DOUBLE-STRANDED LOCKED NUCLEIC ACID PROBES FOR REVEAL-ING INTRACELLULAR GENE EXPRESSION DYNAMICS OF CANCER

CELLS NEAR MECHANICAL WOUNDS Reza Riahi and Pak Kin Wong*

The University of Arizona, USA

ABSTRACT We have developed a double-stranded locked nucleic acid (dsLNA) probe for monitoring the intracellular gene expression

toward the investigation of the role of mechanical injury in cancer metastasis. Specifically, the dsLNA probe is shown to al-low real-time detection of intracellular mRNA in living cells. Using the dsLNA probes, the spatiotemporal gene expressions of cancer cells after mechanical injury are monitored for days. We show that the release of contact inhibition is sufficient to induce the migratory behavior of cancer cells while mechanical injury triggers the cellular antioxidant response in a region ~100 µm away from the wound.

KEYWORDS: dsLNA probe, MDA-MB-231 cancer cells, wound healing assay, HO-1 mRNA expression.

INTRODUCTION

Currently, one of the preferred treatments for breast cancer is surgical removal of the primary tumor. The surgical proce-dure, however, is known to increase the potential of metastatic tumor outgrowth and the circulating tumor cell count. Few systematic studies have been performed to determine the molecular mechanisms that promote metastatic spread after surgery. In particular, little is known about the influences of mechanical injury on the response of cancer cells. To elucidate these fundamental questions in cancer biology, innovative biosensors are required to monitor the response of cancer cells near a wound. We have previously developed a dsDNA probe for rapid detection of specific nucleic acid sequences [1]. The dsDNA probes, nevertheless, are vulnerable to nuclease digestion and nonspecific binding with DNA binding proteins. By incorporating double stranded probes with DNA/LNA alternating monomers (dsLNA) [2-3] into in vitro wound healing as-says, the responses of breast cancer cells, MDA-MB-231, that experienced different extents of mechanical injury were inves-tigated in this study. The ability of dsLNA probes for quantitative measurement were first evaluated by treating the breast cancer cells with tert-butylhydroquinone (tBHQ). The spatiotemporal expressions of β-actin and heme oxygenase-1 (HO-1) mRNA were monitored in cancer cells near the wounding sites for days. This study reveals the influences of mechanical in-jury in cancer cell metastasis.

THEORY

A dsLNA probe is a double-stranded oligonucleotide probe labeled with a fluorophore and a quencher which are in vicini-ty to diminish the fluorescence signal in the absence of a target. The probe is designed based on the specific target nucleic acid sequence. When the dsLNA probe is transfected into a cell, it generates a fluorescence signal upon hybridizing to the target mRNA by thermodynamically replacing the quencher sequence as schematically shown in Figure 1a. In this study, three dsLNA (β-actin, HO-1, and random) probes were designed and transfected into the cells to study the sensitivity and specificity of the probe (Figure 1b-c). The β-actin and HO-1 probes were designed to study the motility and the antioxidant response of the cancer cells and the random probe served as a negative control in the study.

Figure 1. (A) Schematic of dsLNA probes for detecting intracellular mRNA. (B) Molecular structure of dsLNA

probes for transfection using lipofectamine 2000. (C) Intracellular gene expression monitored using dsLNA probes.

978-0-9798064-4-5/µTAS 2011/$20©11CBMS-0001 109 15th International Conference onMiniaturized Systems for Chemistry and Life Sciences

October 2-6, 2011, Seattle, Washington, USA

EXPERIMENTAL The dsLNA probes were prepared by mixing fluorophore and quencher probe sequences at a 1-to-3 ratio. After 5 minutes

of incubation in a water bath at 95oC, the dsLNA probes were cooled down for 3 hours to reach room temperature. Breast cancer cells, MDA-MB-231, were plated in 24-well tissue culture plates for 24 hours to reach 90% confluency. The cells were transfected with 0.8 g dsLNA probe using lipofectamine 2000 in opti-MEM (Invitrogen). The cells were monitored using an inverted fluorescence microscope (TE2000-U, Nikon) and fluorescence images were taken using a CCD camera at different time points for three days. To evaluate the stability of the probe, the intensity levels of the dsLNA and dsDNA probes in the cytoplasms were measured 72 hours after transfection. To induce HO-1 mRNA expression for quantitative stu-dies, cells were treated for 24 hours with 20 M tBHQ after they were transfected with dsLNA probes. The dsLNA probe was employed for studying mRNA gene expression near wounding sites. Both mechanical scraping with a micropipette and PDMS “lift-off” wound healing assays (Figure 2) were performed to introduce different degrees of cell damage for evaluating the role of mechanical injury in the cellular response of the cancer cells. RESULTS AND DISCUSSION

Figure 3 compares the performance and stability of the dsLNA probe with the dsDNA probe. The dsLNA probe successful-ly detects the constitutive gene, β-actin, while the dsDNA probe displays a poor signal-to-noise ratio. These results suggest that the dsLNA probe is capable of intracellular detection of mRNA expression after 72 hours of incubation. This is consistent with the high resistance of LNA to non-specific protein binding and nuclease digestion compared to DNA [2-3]. Figure 4 shows in-duction of HO-1 mRNA in cells treated with tBHQ, which is known to upregulate HO-1 mRNA. Based on our calibration, the result indicates a 7-fold increase in the HO-1 mRNA level, which is in reasonable agreement with quantitative PCR [4]. We fur-ther applied dsLNA probes to cancer cells for studying the kinetics of gene expression near the wounding edges after performing mechanical scratch and PDMS lift-off assays (Figure 5). The intensity levels of HO-1 and β-actin mRNA were maximized on day 3 in both assays. The intensity of the random probe was maintained at a low level suggesting the specificity of the dsLNA assay. To evaluate the impact of wounding sites on the response of cells at different locations from the wounding edge, the spa-tial distribution of gene expressions were measured (Figure 6-7). The mRNA levels of β-actin and HO-1 increase in cells ~150 μm and ~100 μm from the wounding edges, respectively. The distributions and intensities of β-actin mRNA are similar in both wounding assays with and without significant cell injury. Cells were also observed to migrate with similar velocities (data not shown). These results suggest the release of contact inhibition without mechanical injury (PDMS lift-off) is sufficient to induce intracellular β-actin expression and cell migration. On the other hand, a higher level of HO-1 mRNA intensity was observed in the scratch assay. This correlates with the observation that reactive oxygen species (ROS) are generated near the site of injury after mechanical scratching (data not show) [5]. This is consistent with the protective role of HO-1 mRNA against ROS during wound repair [6]. Collectively, our results suggest mechanical injury and contact inhibition play different roles in cancer cell metastasis. CONCLUSION

In this study, we demonstrate a dsLNA assay for detection of intracellular gene expression in individual living cells. The superior resistance of LNA to nuclease digestion enables the dsLNA for long-term monitoring of mRNA expressions. By combining the dsLNA probe with in vitro wound healing assays with different degrees of cell damage, our result sheds light on the role of cell injury in cancer metastasis. With its effectiveness and general applicability for measuring intracellular mRNA expression, the dsLNA probe can be adopted for investigating a wide spectrum of spatiotemporally regulated biologi-cal processes, such as embryogenesis and tissue regeneration.

Figure 2. Wound healing assays with different levels

of mechanical injury. A) Peeling- off the PDMS physical blockage. B) Scraping the cells off with a micropipette.

Figure 3. Normalized fluorescence intensity changes of

-actin (positive), HO-1, and Random (negative) genes in the MDA-MB-231 cell cytoplasm utilizing LNA and DNA probes.

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ACKNOWLEDGEMENTS This work was supported by National Institutes of Health Director's New Innovator Award (1DP2OD007161-01). REFERENCES [1] V. Gidwani, R. Riahi, D.D. Zhang, and P.K. Wong, "Hybridization kinetics of double-stranded DNA probes for rapid

molecular analysis", Analyst, 134, pp. 1675-1681, (2009). [2] Y. Wu, C. James, L. Moroz, and W. Tan, "Nucleic Acid Beacons for Long-Term Real-Time intracellular monitoring"

Analytical Chemistry, 80, pp.3025-3028, (2008). [3] C. J. Yang, L. Wang, Y. Wu, Y. Kim, C.D. Medley, H. Lin, and W. Tan, "Synthesis and investigation of deoxyribo-

nucleic acid/locked nucleic acid chimeric molecular beacons", Nucleic Acids Research, 35, pp. 4030-4041, (2007). [4] Y. Du, N.F. Villeneuve, X.J. Wang, Z. Sun, W. Chen, J. Li, H. Lou, P.K. Wong, and D.D. Zhang, "Oridonin confers

protection against arsenic-induced toxicity through activation of the Nrf2-mediated defensive response", Environ Health Perspect, 116, pp.1154-1161, (2008).

[5] A. Grochot-Przeczek, R. Lach, J. Mis, K. Skrzypek, M. Kozakowska, P. Sroczynska, M. Dubiel, M. Gozdecka, J. Walc-zynski, and J. Kotlinowski, "Heme oxygenase-1 accelerates cutaneous wound healing in mice", Hum Gene Ther, 19, pp.1111-1112, (2008).

[6] C. Hanselmann, C. Mauch, and S. Werner, "Haem oxygenase-1: a novel player in cutaneous wound repair and psoria-sis? ", Biochem J, 353, pp.459-466, (2001).

CONTACT *P.K. Wong, tel: +1-520-6262215; pak@email.arizona.edu

Figure 4. HO-1 upregulation. A) Normalized intensity of HO-1 after tBHQ treatment. B) Illustration of HO-1 mRNA upregulation in the cytoplasm. C) Control cell without treatment showing low intensity level of HO-1 expression in the cytoplasm.

Figure 5. Kinetics of gene expression near the wounding

edge. A) PDMS peel-off. B) Scrapped cells indicating HO-1 mRNA upregulation in day 2 and day 3.

Figure 6. Spatial distribution of gene expression from

wound. Little or none HO-1 mRNA upregulation due to low injury induced by PDMS peel-off.

Figure 7. Spatial distribution of gene expression from

wound after scratching. HO-1 mRNA upregulation has been observed near the wounding edge.

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