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Direct monitoring of cancer-associated mRNAs in living cells to evaluate the therapeutic RNAi efficiency using fluorescent nanosensor Seong Min Ahn, Seounghun Kang, and Dal-Hee Min ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b01498 • Publication Date (Web): 19 Apr 2019 Downloaded from http://pubs.acs.org on April 19, 2019

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ACS Sensors

Direct monitoring of cancer-associated mRNAs in living cells to evaluate the therapeutic RNAi efficiency using fluorescent nanosensor Seong Min Ahn,†,‡ Seounghun Kang,†,‡ and Dal-Hee Min*,†,‡,§ †

Department of Chemistry, Seoul National University, Seoul, 08826, Republic of Korea

‡

Center for RNA Research, Institute for Basic Science (IBS), Seoul, 08826, Republic of Korea

§

Institute of Biotherapeutics Convergence Technology, Lemonex Inc., Seoul, 08826, Republic of Korea

ABSTRACT: Cancer-associated messenger RNA (mRNA) is an important biomarker for early diagnosis, prognosis, and prediction of treatment responses. Despite recent developments in fluorescence live cell imaging, reliable detection and quantification of mRNA in living cells still remains challenging due to a complicated intracellular environment. Herein, we present a fluorescent nanosensor for live-cell monitoring of cancer-related mRNAs involved in the canonical Wnt/β-catenin signaling pathway. The nanosensor enables rapid and accurate assessment of gene down regulation efficiency in a dose- and time-dependent manner by measuring quantitative fluorescence signal corresponding to βcatenin or its target mRNA levels in living cells. It is expected that the fluorescent nanosensor will be applicable to highthroughput screening for the efficient drug discovery and insightful understanding of the molecular mechanisms of potential drug candidate for cancer treatment. KEYWORDS: fluorescent nanosensor, graphene oxide, livecell imaging, mRNA sensing, peptide nucleic acid, siRNA Of the topics in cancer research, understanding of cancerrelated signaling pathways has become increasingly important, which provides insights to elucidate their roles in tumorigenesis and to identify potential targets for cancer therapy.1-3 The canonical Wnt/β-catenin signaling pathway, one of the fundamental mechanisms involved in numerous cellular and physiological functions, regulates cell fate specification, proliferation, migration and apoptosis.4,5 It has been reported that aberrant Wnt/β-catenin signaling pathway was observed in many cancers, including liver, colon, breast, lung, and pancreas cancer.6-8 Hence, many studies have endeavored to elucidate pathophysiological mechanisms by which cancer initiation and progression is tightly linked to dysregulation of Wnt/β-catenin signaling pathway. In particular, the key components in Wnt/β-catenin signaling pathway have been suggested as biomarker or therapeutic target candidates for cancer diagnosis or therapy. Cancer-related mRNA has served as a biomarker to diagnose cancer, monitor cancer progression, and predict thera-

peutic response in cancer treatment.9,10 Various molecular biology technique such as reverse transcription polymerase chain reaction (RT-PCR), northern blot, and microarray have commonly been used to measure the mRNA expression level. However, these techniques are labor-intensive, timeconsuming, invasive, and non-intuitive. Accordingly, fluorescence imaging-based approaches have been developed for the detection and quantification of intracellular mRNAs in living cells with high spatial and temporal resolution.11-14 Recently, versatile nanoplatforms, composed of functional nanomaterials with quenching ability and novel fluorescent probes, have been proposed for early diagnosis, rapid prognosis assessment, and drug discovery.15-19 Despite the recent advances in live-cell visualization of intracellular mRNA, however, there remain issues to be solved, including background fluorescence signal, low-throughput processing, and biocompatibility. Herein, we designed fluorescent nanosensor for evaluating efficacy of siRNA or small molecule inhibitor as potential cancer therapeutics through direct monitoring of cancerrelated mRNAs involved in the Wnt/β-catenin signaling pathway (Figure 1). Our nanosensor consists of dextrancoated graphene oxide nanocolloid (D-GON) as effective quencher and carrier with low cytotoxicity and fluorescent peptide nucleic acid (PNA) probe with high affinity and spec-

Figure 1. Schematic illustration of the fluorescent nanosensor to evaluate the efficacy of potential therapeutics by visualizing and quantifying cancer-associated mRNA in living cells.

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ificity for target mRNA. The PNA probes can be absorbed onto D-GON via hydrogen bonding and pi-pi stacking interaction between aromatic ring of nucleobase in the probe and sp2 domain of D-GON surface. Meanwhile, the fluorescence of the probe is quenched by D-GON. After introduction of the nanosensor into live cells, the detachment of the probe occurs with the recognition of target mRNA and leads to restoration of the fluorescence. On the other hand, when target mRNA is selectively downregulated in response to siRNA or small molecule inhibitor treatment, the quenching of the fluorescence is kept constant Therefore, the fluorescence intensity should be attributed to variations in the expression level of target mRNA. First of all, D-GON was synthesized according to our previously reported method.20,21 UV-vis and Fourier-transform infrared (FT-IR) spectrum showed partial deoxygenation and restoration of sp2 domain, indicating that GON was functionalized by dextran polymer during reduction process (Figure S1A, B). The synthesized D-GON exhibited remarkably enhanced stability in comparison to bare GON in phosphate-buffered saline (PBS) solution and showed low cytotoxicity against cultured HepG2 cells (Figure S1C, D). The average hydrodynamic diameter was determined by dynamic light scattering (DLS) as 50.4 ± 0.5 nm and zeta potential was -37.8 ± 1.1 mV. In order to verify the selectivity and sensitivity of our nanosensor, we examined the ability to recover fluorescence in 1x PBS solution after adding the target RNA fully complementary to PNA probe (Figure 2, S2). After the fluorescence

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of PNA probe was quenched down to 5% compared to initial value upon addition of D-GON, the recovery of fluorescence in the presence of specific target RNA was comparable to that in the presence of scrambled RNA as negative control. With addition of the target RNA entirely complementary to β-actin PNA probe, the fluorescence intensity increased rapidly up to 1 hour, and then plateaued within 6 hours. In contrast, the existence of scrambled RNA did not change the fluorescence intensity (Figure 2A, inset). These results confirm that the nanosensor is capable of recognizing singlestranded RNA in a sequence-specific manner. Moreover, we explored the responsiveness and dynamic range of the nanosensor. The fluorescence intensity was linearly correlated to target RNA concentration from 0 to 1.56 nM with a limit of detection of 169 pM (Figure 2B). Then, we studied whether our nanosensor is feasible in live-cell monitoring of specific mRNAs (Figure 2C). β-actin mRNA, the most commonly used housekeeping gene, was selected as a model target. After the nanosensor was introduced into cells, the fluorescence signal of the probe targeting β-actin mRNA was mostly observed in perinuclear region, whereas the fluorescence of the scrambled probe was hardly restored.22 The quantification of the fluorescence signal was determined using intracellular multi-target analysis (MTA) software (mean fluorescence intensity per cell = sum of all pixel intensities forming spots / number of cells). The fluorescence signal of the probe targeting β-actin mRNA was about 10 times higher than that of the scrambled probe (Figure 2D). This finding validated that the nanosensor enables sequence-specific detection of intracellular mRNA in living cells with high signal-to-noise ratio. Based on the above-mentioned results, we investigated the

Figure 2. (A) Fluorescence emission spectra (excitation wavelength 650 nm) of the nanosensor in 1X PBS solution at 6 hours after adding synthetic RNAs with different sequence (target RNA; entirely complementary sequence to β-actin PNA probe). Inset: time-dependent kinetics of fluorescence recovery. (B) Fluorescence response of the nanosensor to various concentration of target mRNA manner (F / F 0: fluorescence intensity in the presence of target RNA / fluorescence intensity in the absence of target RNA). (C) Representative fluorescence imaging of HepG2 cells at 9 hours after incubation of the nanosensor with different PNA probes. (D) Relative quantification of intracellular red signal in the fluorescence images using MTA software (n = 3).

Figure 3. (A) Representative fluorescence images of β-catenin mRNA in HepG2 cells at 24 hours after treatment of siRNA in concentrationdependent manner. (B) Relative quantification of intracellular red signal corresponding to β-catenin mRNA in the fluorescence images using MTA software. (C) The linear correlation between the nanosensor and RT-PCR for evaluating siRNA-mediated silencing of βcatenin mRNA (n = 3).

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ACS Sensors use of our nanosensor to evaluate siRNA efficacy by providing quantitative assessment of intracellular fluorescence signals corresponding to target mRNA level in living cells (Figure 3). To verify effectiveness of siRNA targeting β-catenin mRNA, fluorescence images of HepG2 cells incubated with the nanosensor were obtained at different siRNA concentrations (Figure 3A). The mean fluorescence intensity of red spots visible in perinuclear region was quantified by MTA analysis. The fluorescence signal corresponding to β-catenin mRNA was gradually reduced with increasing concentration of siRNA (Figure 3B), whereas the signal corresponding to βactin mRNA remained constant (Figure S3). These results implied that the nanosensor could monitor changes in intracellular mRNA level in response to siRNA treatment by quantifying fluorescence signal in a sequence-specific manner. Furthermore, we performed conventional RT-PCR assay under the same conditions to verify the reliability of the nanosensor (Figure S4). The fluorescence signal based on the nanosensor exhibited a strong linear correlation (R2 = 0.978) with the mRNA expression level obtained by RT-PCR (Figure 3C), indicating that there was a similar tendency between the nanosensor and RT-PCR. Thus, we demonstrated that the

nanosensor can directly evaluate dose-dependent silencing effect of siRNA with reliable quantification of target mRNA levels in living cells. Subsequently, to examine downregulating effect of siRNA on the canonical Wnt/β-catenin signaling path-way over time, we measured simultaneously β-catenin and its target mRNA levels in living cells using our nanosensor. As shown in Figure 4A, fluorescence images were obtained at different times post-transfection. The fluorescence signal corresponding to β-catenin mRNA, which is directly targeted by siRNA, was kept in sufficiently low levels after 48 hours. Meanwhile, a decrease in fluorescence signal corresponding to cyclin D1 mRNA, a downstream target gene regulated by β-catenin protein, occurred gradually between 48 and 72 hours (Figure 4B). This finding revealed that continuous knock-down of βcatenin mRNA by siRNA affected protein expression level (Figure S5), which led to time-dependent downregulation of β-catenin target gene expression. Moreover, RT-PCR analysis under the same conditions showed that changes in expression levels of β-catenin and cyclin D1 mRNA were similar to those obtained using the nanosensor (Figure S6A). Additionally, we found that fluorescence signal corresponding to cMyc mRNA, another β-catenin target gene, was correlated with mRNA expression levels estimated by RT-PCR assay (Figure S6B, C). Taken together, these results suggest that the nanosensor could simultaneously monitor changes in expression levels of several mRNAs over time by means of quantitative assessment of fluorescence signals in living cells. Finally, we tested the ability of our nanosensor to verify efficacy of small-molecules as potential therapeutics to inhibit a signaling pathway implicated in cancer (Figure 5). It has been reported that PNU74654, known as an anticancer agent, inhibits Wnt/β-catenin signaling pathway by interrupting the interaction between β-catenin protein and T-cell factor/lymphocyte enhancer factor (TCF/LEF) transcription factors, which regulates the expression of genes associated

Figure 4. (A) Representative fluorescence images of β-catenin and cyclin D1 mRNA in HepG2 cells at 48 and 72 hours after transfection of siRNA against β-catenin mRNA (50 nM). (B) Relative quantification of intracellular red signal corresponding to β-catenin and cyclin D1 mRNA in the fluorescence images using MTA software (P-value was calculated by Student’s t-test: * for p < .05, n = 3).

Figure 5. (A) Scratch wound healing assay in HepG2 cells treated with PNU74654. (B) Representative fluorescence images of β-catenin and Cyclin D1 mRNA in HepG2 cells at 48 hours after treatment of PNU74654. (C) Relative quantification of intracellular red signal corresponding to β-catenin and Cyclin D1 mRNA in the fluorescence images using MTA software (n =3).

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with cell proliferation and differentiation.23-25 A scratch wound healing as-say showed lower wound closure rate of the PNU74654-treated HepG2 cells than that of untreated cells, indicating that PNU74654 treatment prevented migration and proliferation of HepG2 cells (Figure 5A). To further investigate the impact of PNU74654 on regulation of gene expression involved in Wnt/β-catenin signaling pathway, the nanosensor was introduced into HepG2 cells pre-treated with PNU74654. The fluorescence signal corresponding to cyclin D1 mRNA in the cells treated with PNU74654 was significantly decreased compared to un-treated cells, whereas the signal corresponding to β-catenin mRNA maintained constant high level (Figure 5B). These results implied that PNU74654 did not affect β-catenin mRNA level but only caused a decline in cyclin D1 mRNA level by interfering with binding of β-catenin protein to TCF/LEF transcription factors. Like-wise, mRNA expression levels measured by RTPCR assay were significantly correlated with those monitored according to the variation of fluorescence signal of the nanosensor (Figure 5C, S7). In summary, we developed a fluorescent nanosensor for direct monitoring intracellular level of cancer-associated mRNAs, involved in the canonical Wnt/β-catenin signaling pathway, in living cells for highly accurate non-invasive diagnosis and prognosis of cancer. Quantitative fluorescence imaging analysis for evaluating mRNA expression levels determined cancer therapeutic implications of siRNA and small-molecule inhibitor modulating Wnt/β-catenin signaling pathway. Based on these results, we expect that the nanosensor could be applied to high throughput screening for the efficient drug discovery and insightful understanding of the molecular mechanisms of potential drug candidates for cancer treatment in the near future.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Materials and methods, characterization of D-GON, live-cell fluorescence imaging, RT-PCR analysis (PDF)

AUTHOR INFORMATION Corresponding Author E-mail: [email protected]

Notes D.-H.M. is named on patents that describe the use of graphene derivatives and nucleotides as a platform for fluorescent biosensing of biomolecules. The remaining authors declare no competing interests.

ACKNOWLEDGMENT This work was supported by the Basic Science Research Program (2016R1E1A1A01941202, 2016R1A4A1010796), International S&T Cooperation Program (2014K1B1A1073716), and the Research Center Program (IBS-R008-D1) of IBS (Institute for Basic Science) through the National Research Foundation of Korea (NRF).

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