × oil lens on a Nikon (A1R) confocal microscope.

To stain the membrane, around 50,000 HeLa cells were seeded overnight in a 8 well chamber slides. The cells were treated with 1 mg PS-21 FITC NPs and 1 mg PS-155 TAMRA NPs. The cells were washed with media twice after the treatment duration. The cells were incubated in membrane stain (CellMask plasma membrane stain, Invitrogen) at 37°C for 20 min. The cells were then washed with media twice and then incubated in hoechst dye at 37°C for 20 min. The cells were then washed with media twice and then imaged using 60 × oil lens on a Nikon (A1R) confocal microscope.

Flow Cytometry Analysis

Approximately 50,000 HeLa cells were seeded overnight in a 24 well plate. The cells were treated with 1 mg/0.5 ml NPs for 24 h. The cells were washed with PBS twice to remove the excessed NPs and then trypsinzed. The cells were fixed using 4% formaldehyde and analyzed using LSR Fortessa X-20 Analyzer (BD Bioscience, Franklin Lakes, NJ, USA ). The data were processed in FlowJo analysis software.

Real Time PCR (RT-PCR)

Approximately 100,000 DLBCL cells (SUDHL-2 or SUDHL-5 cells) were seeded in a 12 well plate. The cells were treated with 1 mg/0.5 ml NPs for 24 h. In case of simultaneous treatment, 1 mg antimiR-21 NPs and 1 mg antimiR-155 NPs were added. The cell pellet was collected by centrifuging at 1,000 rpm for 4 min at 4°C. The RNA was extracted from the cell pellet using a Qiagen RNA extraction kit following the manufacturer protocol. The cDNA library was prepared using RT buffer, reverse transcriptase (50 U/ml), RNase inhibitor (20 U/μl), dNTPs (100 mM), nuclease free water, and the RT primers for miR-21, miR-155, and U6.

The cDNA was amplified using the miR-21, miR-155, U6 assays and universal master mix, and detected using CFX Connect RT-PCR detection system (Bio-Rad, Hercules, CA, USA). RT random primers were used for the cDNA preparation of downstream target (BACH1, FOXO3, SHIP, PTEN, FOXO1, PDCD4) and the cDNA was amplified using the respective mRNA assays.

Cell Viability by Trypan Blue Assay

Approximately 2000 DLBCL cells (SUDHL-2 or SUDHL-5 cells) were seeded in a 96 well plate and treated with 0.1 mg/100 μl NPs for 24 h. For the simultaneous treatment, the cells were treated with 0.1 mg antimiR-21 NP and 0.1 mg antimiR-155 NP dose. After 24 h, the dead cells were stained using trypan blue dye and counted using cell counter (Bio-Rad, USA).

Cytotox Assay

Approximately 2000 live SUDHL-5 cells were seeded in a 96 well plate and treated with 0.2 mg/100 μl of NPs for 24 h. The cells were treated simultaneously with 0.2 mg/100 μl of each antimiR-155 and antimiR-21 NP dose. After 24 h, the CytoTox-assay reagent was added and incubated for 15 min at room temperature. The luminescence signal was read on a Tecan microplate reader (infinity 200 pro).

Statistical Analysis

The statistical analysis was performed using GraphPad prism 9 software (version 9.2). Unpaired two tailed t test was used for statistical significance.

Results

Design of antimiR PS and PNA Oligomers

We purchased full-length PS targeting miR-21 and miR-155 (Fig. 1B. PS-21 and PS-155) commercially. We used three PS modifications at each end of the purchased oligomers. To determine the transfection efficiency of formulated NPs, we also used fluorophores, FITC, and TAMRA conjugated PS-21 and PS-155, respectively. We synthesized PNAs targeting miR-21 and miR-155 (Fig. 1B. PNA-21 and PNA-155) using solid-phase synthesis-based protocols and performed quality control analysis by reverse phase HPLC. We also used scrambled PS (PS-21 scramble and PS-155 scramble) and scramble PNA (PNA-21 scramble and PNA-155 scramble) for our study.

Fig. 1.

(A) Chemical structure of DNA, phosphorothioate (PS), and peptide nucleic acid (PNA) monomeric units. (B) The PS and PNA sequences used in this study are shown. PS-21 and PNA-21 are designed to bind to miR-21 whereas the PS-155 and PNA-155 are designed to bind to miR-155. The PS modifications are present on the terminal three positions. PS-21 scramble, PNA-21 scramble, PS-155 scramble, and PNA-155 scramble are the controls with mismatched sequences.

PLGA NP Formulation and its Characterizations

To ensure cellular delivery of the antimiRs, we encapsulated the antimiRs in PLGA polymer. A prior study from our lab established that the acid terminated PLGA could efficiently encapsulate PS-based antimiRs and generate NPs of optimal physico-chemical properties(35). We formulated PLGA NPs containing PNAs using ester terminated PLGA NPs. The NPs showed spherical morphology as analyzed by scanning electron microscopy and an average dry size ~ 140 nm, measured by Image J software (Fig. 2A). Further, we performed dynamic light scattering studies and determined the hydrodynamic diameter of the PLGA NPs containing antimiR PS and PNAs in the range of 300–100 nm (Table I). Sonicating the NPs during sample preparation for DLS measurement did not influence the NP size (Fig. S1). The formulated NPs also showed a narrow size distribution poly dispersity index in the range of 0.2–0.3. Further, we assessed the surface charge density of PLGA NPs containing PS and PNAs. As expected, PLGA NPs containing PS and PNA antimiRs possess a negative zeta potential of −21 to −26 mV. We next evaluated the release of the PS and PNA antimiR-155 and −21 by incubating the NPs in the buffered saline at physiological temperature and measuring their UV–Vis absorbance at 260 nm. As expected, NPs showed a sustained release for 24 h (Fig. 2B). We also performed the loading studies to determine the amount of the antimiRs encapsulated in these NPs. The average loading was found to be in the range of 260 – 330 pmol/mg (Fig. S2, Table S8).

Fig. 2.

(A) SEM images of PLGA NPs containing indicated PS and PNA-based encapsulants. These images were taken at 10,000 magnification. The scale bar is 5 μm. (B) Cumulative release of PS or PNA antimiRs from PLGA NPs at given time points. N = 3, data plotted as mean ± standard error mean.

Table I.

Characterization of PLGA NPs containing PS and PNA for size, PDI and zeta potential

NP	DLS (nm)	PDI	Zeta potential (mV)
Control	367.3 ± 9.07	0.32 ± 0.08	−21.0 ± 2.55
PS-21	354.4 ± 26.8	0.33 ± 0.02	−21.2 ± 1.29
PS-155	378.2 ± 10.4	0.25 ± 0.02	−25.5 ± 1.97
Control	347.8 ± 6.78	0.21 ± 0.04	−22.9 ± 1.21
PNA-21	376.1 ± 43.2	0.25 ± 0.02	−25.6 ± 2.44
PNA-155	329.2 ± 22.4	0.17 ± 0.04	−23.9 ± 1.55

Cellular Uptake

Next, we studied the transfection efficiency of the PLGA NPs containing fluorophore-conjugated PS by confocal microscopy in HeLa cells. HeLa cells were incubated with indicated NPs for 24 h and then imaged (Fig. 3A). We observed substantial uptake of NPs containing PS-21 FITC and PS-155 TAMRA. We also noted the uniform distribution of green and red fluorescence of the cells treated with NPs containing PS-21 FITC and PS-155 TAMRA, respectively (Fig. S3). These results indicated that both PS-21 FITC and PS-155 TAMRA encapsulated NPs can simultaneously undergo cellular uptake (Fig. 3A). Further, we quantified these results in HeLA cells (Fig. 3B) by flow cytometry-based methods.

Fig. 3.

(A) Confocal images of HeLa cells. The images were taken after 24 h treatment with both PS-21 FITC NPs (green) and PS-155 TAMRA NPs (red). DAPI (blue) was used for nucleus staining and cell mask (pink) was used for membrane staining. (B) Flow cytometry analysis of the cellular uptake of indicated NPs in HeLa cells as shown by TAMRA and FITC intensity in the X and Y axis, respectively.

Effect on miR-155 and miR-21 Gene Expression

Next, we determined the effect of downregulating miR-155 and miR-21 in the SUDHL-2 and SUDHL-5 lymphoma cell lines treated with PLGA NPs by RT-PCR. (Fig. 4). In addition to treatment with individual NPs, we did simultaneous treatment with mixture of NPs containing both antimiR-155 and −21 to ensure that both antimiRs did not impact each other’s efficacy when treated together. After treatment of SUDHL-2 cell line with NPs containing PS-21, we noted ~ 60% downregulation of miR-21 levels. Similarly, SUDHL-2 cells treated simultaneously with PS-21 + PS-155 NPs also showed ~ 58% downregulation of miR-21. The cells treated with PLGA NPs containing scramble PS-21 did not show downregulation of miR-21 (Fig. 4A). We also assessed the gene expression level of miR-155 after incubating the SUDHL-2 cells with PLGA NPs containing PS-155. RT-PCR showed ~ 90% reduced miR-155 expression in the SUDHL-2 cells treated with PS-155 NPs and in the group treated simultaneously with PS-21 NPs and PS-155 NPs (Fig. 4B). These results indicate that treating cells with multiple antimiRs does not impact their cross-reactivity.

Fig. 4.

Knockdown efficacy of gene expression of miR-21 and miR-155 by PS and PNA NP treatments. (A) Relative gene expression of miR-21 levels in RNA extracted from SUDHL-2 cells after PS treatment. (B) Relative gene expression of miR-155 levels in RNA extracted from SUDHL-2 cells after PS treatment. (C) Relative gene expression of miR-21 levels in RNA extracted from SUDHL-2 cells after PNA treatment. (D). Relative gene expression of miR-155 levels in RNA extracted from SUDHL-2 cells after PNA treatment. (E) Relative gene expression of miR-21 levels in RNA extracted from SUDHL-5 cells after PS treatment. (F) Relative gene expression of miR-155 levels in RNA extracted from SUDHL-5 cells after PS treatment. (G) Relative gene expression of miR-21 levels in RNA extracted from SUDHL-5 cells after PNA treatment. (H) Relative gene expression of miR-155 levels in RNA extracted from SUDHL-5 cells after PNA treatment. Three replicates were used for this study and the data plotted as standard error mean. Unpaired t test was used for statistical analysis with *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Similarly, we evaluated gene expression of miR-21 and miR-155 in SUDHL-2 cells treated with PNA NPs (Fig. 4C and 4D). We observed a ~ 50% and ~ 60% decrease in miR-21 gene expression levels after treatment with PLGA NPs containing PNA-21 only and simultaneous treatment with PLGA NPs containing PNA-21 and PNA-155, respectively (Fig. 4C). We noted ~ 90% and ~ 95% miR-155 level knockdown for NPs containing PNA-155 only and simultaneously treatment of PNA-21 NPs and PNA-155 NPs, respectively (Fig. 4D). Consistent with PS based studies, we observed a similar miRNA knockdown for the individual treatment groups and the simultaneous treatment groups (Fig. 4C and 4D).

Further, we evaluated gene expression of miR-21 and −155 in SUDHL-5 cell lines treated with antimiRs. We observed ~ 70% and ~ 65% reduced miR-21 levels after treatment with PLGA NPs containing PS-21 only and after simultaneous treatment with PS-21 NPs and PS-155 NPs, respectively (Fig. 4E). We observed ~ 95% reduction in miR-155 after individual treatment of PS-155 NPs and ~ 90% reduction after simultaneous treatment with NPs containing PS-21 NPs and PS-155 NPs (Fig. 4F). Similarly, when SUDHL-5 cells were treated with PNA containing NPs, we observed ~ 50% and ~ 70% miR-21 knockdown for PLGA NPs containing PNA-21 only and simultaneous treatment with PNA-21 NPs and PNA-155 NPs, respectively (Fig. 4G). We observed ~ 90% miR-155 knockdown for individual treatment of PNA-155 NPs and concurrent treatment of PNA-21 NPs and PNA-155 NPs (Fig. 4H). We did not notice significant inhibition of miR-21 and miR-155 in cells treated with scrambled controls. We did not notice significant knockdown in miR-21 and miR-155 levels after treatment of SUDHL-5 cells with naked antimiR-21 and naked antimiR-155 (Fig. S4). This highlights the importance of encapsulating the antimiRs in NPs for facilitating cellular uptake.

Effect on Downstream Targets of miR-155 and miR-21

Further, we investigated the effect on the downstream targets after miR-21 and miR-155 inhibition. BACH1, FOXO3 and SHIP1 are the direct downstream targets of miR-155 [36, 37]. PTEN, FOXO1, and PDCD4 are the direct downstream targets of miR-21 [17, 38]. We noticed a 30% increase in the BACH1 levels, 30% increase in FOXO3 levels, and a 70% increase in SHIP1 levels in the SUDHL-2 cell lines treated simultaneously with PS-21 PLGA NPs and PS-155 PLGA NPs (Fig. 5A). We also noticed a 10% increase in the PTEN levels, 10% increase in FOXO1 levels and 30% increase in PDCD4 levels in the SUDHL-2 cell lines treated simultaneously with PS-21 NPs and PS-155 NPs (Fig. 5A). We observed a 30% increase in BACH1 levels, 40% increase in FOXO3 levels and a 10% increase in SHIP1 levels in SUDHL-2 cells simultaneously treated with PNA-21 NPs and PNA-155 NPs (Fig. 5B). We also observed a 20% increase in the PTEN levels, 10% increase in FOXO1 levels and 30% increase in PDCD4 levels after concurrent treatment with PNA-21 NPs and PNA-155 NPs (Fig. 5B). Next, we also evaluated the effect on downstream targets after treatment of NPs containing PS (Fig. 5C) and PNA (Fig. 5D) in the SUDHL-5 cell line. We observed a 30% increase in the BACH1 levels, 80% increase in FOXO3 levels, and a 30% increase in SHIP1 levels for the SUDHL-5 cells treated with PS-21 NPs and PS-155 NPs (Fig. 5C). We noted a 40% increase in the PTEN levels, 10% increase in FOXO1 levels and 10% increase in PDCD4 levels for the simultaneous treatment group of PS-21 NPs and PS-155 NPs (Fig. 5C). Similarly, with the concurrent treatment of PNA-21 NPs and PNA-155 NPs, we observed a 40% increase in the BACH1 levels, 10% increase in FOXO3 levels and a 30% increase in SHIP1 levels (Fig. 5D). We also observed a 20% increase in the PTEN levels, 30% increase in FOXO1 levels and 30% increase in PDCD4 levels for the simultaneous treatment group of PNA-21 NPs and PNA-155 NPs (Fig. 5D). Thus, simultaneous inhibition of miR-21 and miR-155 results in de-repression of multiple tumor suppressor target genes.

Fig. 5.

Gene expression levels of downstream targets of miR-155 (BACH1, FOXO3, and SHIP1) and miR-21 (PTEN, FOXO1, and PDCD4). (A) Relative gene expression of downstream targets in RNA extracted from SUDHL-2 cells after PS treatment. (B) Relative gene expression of downstream targets in RNA extracted from SUDHL-2 cells after PNA treatment. (C) Relative gene expression of downstream targets in RNA extracted from SUDHL-5 cells after PS treatment. (D) Relative gene expression of downstream targets in RNA extracted from SUDHL-5 cell after PNA treatment. Three replicates were used for this study and the data was plotted as standard error mean. Unpaired t test was used for statistical analysis *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Cell Viability

Next, we assessed the cell viability in SUDHL-2 and SUDHL-5 cell lines after treatment with PLGA NPs containing PS and PNA-based antimiRs using the trypan blue-based cell viability assay. Treatment with PLGA NPs containing PS-21 or PS-155 in SUDHL-2 cells showed a 65% and 75% decrease in cell viability, respectively. However, the simultaneous treatment of PS-21 NPs and PS-155 NPs showed an 85% decrease in cell viability (Fig. 6A). Consistent with the miR-155 and miR-21 gene expression analysis (Fig. 4) concurrent treatment of the two antimiRs showed a more significant reduction in cell viability. In the SUDHL-2 cell line, PLGA NPs containing only antimiR PNA-21 or PNA-155 antimiR reduced the cell viability by 20%. Whereas in the case of concurrent treatment with PNA-21 NPs and PNA-155 NPs, it results in a ~ 35% decrease in the cell viability (Fig. 6A). Similarly, in SUDHL-5 cells, treatment with PS-21 NPs or PS-155 NPs alone reduced the cell viability ~ 25% and 45%, respectively. Whereas simultaneous treatment with PS-21 NPs and PS-155 NPs reduced cell viability to 80% (Fig. 6B). Similarly, PNA-21 NPs and PNA-155 NPs reduced cell viability by 50% and 60%, respectively. Simultaneously, treatment with PNA-21 NPs and PNA-155 NPs showed a decrease in cell viability by ~ 75% (Fig. 6B). We also evaluated the cellular viability in SUDHL-5 cells after equal dose treatment for the individual treatment group and the simultaneous treatment group. The results suggest that simultaneous targeting of two different miRNAs results in decreased cell proliferation of lymphoma cells (Fig. S5). We also performed the cytotoxicity assay, which detects the luminescence signal from the dead cells. PS-21 and PS-155 showed ~ 70% and ~ 60% cell viability in SUDHL-5 cells. The simultaneous treatment with PS-21 NPs and PS-155 NPs showed a cell viability of 50% (Fig S6A). Similarly, PNA-21 NPs and PNA-155 showed a cell viability of ~ 65% in SUDHL-5 cells. Simultaneous treatment with PNA-21 and PNA-155 NPs showed a cell viability of ~ 60% (Fig. S6B). Similar to the gene expression results for the naked oligos, the treatment of SUDHL-5 cells with naked PS or PNA oligos did not show a reduction in cellular viability (Fig. S7A, Fig. S7B).

Fig. 6.

Cell viability by the trypan blue assay. (A) Cell viability after PS NP and PNA NP treatment in SUDHL-2 cells. (B) Cell viability after PS NP and PNA NP treatment in SUDHL-5 cells. Three replicates were used for the study and the data plotted as standard error mean. Unpaired t-test was used for statistical analysis with *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Discussion

Diffuse large B cell lymphoma (DLBCL) is one of the aggressive forms of Non-Hodgkin lymphoma. A combination of a cocktail of chemotherapeutic drugs, rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP), is the standard therapy for treating lymphoma [39]. Options for patients refractory to the chemotherapy treatment and with relapse of lymphoma still need to be addressed [40]. Therefore, there is an urgent need for alternative treatment options to treat fast proliferating and aggressive lymphoma.

Several anti-miRs have gained significant attention in precision medicine-based therapeutics. AntimiR-21 is in phase I clinical trial for the treatment of Alport syndrome [10, 41]. Similarly, Cobomarsen is a locked nucleic acid antimiR-155 currently in Phase II clinical trial for the treatment of cutaneous T cell lymphoma (CTCL) and mycosis fungoides (MF) [42, 43]. A single miRNA can bind to multiple mRNA targets and control the gene expression. To date, several studies have shown a single miRNA inhibition for therapeutic interventions [43-45]. However, various miRNAs are involved in signaling pathways that regulate neoplastic transformation. Therapies aimed at inhibiting multiple oncomiRs allow the de-repression of several downstream targets compared to those that target a single oncomiR. In addition, considering tumor heterogeneity, single miRNA targeting can saturate the miRNA-based therapeutics as other oncomiRs can still increase the disease progression. Hence, it would be adequate to target multiple miR-based targets instead of single miRNAs. This strategy can inhibit other oncomiRs in other cancers, thus providing a means to tailor the treatment to the oncomiRs overexpressed in a particular cancer patient. Therefore targeting multiple miRNAs seems to be a promising approach to suppressing tumor growth [46].

Here, we tested the hypothesis that targeting two miRNAs simultaneously de-represses the expression of multiple tumor suppressive proteins that eventually lead to better phenotypic outcomes. Therefore, in this study, we targeted miR-21 and miR-155 simultaneously that could be useful for potential lymphoma therapy. We designed anti-miR-21 and antimiR-155 complementary to miR-21 and miR-155, respectively. We evaluated two classes of nucleic acid analogs; PS and PNA. PS exerts its antisense action by RNase-mediated cleavage, while PNA exerts action by steric hindrance. Our and other results indicated that naked oligomers are challenging to transfect in the cells. For cellular delivery, we encapsulated antimiRs in PLGA NPs by the double emulsion solvent evaporation technique. Encapsulating antimiRs in the nanoparticles protect them from enzymatic degradation and facilitates their cellular uptake. The formulated NPs had a spherical morphology and were uniform in size. We extensively characterized these NPs for hydrodynamic size, size distribution, and surface charge density. The NP size was found to be in the size range of 300–400 nm, and the surface charge density was found to be in the range of −21 to −26 mV.

We found that NPs containing antimiRs in cell culture experiments were effective at delivery and engaged their targets to affect gene expression in two DLBCL cell lines: SUDHL-2 and SUDHL-5. By comparing the targeting of a single antimiR treated group's activity with the simultaneously targeted multiple antimiRs treated groups, we found that simultaneous targeting of two miRNAs is more effective as compared to targeting single miRNAs. We also noticed that BACH1, FOXO3, SHIP1, PTEN, FOXO1 and PDCD4 expression was de-repressed in the simultaneous treatment group.

Conclusions

In this study we evaluated phosphorothioate and peptide nucleic acid for targeting miR-21 and miR-155 and we observed a synergistic decrease in the viable lymphoma cells for the simultaneous treatment group. We tested that novel synthetic nucleic acids in conjunction with nanotechnology can be employed to simultaneously target multiple miRNAs with complementary functions for effective cancer therapy. Hence, here we establish that targeting multiple onco-miRNAs (miR-155 and miR-21) may achieve greater efficacy for potential lymphoma therapy. The future involves evaluating the efficacy of this treatment strategy in the lymphoma mouse model in preclinical studies. In addition to the xenograft mouse model, the safety and efficacy need to be tested in the transgenic mouse model. This work can provide novel avenues for treatment approaches for hard-to-treat cancers in the future.