Tracking of mitochondrial endogenous ribonucleic acid in the cancer

Jan 8, 2019 - The expression of the genetic information on protein is achieved by mitochondrial RNA (mtRNA). However, mtRNA tracking in biological ...
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Tracking of mitochondrial endogenous ribonucleic acid in the cancer cells and macrophages using a novel small-molecular fluorescent probe Yong Liu, Jie Niu, Weishan Wang, and Weiying Lin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05305 • Publication Date (Web): 08 Jan 2019 Downloaded from http://pubs.acs.org on January 8, 2019

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Analytical Chemistry

Tracking of Mitochondrial Endogenous Ribonucleic Acid in the Cancer Cells and Macrophages Using a Novel Small-molecular Fluorescent Probe Yong Liu, Jie Niu, Weishan Wang and Weiying Lin * Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and Chemical Engineering, School of Materials Science and Engineering, University of Jinan, Jinan, Shandong 250022, P. R. China. Fax: (+) 86-53182769031, E-mail: [email protected]. ABSTRACT: The expression of the genetic information on protein is achieved by mitochondrial RNA (mtRNA). However, mtRNA tracking in biological systems is bound due to the lack of an effective method. To solve this pressing problem, we construct a low molecular weight probe MR-IDE to track endogenous mtRNA in the cancer cells and macrophages for the first time.

Nucleic acid transcription plays an important role in the gene expression process. For example, mitochondrial ribonucleic acid (mtRNA) can express genetic information on protein in living biological systems.1 mtRNA controls RNA stability, modification, and degradation in living cells.2 Nucleic acid transcription also occurs in the mitochondria, because the post-transcriptional process is essential in correcting mtRNA expression in the biological system. All proteins involved in RNA metabolism are encoded into the mitochondria and other organelles.3 Mutations leads to mtRNA decay and further causes some human disease.4 Tracking endogenous mtRNA in biological systems is necessary to understand the role of mtRNA in genetic expression. As effective molecular tools, small molecular probes are attractive in detecting active molecules in the field of biological imaging.5 Many researchers have focused on fluorescent and aggregation–disaggregation methods to develop new RNA probes. Various low molecular weight probes have been developed in sensing RNA in living biological systems. The emergence of a series of low molecular weight RNA probes is sufficient to demonstrate the status of RNA in the biological research field.6 Fluorescent probes that can target the mitochondria for active molecule detection have been widely used, including mitochondrial acid base,7 reactive oxygen species,8 sulfite radical,9 metal cations,10 amidoxime,11 amino acids,12 and hydrogen sulfide.13 However, research on a low molecular weight mtRNA probe that can target the mitochondria has never been conducted. In the reported nucleic acid probes, the 3,6 position-substituted carbazole dyes can sense nucleic acids on the basis of intercalative binding.14 In terms of this sensing mechanism, indole double ionic salts with a V structure also detects RNA in living cells.15 The structural characteristics of indole double ionic salts is equivalent to the compression of the conjugated system of carbazole salts with a V structure. According to the structural characteristics above, we further constructed a new conjugated system and developed the pyrrole monoionic salt MR-IDE. We regard this kind of ionic

salt as a new fluorescence platform, in which a mitochondria site is introduced (Figure 1). Compared with conventional RNA probes, MR-IDE can track endogenous mtRNA in cancer and macrophage cells. MR-IDE also exhibits different fluorescence signals in cancer and macrophages cells The synthetic detail of MR-IDE is described in the supporting information.

Figure 1. Strategies for sensing RNA. Using MR-IDE, we first examined its optical properties in different solutions (Figure S1). The photophysical properties of MR-IDE in different solutions are shown in Table S1. The optical data above demonstrated that MR-IDE showed strong absorption and emission spectra at 425 and 525 nm in a buffer solution, respectively. A large Stokes shift of 100 nm in a pure water system was observed. The compound also exhibited higher fluorescence quantum yield in an organic system than that in pure water systems. This result was consistent with the photophysical properties of conventional mitochondria and nucleic acid fluorescent probes.16, 17 To verify whether the MR-IDE can identify RNA in living systems, we carried out fluorescent titration in buffer solution. MR-IDE showed weak fluorescence emission intensity at 530 nm (λex: 488 nm) in the water phase. However, with the addition of RNA, the fluorescence intensity of the probe increased gradually under the same conditions. The amplitude of the rise reached nine-fold higher than the fluorescence intensity of the probe itself (Figure 2A). The trend of fluorescence titration

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demonstrated that the fluorescence intensity of the probe from increased gradually to balance with the increase in RNA concentration (Figure 2B). These results demonstrated that MR-IDE can sense RNA on the basis of an intercalative binding mechanism. Considering that DNA and RNA are groove structures, DNA interference still existed during RNA detection. In the presence of DNA and RNA, MR-IDE possessed stronger fluorescence response signals to RNA than DNA in specific concentrations (Figure 2C). The trend of DNA fluorescence titration exhibited a similar titration trend with RNA. However, the probe showed stronger fluorescence signal to RNA than DNA in the buffer solution (Figure 2D).

RNA-Select to RNA and DNA (Figure 2F). Thus, MR-IDE should selectively identify RNA in biological systems. To verify further whether MR-IDE can image mitochondria in biological systems, we investigated mitochondrial colocalization. Before the experiments, the toxicity of the probe was tested by MTT (Table S2). The toxicity experiment showed that MR-IDE possessed an imaging ability in living biological systems. As shown in Figures 3AI−AIV, we selected the nucleus region (ROl 1), in which the fluorescence signal was weak in the cell image. Studying the occurrence frequency of fluorescence signals in this region showed that both probes possessed low colocalization effect in living cell nuclei (Figure 3AV). However, both probes exhibited high colocalization effects by studying the occurrence frequency of the fluorescence signals in ROl 2 in the cytoplasm of living cells (Figure 3AVI). Mitochondria is distributed in the cytoplasm. Thus, MR-IDE can image mitochondria in living cell cytoplasm. To prove this inference, we further studied the colocalization of the whole region (Figures 3BII–BIV). Studying the interrelationship between the frequency and fluorescence signals of the probe and the MTR in whole cells, as well as the localization coefficient (Figures 3BV–BVI), indicated that MR-IDE can target the mitochondria in living cell cytoplasm. MR-IDE also possessed favorable counterstain compatibility with other commercial probes (Figure S2). The results showed that MR-IDE was located in the cytoplasm. The above results further demonstrated that MR-IDE can locate mitochondria in the cytoplasm.

Figure 2. (A) The fluorescence emission intensities of the probe MR-IDE with the increasing of RNA. [MR-IDE]: 1 μM; (B) Fluorescence variation of the probe MR-IDE (1 μM) at 530 nm with the increasing of RNA; (C) Fluorescence responses (at 530 nm) of MR-IDE (1 μM) to RNA and DNA; (D) Fluorescence variation of the probe MR-IDE (1 μM) to RNA and DNA at 530 nm. λex = 460 nm; (E) Fluorescence properties of probe in different analyses. From left to right: 1) MR-IDE, 2) Arg, 3) Ser, 4) IIe, 5) Phe, 6) ASP, 7) Val, 8) Ala, 9) His, 10) Thr, 11) GSH, 12) Hcy, 13) Glu, 14) S2O32-, 15) CH3COO-, 16) NO2-, 17) N3-, 18) Br-, 19) Ca2+, 20) K+, 21) Na+, 22) Al3+, 23) Fe2+, 24) Cu2+, 25) GM, 26) LPS, 27) dNTPs, 28) NTPs; 29) FBS, 30) DNA and 31) RNA; (F) Fluorescence responses of commercial probe (2 μM) to RNA and DNA in PBS solution. [RNA] = [DNA] = 155 mg/mL. Selectivity plays a key role in fluorescence probe fields.18 To prove that the probe can achieve high RNA selectivity recognition, we further studied the fluorescence properties of the probe to various relevant species (i.e., Arg, Ser, IIe, Phe, ASP, Val, Ala, His, Thr, GSH, Hcy, Glu, S2O32−, CH3COO−, NO2−, N3−, Br−, Ca2+, K+, Na+, Al3+, Fe2+, Cu2+, GM, LPS, dNTPs, NTPs, FBS, and DNA). The results demonstrated that MRIDE exhibited stronger fluorescent intensities to RNA than other analyses (Figure 2E). The results above further indicated that MR-IDE exhibited high selectivity to RNA in the aqueous phase. For selective experiments, although DNA exhibited some interference during RNA detection, this interference cannot be ignored. These results were consistent with the response trend of the commercialized RNA probe

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Figure 3. Confocal images of HeLa cells treated with both MTR (0.5 μM) and MR-IDE (5 μM) for 30 min. (A) (AI and BI): Bright-field image; (AII and BII): Fluorescence image of MR-IDE collected between 500 and 525 nm upon excitation at 488 nm; (AIII and BIII): Fluorescence image of MTR collected between 575 and 595 nm upon excitation at 561 nm; (AIV and BIV) Merged image of AII and AIII; (AV-VI): The interrelation between the frequency and fluorescence signals of probe and MTR in ROl 1 (nucleus) (AV) and ROl 2 (Cytoplasm) (AVI). (BV): The interrelation between the frequency and fluorescence signals of probe and MTR in whole cells. (BVI): Correlation plot of MR-IDE and MTR intensities.

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Analytical Chemistry We further studied the cellular imaging features in cancer cells (HeLa and HepG2 cells) and macrophages (RAW264.7 cells) by using MR-IDE. For HeLa cells, the fluorescence signal of MR-IDE mainly appeared in the cytoplasm and nucleolus (Figures 4A–C). Ribosomal RNA was distributed in the nucleolus of living cells. Thus, MR-IDE should image RNA in cancer cells. We further studied the mean fluorescence intensity in the cytoplasm (a1), nucleolus (b1), and nucleus regions (c1). As shown in Figure 4D, the results showed that the fluorescence signal of the cytoplasm was stronger than those of the nucleolus and nucleus. Similar to that of HeLa cells, the fluorescence signal of the probe mainly focused on the cytoplasm and nucleolus of HepG2 cells (Figures 4E–H). However, the nucleolus exhibited weaker fluorescence signals than HeLa cells. Compared with cancer cells, regardless of cytoplasm and nucleolus, the fluorescence intensities of macrophages were weaker than those of cancer cells (Figures 4I–L). In the cytoplasm, three cell lines treated with MR-IDE exhibited stronger mean intensities than the other regions (Figure S3). Thus, MR-IDE may be able to image RNA in living cell cytoplasm.

However, the mean fluorescence intensity of the RNasetreated cytoplasm was much weaker than that of the nucleus (Figure 5G). The results demonstrated that MR-IDE can detect endogenous RNA in cancer cells.6, 19 Given that MRIDE targets mitochondria in the cytoplasm, it can image mtRNA in HeLa cancer cells. For the cancer cell HepG2, similar to HeLa cells, the control group exhibited strong fluorescence in the nucleus and nucleolus (Figures 5H–M). The mean fluorescence intensities of the cytoplasm and nucleus were similar with HeLa cells (Figure 5N). Thus, the RNA digestion test results demonstrated that MR-IDE can image mtRNA in different cancer cells. As shown in 5O-U, the intracellular fluorescence signals of the experiment group were from the nucleus in RAW264.7. This result was consistent with that of a commercial RNA fluorescent probe.20 These results demonstrated that MRIDE can sense mtRNA in RAW264.7 macrophages. Compared with the digestion experiment of cancer cells, the fluorescence signals of RAW264.7 cells were localized in the nucleus, with faint nuclei distribution. However, the intracellular fluorescence signals of cancer cells were mainly from the nucleus and nucleolus (Figure S4). Therefore, MRIDE can target the mitochondria in living cell cytoplasm. Combined with the digestion experiment, this novel probe can image mtRNA in cancer cells and macrophages.

Figure 4. Confocal images of the cancer cells (HeLa and HepG2 cells) and macrophages (RAW264.7 cells) treated with MR-IDE (5 μM) for 30 min. (A, E, I): Bright-field image; (B, F, J): Fluorescence images pictures of MR-IDE. Emission wavelength: 500-525 nm ; Excitation : 488 nm; (C, G, K): Merged images of (A and B), (E and F) and (I and J); (D, H, L): The mean fluorescence intensity in cytoplasm, nucleus and nucleoli regions. a: Cytoplasm, b: Nucleolus, c: Nucleus. Bar = 10 μm. To verify the inference above, we further performed RNA digestion test, we prepared HeLa, HepG2, and RAW 264.7 macrophages to prove this point. Before the digestion test, cells were treated as follows. First, fixed cells were obtained by paraformaldehyde (4%). Second, the fixed cells above were punched by Trition X-100 (0.5% ). Third, the cells were treated with 20 µg/mL of RNase for 120 min. Fourth, the system above was further washed with PBS, and then the cells were treated with MR-IDE (10 μM) for 0.5 h. Finally, the fixed cell images were obtained (Figure 5). For cancer cells, we prepared two fixed HeLa cell groups, as follows: one group was treated with RNase as the experiment group, and another group was chosen as the control group. After treating with RNase, the intracellular fluorescence concentrated on the nucleus and nucleolus, while thegreen fluorescence emission of the control group was distributed in the nucleolus and cytoplasm. The mean fluorescence intensity of the cytoplasm untreated with RNase was stronger than that of the nucleus (Figures 5A–F).

Figure 5. The RNase digest experiment. All fluorescence imaging pictures of fixed cells stained with MR-IDE (5 μM). Imaging pictures of HeLa (A-C), HepG2 (H-J) and RAW264.7 (O-Q) cells untreated with RNase; Imaging pictures of HeLa (D-F), HepG2 (K-M) and RAW264.7 (R-T) cells treated with RNase; (G, H, L): The mean fluorescent intensity of cytoplasm and nucleus before or after treatment with RNase. Ex = 488 nm, Em = 500-525 nm. Bar = 10 μm. To verify whether MR-IDE can track mtRNA, we further studied the stability of this probe. The mean intensities of MR-IDE remained unchanged for a long period of time. The results indicated that MR-IDE showed high photostability (Figure S5). We further observed dynamic changes in mtRNA in living cell cytoplasm for a long period of time (Figure S6). The results indicated that the probe can dynamically monitor mtRNA in the cytoplasm of cancer cells and macrophages for a long period of time (Movie S1). We further investigated the photostability of MR-IDE and “SYTO RNA-Select”. As shown in Figure S7, MR-IDE showed higher photostability than the

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commercial probe SYTO-RNA-Select. SYTO-RNA-Select cannot dynamically detect mtRNA in cancer cells and macrophages. In summary, we constructed a new conjugated system and developed the pyrrole mono-ionic salt MR-IDE. We considered this kind of ionic salt as a new fluorescence platform, in which a mitochondria site is introduced to afford a novel small-molecular mtRNA fluorescent probe. MR-IDE possesses excellent properties, including high selectivity and photostability, low cytotoxicity, and good cell-membrane permeability. In terms of the properties mentioned above, MR-IDE can dynamics image endogenous mtRNA in the cancer cell and macrophages for the first time. The phenomenon mentioned above provides important information for studying the distribution and changes of nucleic acid in cancer cells and macrophages.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Synthesis of the probes, absorption and fluorescence spectra, imaging assays, 1H NMR and 13 C NMR spectra. (PDF)

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

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Funding was partially provided by NSFC (21472067, 51503077, 21672083, 21877048); Taishan Scholar Foundation (TS 201511041), NSFSP (ZR2015PE001).

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