356 | Chem. Commun., 2017, 53, 356--359 This journal is©The Royal Society of Chemistry 2017

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Simultaneous detection of multiple targetsinvolved in the PI3K/AKT pathway forinvestigating cellular migration and invasion witha multicolor fluorescent nanoprobe†

Mingming Luan,‡ Na Li,‡ Wei Pan, Limin Yang, Zhengze Yu and Bo Tang*

We develop a multicolor fluorescent nanoprobe for assessing cellular

migration and invasion by simultaneously imaging miRNA-221,

PTEN mRNA and MMP-9 involved in the PI3K/AKT pathway which

can regulate cellular mobility and invasiveness.

Cancer in humans is a highly fatal disease with poor prognosisand the most life-threatening aspect of cancer is tumor metastasiswhich requires cellular migratory and invasive capacities.1 Cellmotility is regarded as the predominant regulator of tumorinvasion.2 There has been increasing interest in studying cellmigration and invasion because this is pivotal to establish effectivestrategies for cancer diagnosis, prognosis and treatment promotingcontrol in cancer metastasis.3 Many migration/invasion assayssuch as wound healing and transwell invasive assays have beenestablished to evaluate migration and invasion; however, theseapproaches are labor intense and time consuming to manage.4

Hence, it is necessary to develop a quicker, simpler and moreaccurate approach to assess cellular migration and invasion. Themigration and invasion of cancer cells were reported to beregulated through a network of signaling pathways which canbe cooperatively influenced by microRNAs (miRNAs), messengerRNAs (mRNAs) and matrix metalloproteinases (MMPs).5 It wasreported that miR-221 promoted cellular migration and invasionvia directly repressing PTEN and TIMP3 expression leading tothe aberrant activation of the PI3K/AKT pathway and MMP-9expression.6

The current widely used approaches to analyze genes andproteinases in signaling pathways are based on traditionaltechniques such as northern blotting, real-time reversetranscription-PCR (RT-PCR), microarray hybridization, western

blotting and immunohistochemistry.7 However, these approachesrequire substantial time, many steps and a large number of cellsamples. Moreover, the cell lysate or immobilization process usedin these methods makes it impossible to study the dynamicchanges and natural situation of these molecules. Thus, it isimportant to develop a noninvasive method for detecting multiplemolecules in signaling pathways. And we anticipate that cellularmigration and invasion could be assessed by identifying theexpressions of genes and proteinases involved in signalingpathways. Fluorescence imaging analysis could have greatpotential in simultaneously monitoring genes and proteinasesinvolved in signaling pathways of living cells and providinguseful information about cellular migration and invasion.

Here, we construct a multicolor fluorescent nanoprobe forinvestigating cellular migration and invasion through detectingmiRNA, mRNA and MMP involved in the PI3K/AKT pathway ofliving cells (Scheme 1). The nanoprobe was composed of gold

Scheme 1 Schematic diagram of the multicolor nanoprobe for simultaneousdetection of miRNA, mRNA and MMP involved in the signaling pathway.

College of Chemistry, Chemical Engineering and Materials Science, Collaborative

Innovation Center of Functionalized Probes for Chemical Imaging in Universities of

Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education,

Institute of Molecular and Nano Science, Shandong Normal University,

Jinan 250014, P. R. China. E-mail: tangb@sdnu.edu.cn

† Electronic supplementary information (ESI) available: Experimental details andsupplementary figures. See DOI: 10.1039/c6cc07605j‡ These authors contributed equally to this work.

Received 19th September 2016,Accepted 23rd November 2016

DOI: 10.1039/c6cc07605j

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nanoparticles (AuNPs) anchored with a dense layer of oligo-nucleotide molecular beacons (MBs) and a peptide, modifiedstep-by-step via Au–S bond formation.8 Firstly, the AuNPs wereassembled with MBs folding into a hairpin conformation.9 TheMBs could respond specifically to miR-221 and PTEN mRNAtargets. Next, the synthetic peptide was assembled on thesurface of AuNPs. The peptide could be specifically cleaved byMMP-9.10 In the absence of the targets, the fluorescence of threefluorophores linked with the MBs and the peptide was efficientlyquenched by AuNPs. In the presence of the correspondingtargets, the MBs were forced to unwind and the peptide wascleaved by MMP, thus leading to fluorescence recovery.

The nanoprobe was prepared using 20 nm AuNPs, becauselarge-sized AuNPs can accommodate more recognition unitsand cause higher surface plasmon resonance (SPR) absorptionthan small-sized AuNPs. The morphologies of AuNPs and thenanoprobe were characterized by transmission electron micro-scopy (TEM) (Fig. S1, ESI†). The results showed that they had aspherical morphology. The UV/Vis spectra (Fig. S2, ESI†)showed that the AuNPs were successfully functionalized owingto the red shift of the peak from 519 to 524 nm. According to awell-established assay,11 each AuNP was determined to carry19 � 1 Cy5 labeled MB1 targeting miR-221, 20 � 1 Alexa Fluor488 labeled MB2 targeting PTEN mRNA, 82 � 1 RhB labeledpeptide targeting MMP-9 (Fig. S3, ESI†).

The capability of the nanoprobe to recognize the two DNAtargets and MMP target was evaluated. The nanoprobe wasincubated with the complementary miR-221 target, PTENmRNA target and MMP-9 target, respectively. The fluorescenceof each dye was intensified with the addition of respectivetarget in a concentration-dependent manner (Fig. 1), suggestingthat the fluorescence recovery should be attributed to theresponse of the nanoprobe towards the targets.

Selectivity is an important aspect for simultaneously monitoringdifferent targets in living cells. Fig. S4 (ESI†) shows that every MBcould generate notable fluorescence upon meeting its own DNAtarget. However, the fluorescence signals were faint in the presenceof single-base mismatched DNA target or the other two targets andcomparable to the background fluorescence. Similar results werefound when the nanoprobe was incubated with the MMP target.These results confirmed that the nanoprobe could sense differenttargets with high specificity.

A dynamics study of the nanoprobe toward three differenttargets was performed. The nanoprobe could respond to DNA

targets within 15–20 min and MMP-9 within 20–30 min (Fig. S5,ESI†). The matched response time for DNA and MMP targets makesthe nanoprobe hold great promise for simultaneously monitoringdifferent biomolecules in living cells. Further experiments showedthat the nanoprobe had good stability and reproducibility (Fig. S6and S7, ESI†).

Cell toxicity of the nanoprobe was measured by MTT assay(Fig. S8, ESI†) in MCF-7 cells (human breast cancer cell line).Cell survival rates were over 95% after treatment with nakedAuNPs and the nanoprobe for various times, indicating that thenanoprobe was almost noncytotoxic.

The multicolor nanoprobe was then used to study cellularmobility and invasiveness by measuring the relative fluorescenceof multiple biomarkers in the signaling pathway. MiR-221, PTENmRNA and MMP-9 in less invasive MCF-7 cells were selected asan example. It was reported that anisomycin induced miR-221and SU11274 reduced miR-221. MCF-7 cells were divided intothree groups. One group was treated with anisomycin andanother group was treated with SU11274. The untreated groupserved as control. Then the three groups of MCF-7 cells wereincubated with the nanoprobe and imaged using confocal laserscanning microscopy (CLSM). The relative levels of miR-221,PTEN mRNA and MMP-9 were reflected by fluorescence inten-sities (Fig. 2).

The results showed that the red fluorescence for miR-221was stronger in the anisomycin-treated cells and weaker in theSU11274-treated cells. The green fluorescence for PTEN mRNAdisplayed opposite fluorescence changes because miR-221 negativelyregulated PTEN mRNA. RT-PCR further demonstrated therelative levels of miR-221 and PTEN mRNA after drug treatment(Fig. S9 and S10, ESI†). The yellow fluorescence for MMP-9was also observed and the fluorescence changes were similarto those of miR-221 because miR-221 could activate MMP-9.

Fig. 1 Fluorescence recovery of the nanoprobe upon addition of variousconcentrations of: (a) miR-221 target, (b) PTEN mRNA target and (c) MMP-9in a concentration-dependent manner. The fluorescence of the nanoprobewas measured with 648 nm, 488 nm and 554 nm excitation wavelengths,respectively.

Fig. 2 Intracellular imaging of different levels of miR-221, PTEN mRNAand MMP-9 under CLSM. The MCF-7 cells were incubated with thenanoprobe (1 nM) for 4 h at 37 1C. The three channels were recordedwith 633 nm excitation, 543 nm excitation, and 488 nm excitation, fromleft to right. Scale bars: 100 mm.

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358 | Chem. Commun., 2017, 53, 356--359 This journal is©The Royal Society of Chemistry 2017

The relative abundance of MMP-9 concurred well with westernblot analysis (Fig. S11, ESI†).

To test the effects of miR-221, PTEN mRNA and MMP-9levels on cellular migration and invasion, wound healing andtranswell invasion assays were studied. The cellular migrationcapability was strongest and the wound was completely closedat 48 h with anisomycin treatment, while the capability wasweakest with SU11274 treatment (Fig. 3a and b). Similar to themigration properties, the number of invasive cells was increasedwith anisomycin treatment and conversely, decreased with SU11274treatment (Fig. 3c and d). The invasive ability could be furtherverified by measuring the absorbance at 595 nm (Fig. S12, ESI†).These results implied that the cellular migration and invasion couldbe influenced by miR-221, PTEN mRNA and MMP-9 and be assessedby the fluorescence changes of these components.

Next, the nanoprobe was applied for analyzing the migrationand invasion of normal cells influenced by cancer cells. Twodifferent cell types were chosen: MCF-10A (normal mammaryepithelial cells) and MDA-MB-231 cells (highly metastatic breastcancer cells) which were reported to secrete abundant miR-221via exosomes.12 Furthermore, miRNAs could be internalized byrecipient cells via exosomes and regulate genes and proteinases.13

After the MCF-10A cells were incubated with the nanoprobe, thegreen fluorescence was strong while the red and yellow fluores-cence signals were very weak (Fig. 4). Interestingly, after MCF-10Acells were cultured with MDA-MB-231 medium for six days, thered fluorescence and yellow fluorescence were increased, whilethe green fluorescence was decreased. The results indicatedthat after being treated with the cancer medium, miR-221 andMMP-9 expressions increased and PTEN mRNA expressiondecreased. Therefore, based on the imaging results, the migrationand invasion of the treated MCF-10A cells were predicted to beenhanced. Then the migration and invasion assays of the treatedand untreated MCF-10A cells were further studied. The mobilityof the treated MCF-10A cells was enhanced (Fig. 5a and b).

Similar to migration assay, the invasiveness of the treated MCF-10A cells was improved, whereas the untreated MCF-10A cellsshowed negligible invasiveness (Fig. 5c and d and Fig. S13,ESI†). The migration and invasion of MCF-10A cells wereconsistent with the CLSM results. This reveals that the multicolornanoprobe could investigate cellular migration and invasion bydetecting the expression changes of miR-221, PTEN mRNA andMMP-9.

In conclusion, we have reported a multicolor fluorescentnanoprobe which can investigate cellular migration and invasionby simultaneously monitoring multiple components involved inthe PI3K/AKT pathway. As far as we know, this is the first time thatcellular migration and invasion have been assessed using afluorescent nanoprobe. The nanoprobe consists of AuNPsanchored with recognition units exhibiting high specificityand rapid response. Confocal imaging demonstrated that thenanoprobe could simultaneously detect the expression changesof miR-221, PTEN mRNA and MMP-9 which can cooperatively

Fig. 3 Effects of the variation in miR-221 expression on MCF-7 cellsmigration and invasion. (a) Representative images of wound-healing assaywere captured at 0 h, 12 h, 24 h, and 48 h after wounding with or withoutdrug treatment. (b) Ratio of wound healing at different closure times. (c) Theinvasive cells were fixed, stained and photographed. (d) Quantitative resultsof invading cells. Cells were counted under a microscope from ten randomfields. No significant differences were found.

Fig. 4 Intracellular imaging of miR-221, PTEN mRNA and MMP-9 in MCF-10A cells cultured with and without MDA-MB-231 medium. The nanop-robe (1 nM) was introduced into MCF-10A cells for 4 h at 37 1C beforefluorescence images were captured. Scale bars: 100 mm.

Fig. 5 Effects of the medium from MDA-MB-231 cells on MCF-10A cellsmigration and invasion. (a) Representative images of wound-healing assaywere captured at different times with or without MDA-MB-231 mediumtreatment. (b) Ratio of wound healing at different closure times. (c) Theinvasive MCF-10A cells were fixed, stained and photographed. (d) Quanti-tative results of invading cells. Cells were counted under a microscopefrom ten random fields. No significant differences were found.

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influence cellular mobility and invasiveness. The imaging resultswere further verified by wound healing and transwell invasiveassays. In cancer cells culture medium-treated and untreatedMCF-10A cells, the cellular migration and invasion were also inaccordance with the analysis of CLSM images. The multicolornanoprobe could provide an easy, noninvasive, instantaneousand intuitive way to evaluate cellular mobility and invasiveness.We expect that the nanoprobe will be a promising tool toinvestigate cellular migration and invasion and further establisheffective targeted therapeutic strategy to limit cancer metastasis.

This work was supported by the 973 Program (2013CB933800)and the National Natural Science Foundation of China (21390411,21535004, 21422505, 21375081 and 21505087), and the NaturalScience Foundation for Young Scholars of Shandong Province(JQ201503).

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