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Proteomic analysis and NIR-II imaging of MCM2 protein in hepatocellular carcinoma Jing Yang, Qi Xie, Hui Zhou, Lei Chang, Wei Wei, Yin Wang, Hong Li, Zixin Deng, Yuling Xiao, Junzhu Wu, Ping Xu, and Xuechuan Hong J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00181 • Publication Date (Web): 11 May 2018 Downloaded from http://pubs.acs.org on May 12, 2018

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Journal of Proteome Research

Proteomic analysis and NIR-II imaging of MCM2 protein in hepatocellular carcinoma Jing Yang†,#,※ , Qi Xie†,#,§,※, Hui Zhou†,※, Lei Chang#,※, Wei Wei#, Yin Wang†,#, Hong Li⊥, Zixin Deng†, Yuling Xiao†, Junzhu Wu §,*, Ping Xu†,#,Ψ,* and Xuechuan Hong†,∇ ,Π,*

†

State Key Laboratory of Virology, Key Laboratory of Combinatorial Biosynthesis and Drug

Discovery (MOE) and Zhongnan Hospital of Wuhan University, Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China #

State Key Laboratory of Proteomics, National Center for Protein Sciences (Beijing), Beijing

Proteome Research Center, Beijing Institute of Lifeomics, Beijing 102206, China §

Center for Experimental Basic Medical Education, School of Basic Medical Sciences, Hubei

Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals and Hubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan University, Wuhan 430071, China ∇ Medical College, Tibet University, Lasa, 850000, China ⊥

Ψ

Pathology Department, Binzhou Medical University Hospital, Binzhou, 256600, China

Anhui Medical University, Hefei 230032, China

Lead Corresponding Author *Tel: (86)-027-68759887. Fax：(86)-027-68759987. Email: [email protected].

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ABSTRACT Targeted therapy of hepatocellular carcinoma (HCC) is essential for improved therapies. Therefore, identification of key targets specifically to HCC is an urgent requirement. Herein, an iTRAQ quantitative proteomic approach was employed to identify differentially expressed proteins in HCC tumor tissues. Of the up-regulated tumor related proteins, minichromosome maintenance 2 (MCM2), a DNA replication licensing factor was one of the most significantly altered proteins, and its overexpression was confirmed using tissue microarray. Clinicopathological analysis of multiple cohorts of HCC patients indicated that overexpression of MCM2 was validated in 89.8% tumor tissues, and strongly correlated with clinical stage. And, siRNAs-mediated repression of MCM2 expression resulted in the significant suppression of HepG2 cell cycle and proliferation through cyclin D-dependent kinases (CDKs) 2/7 pathway. Finally, the first small-molecule based MCM2-targeted NIR-II probe CH1055-MCM2 was concisely generated and subsequently evaluated in mice bearing HepG2 xenograft. The excellent imaging properties such as good tumor uptake, high tumor contrast and specificity were achieved in the small animal models. This analytical strategy can uncover novel accessible targets of HCC useful for imaging and therapy.

Key words: Proteomics, NIR-II imaging, Targets, Minichromosome maintenance proteins

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■ INTRODUCTION HCC is the commonest primary hepatic malignancy and globally the third leading cause of cancer-related death. Annually over 748,000 new cases develop worldwide, and an estimated 700,000 die from this disease1,2. Many patients are either inoperable at diagnosis or have recurrent cancer after hepatic resection with curative intent. The 5-year survival rate of HCC is less than 50% and a recurrence rate is as high as 70% 3. In spite of increased research and new treatment modalities, the strategies are still insufficient for improving the prognosis of patients. Recent advances in understanding HCC carcinogenesis and progression from genetic, epigenetic and pathophysiological perspectives have brought to target-oriented therapies, which could greatly improve the clinical management of HCC patients4. Proteomic technologies have greatly improved in the past decades5,6. A proteomic approach has been applied with success in the studies of tumors7–9, via identification of new proteins or peptides specifically expressed in fluids, cancer cells or tissues10–13. Based on its high-throughput capacity, it was suitable to overcome some limitations of current approaches for discovering novel diagnostic and prognostic biomarkers as well as therapeutic targets. To date, a number of highly sensitive and specific biomarkers for diagnosis and prognosis of HCC have been reported. MCM2-7 have been reported to compose the eukaryotic replicative helicase, unwinding duplex DNA for the initiation and elongation phases during DNA synthesis through improving the progression and maintaining the stability of the replication fork14. Previous studies had reported MCM2 and MCM6 proteins were up-regulated in HCC tissues15 and were the “poor prognostic 3



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signatures” that are linked to adverse clinical outcome15–18. However, the specificity of MCM2 in HCC in vivo and its mechanism were not well defined. It has been reported that the mRNA and protein levels of MCM6 were shown to be higher in the plasma of 61 HCC cases than that in 30 healthy individuals19. In addition, Qu et al. also demonstrated that MCM7 was significantly overexpressed in tumor tissues and was associated with a worse overall survival in 153 HCC samples20,21. Previously, we have identified protein filamin C significantly overexpressed with the development of HCC through iTRAQ proteomics22. However, proteomic analysis of normal liver and diseased tissues has yielded thousands of proteins and peptides for diagnostic and tissue assignment and required overwhelm in vivo target validation process. Reducing tissue data complexity to a manageable subset of candidates most relevant to targeting, imaging and treating disease is clearly desired. Thus, new strategies are required to filter the overabundance of molecular information to allow rapid discovery and validation of specific targets in real-time that can direct molecular imaging and therapy in vivo23. Fluorescence imaging in the second near-infrared window (NIR-II, 1000-1700 nm) with diminished tissue auto-fluorescence, reduced photon scattering, and a deep penetration depth has been widely used in biological and biomedical research recently24–26.

Small-molecule

NIR-II

fluorescently-labeled

probes

such

as

CH105527-30, H131, Q4-132 provide the advantage of superior signal-to-background ratios, the ability to differentiate even subtle cellular differences, which could be useful in the identification and clinical monitoring of potential drug targets in HCC in 4


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vivo. In this study, proteomics and NIR-II imaging were integrated to gain quantitative sights into new molecular targets in a noninvasive manner for HCC treatment (Figure 1). An evolutionarily conserved group of proteins MCM2-7 which are essential for DNA replication were significantly enriched in HCC tumor tissues through iTRAQ proteomics. Especially, the overexpression of MCM2 was confirmed using tissue microarray, HCCs RNAseq data from the Cancer Genome Atlas (TCGA) database33, ex vivo and in vivo NIR-II imaging. Clinicopathological analyses revealed that overexpression of MCM2 was closely associated with hepatocarcinogenesis. This novel integrated analytical strategy described here is broadly applicable to any system and is particularly important to uncover novel accessible targets of HCC useful for imaging and therapy in a noninvasive manner.

Figure 1. Schematic illustration of proteomic and NIR-NIR-II imaging analysis of MCM2 in the present study for uncovering novel accessible targets of HCC.

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■ EXPERIMENTAL METHODS Clinical Specimens. Paired HCC tumor and adjacent samples used for proteomics were provided by Zhongshan Hospital (Shanghai, China), and tumor tissues were classified at stage I by an oncologist of Zhongshan Hospital according to the principle of Tumor-Node-Metastasis (TNM) staging system published by the Union for International Cancer Control (UICC)22. Paraffin sections of HCC tumor and adjacent tissues were provided by Binzhou Medical University Hospital (Shandong, China). Their clinical characteristics were displayed in detail in Table S1. The tissue microarray of 88 HCC cases of tumor and adjacent tissues were acquired from Outdo Biotech Co. LTD (Shanghai, China), and the clinicopathological factors of these cases associated with tissue microarray were described in detail (Table 1) that HCC cases were almost developed from liver cirrhosis with variable levels of α-fetoprotein (AFP), the tumor size and clinical stages. All participants were giving written informed consent for their participation, and this study was approved by the local ethics committee. Table 1 Clinical features of tissue microarray cases (y-is years old, NA-is not apllicable) Clinical variable

Number

Etiology (HBV/HCV/nonviral/NA) Gender (male/female) Age (≥50 y/＜50 y) AFP (＞300 ng/mL/≤300 ng/mL) ALT (＞50 U/L/≤50 U/L) Cirrhosis (yes/no) Tumor size (＞5 cm/≤5 cm) Clinical stage (II-III/I)

77/1/9/1 72/16 50/38 35/53 31/57 76/12 41/47 86/2 6


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HCC Sample Preparation, iTRAQ labeling and LC-MS/MS Analysis. Paired tumor tissues of stage I and adjacent tissues from six HCC cases were analyzed through iTRAQ quantitative proteomics. As shown in Figure S1, tissues were homogenized and lysed with lysis buffer; the total cell lysis (TCL) of each sample was separated with short 10% SDS-PAGE, cut and sliced into small particles, digested into peptides with trypsin. Tryptic peptides of each sample were labeled as follows: tumor tissues labeled with 113 and 115, adjacent tissues were 114 and 116. 115 and 116 were technology duplications. Labeled peptides were mixed and separated into 36 fractions through RP-HPLC and analyzed through a low pH RP chromatography coupled with tandem mass spectrometry (LC-MS/MS) on an LTQ Qrbitrap Velos Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a nanoAcquity Ultra Performance LC (UPLC) system (Waters Corporation, Milford, MA, USA). The exhaustive parameters of the protein extraction, digestion, iTRAQ labeling, RP-HPLC peptide separation and LC-MS/MS analysis had all been described previously22. Cell Culture. HepG2 cells were bought from the National Infrastructure of Cell Line Resource (Beijing, China), and cultured with Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% FBS bought from the same company at 37 °C in a humidified incubator containing 5% CO2. MCM2 Silencing. HepG2 cells were transfected with 100 nM siRNA against MCM2 (siMCM2) or scrambled siRNA oligonucleotides without effects (Scramble) 7



through Lipofectamine® 3000 (Thermo Fisher Scientific, Waltham, MA, USA) when HepG2 cells were up to 60% confluent. The siRNA sequence of the targeting MCM2 was as follows: MCM2 (5’-GGAGCUCAUUGGAGAUGGCAUGGAA-3’)34. Nuclear Protein Sample Preparation and LC-MS/MS Analysis. Cells were harvested and lysed 48 h after transfection with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Nuclear protein samples after siRNA transfection were analyzed through label-free quantitative proteomics and the process was as follows: 50 µg nuclear protein of each sample was separated by 10% SDS-PAGE, cut into 6 fractions, sliced into particles, then digested into peptides with trypsin (10 ng/µL) and followed by analysis using the Eksigent nanoLC 425 LC (AB Sciex, Foster City, CA, USA) system connected to a TripleTOF 6600 system (AB Sciex, Foster City, CA, USA)35. Proteomic Data Analysis. Proteins were identified and quantified through submitting raw files produced by MS to the search engine MaxQuant (v1.5.8.3)36. The target-decoy search strategy was used for evaluating the false discovery rate (FDR). The parameter settings for database searching through MaxQuant were as follows: precursor ion spectra were set at a tolerance of 20 ppm, two missed cleavages were permitted, and a peptide-spectra match (PSM) was performed on a mini peptide length of 7 amino acids. The FDR allowing for credible proteins and peptides was less than 1.0%. For quantification through iTRAQ labeling, carbamidomethylated cysteine, iTRAQ-modified lysine residue and iTRAQ-modified N-terminal residue were set as 8


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fixed modifications, and oxidation of methionine was set as a variable modification. For label-free quantification, carbamidomethylated cysteine was defined as a fixed modification and oxidation of methionine as a variable modification. The quantification of proteins was based on the reporter ion intensity quantified for the identified unique and razor peptides. The global normalization was based on the assumptions that the total intensity of each sample was approximately equal37. Proteomic data of tumor and adjacent tissues of stage I cases of HCC was used in this study, it was normalized as 4-plex iTRAQ labeling before analysis. The p value was calculated by the “significance B” of Perseus (v1.5.8.5)38,39 with intensity and Log2 ratio of total proteins. Differentially expressed proteins were defined as statistically significant based on the threshold of Log2 ratio (tumor/adjacent) of ± 1.0 (t test, p < 0.05). The enrichment analysis of differentially expressed proteins was applied through the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) analysis of the DAVID Functional Annotation Bioinformatics Microarray Analysis (https://david.ncifcrf.gov/) and the ClueGO of the Cytoscape (v3.5.0)40. The analysis of MS/MS spectra was performed through the pBuild of pFind (v3.0)41 and Xcalibur Qual Browser (v2.2) (Thermo Fisher Scientific, Waltham, MA, USA). RNAseq Data Analysis. The RNAseq data from 373 HCC cases including 373 tumor tissues and 50 adjacent tissues were downloaded from the website (http://www.cbioportal.org/) of TCGA database. 373 tumor tissues were divided into four clinical stages: stage I (172 cases), stage II (86 cases), stage III (85 cases) and 9



stage IV (5 cases). Cases of stage IV was omitted for further analysis because of the limited number. For the RNAseq data, Z-scores had been precomputed from the expression values42. Z-scores for mRNA expression were determined for each case by comparing with all cases with mRNA data respectively (Z-score = (expression in tumor sample - mean expression in reference sample) / SD of expression in reference sample)42,43. Immunohistochemistry. Antibodies against MCM2 (Abcam, Cambridge, UK) were used according to provided instructions. Secondary antibodies, goat anti-rabbit and goat anti-mouse IgG conjugated with horseradish peroxidase (HRP), were purchased from Kangwei Century Co. LTD (Beijing, China). Every step, including tissues blocking and incubation with the first and the second antibodies, was processed according to the standard procedure. The staining result was semi-quantitatively using “-/+/++/+++” by two pathologists of Zhongshan Hospital. “-” represented no membranous staining in any of tissues. “+” indicated a weak staining intensity of 10%-30% of cells. A strong staining intensity of 10%-30% of cells or weak intensity of 30%-50% of cells was indicated by “++”. And, “+++” suggested more than 30% of cells were stained with strong intensity or more than 50% of cells were stained with weak intensity44. Quantitative Real time-PCR Analysis. Cells were harvested 48 h after siRNA transfection and the total RNA was extracted with the TRIzol® Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instruction. Reversed transcription was performed with the ReverTra Ace qPCR RT Kit 10


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(TOYOBO, Osaka, Japan). The primer sequence of MCM2 and the calibrator glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was as follows: MCM2 (forward

5’-GCCCGCTACCTTTCATTCC-3’,

5’-TGACGAGCCTTATCCACCAA-3’);

GAPDH

reverse (forward

5’-GAAGATGGTGATGGGATTTC-3’, reverse 5’-GAAGGTGAAGGTCGGAGT-3’). The relative expression level of MCM2 was normalized to GAPDH by using the 2-△△CT method45. Western Blot Analysis. The total cell lysate of LO2, HepG2, HCC 97H and Hep3B were analyzed with MCM2 (Abcam, Cambridge, UK), GAPDH antibodies (Abcam, Cambridge, UK) through Western blot. The total nuclear protein lysate of siMCM2 and Scramble samples was analyzed with antibodies against MCM3, MCM4 (Abcam, Cambridge, UK), CDK2, CDK7 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and GAPDH through Western blot. Cell Cycle Analysis. Cells were collected 48 h after siRNA transfection, fixed in 70% ethanol at 4 °C for 30 min, centrifuged, resuspended with 0.5 mL PBS buffer and stained with propidium iodide (PI) solution (PBS containing 20 µg/mL PI, 100 µg/mL RNase A and 1% Triton-X 100) at room temperature for 30 min in dark. The DNA content was analyzed by Flow cytometry (BD biosciences, San Diego, California, USA) and cell number of each phase of cell cycle was estimated with Modfit software. Cell Proliferation Analysis. For cell proliferation analysis, 8000 cells per well were added into a 96-well plate with two replications per condition. 100 nM siRNA 11



targeting MCM2 or scramble siRNA was added the next day, and cells of each well were detected at 24, 48 and 72 h after siRNA transfection with Cell Counting Kit 8 (Dojindo, Tokyo, Japan). Animal Model. 6-week-old female BALB/c nude mice (body weight 20 g-22 g) to establish the nude mouse tumor model were bought from Charles River Laboratories (Beijing, China). Approximately 2 × 106 HepG2 cells diluted in 70 μL PBS buffer were injected into the right leg of nude mice by subcutaneous injection. Tumor growth was monitored daily through measuring the length (the longest dimension), width (the distance perpendicular to and in the same plane as the length), and height (the distance between the exterior tumor edge and the mouse's body) of each tumor or lobe with vernier calipers46. Animals were imaged about 3 weeks after inoculation when the maximal diameter of tumor was approximately 6.0 mm in size. The animal experiment in NIR-II imaging studies were done in the Center for Animal Experiment of Wuhan University (Wuhan, Hubei, P.R. China), which has been accredited by Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International), and all animal studies were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Chinese Animal Welfare Committee and approved by the Institutional Animal Care and Use Committee (IACUC), Wuhan University Center for Animal Experiment, Wuhan, China. MCM2-targeted of HCC Tumors through NIR-II Imaging. The synthesis of the imaging probe CH1055-MCM2 was described in supporting information. For 12


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tumor size 6.0 mm and 7.2 mm group mice, synthesized imaging probe CH1055-MCM2 were injected into HepG2 tumor-bearing nude mice through tail vein. For the blocking group mice with tumor size 7.0 mm, excessive MCM2 antibodies about 300 µg as blocking agents was firstly injected into nude mice, imaging probe CH1055- MCM2 (50 µg) was injected 30 minutes after injection of MCM2 antibodies. Nude mice were mounted on the imaging stage in the prone position beneath the laser at different time point after injection. The excitation light was provided by an 808 nm diode laser. The emitted light from the animal was filtered through a 1000 nm long-pass filter coupled with the InGaAs camera (Suzhou, China) for NIR-Ⅱ imaging. The exposure time for all images was 120 ms. Ex vivo Biodistribution Analysis. Ex vivo fluorescence imaging of organs and tissues were used Suzhou Optics NIR-II fluorescence imaging system with an InGaAs camera at a power density of approximately 82 mW / cm2 and an exposure time of 120 ms at 808 nm laser diode irradiation. After injection of CH1055-MCM2 for 48 h, HepG2 xenograft mice were sacrificed and NIR-II images of major organs were collected. Statistics analysis. All data in this study were analyzed through Graphpad prism (v5.01) and Perseus (v1.5.8.5). Statistical comparisons were evaluated by the t test analysis and one-way ANOVA analysis for RNAseq data, and the significance of MCM2 expression in tumor tissues was determined through the Fisher's exact test and chi-square test. Summary data were showed as the mean ± SD. It was considered statistically significant when the p value less than 0.05. 13



■ RESULTS AND DISCUSSION Proteomic Profiling of Differentially Expressed Proteins in HCC Tumor Tissues. To identify differentially expressed proteins in HCC at TNM stage I and adjacent HCC tissues from 6 patients (mean age, 53 ± 9 years; range, 44–60 years), a quantitative iTRAQ proteomic study was designed and performed (Figure S1)22. According to these criteria, a total of 4620 proteins were identified, and 3475 proteins with more than one unique peptide were quantified both in HCC tumors and adjacent tissues in our study which is comparable to our previous results. Of these, 112 proteins were identified as up-regulated and 146 proteins were identified as down-regulated in tumor tissues (Table S2). The down-regulated proteins were mainly clustered in metabolism while the up-regulated proteins were involved in diverse biological processes, including ribosome, cell cycle, spliceosome and DNA replication (Figure 2A, Figure 2B and Figure S2). DNA replication proteins and cell cycle regulatory proteins were closely associated with malignant proliferation of tumor cells. These proteins are increased in cell division and rapidly down-regulated in cells undergoing differentiation or quiescence, and serve as specific markers for proliferating cells47. MCMs were identified with most significant up-regulated proteins both in the cell cycle and DNA replication pathways that these MCMs were up-regulated over 1.6-fold in HCC tissues compared to adjacent HCC tissues (p < 0.01) (Figure 2C and Figure 2D). MCMs are divided into 10 subgroups based on amino acid sequences and functional similarities48. The members of the MCM2-7 14


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subgroup form hetero-hexametric channels that contain either identical or different subunits and function as a replicative helicase unfolding duplex DNA into single-stranded DNA during DNA replication14,47. Among MCM proteins, MCM2 was found to show one of the most significant differences in expression between HCC and adjacent tissues (p < 0.01) with a nearly perfect MS/MS peptide matching (Figure S3) and was up-regulated more than 4-fold in tumor tissues compared with adjacent tissues according to the reporter ions of unique peptides (Figure S4).

Figure 2. Differentially expressed proteins in HCC tumor tissues by a quantitative iTRAQ proteomic analysis. (A) KEGG pathway analysis of up-regulated proteins in HCC tumor tissues. (B) Enriched proteins in cluster analysis. (C) The scatter plot of all quantified proteins. Red represented up-regulated proteins and blue represented down-regulated proteins in HCC tumor tissues. (D) MCM2-7 were up-regulated over 1.6-fold in HCC tumor tissues (p< 0.05). Overexpression of MCM2 in HCC Tumor Tissues. To further investigate the 15



oncogenic properties of MCM2 in HCC, and to evaluate its potential diagnostic and therapeutic value, immunohistochemistry was performed by examination of the MCM2 expression pattern in paraffin-embedded tissues. The expression of MCM2 was confirmed in 12 paired tumor and adjacent tissues from HCC cases through immunostaining with MCM2 antibodies (Figure 3A). All tumor tissues (no = 12), showed positive immunostaining while liver tissues adjacent to HCC (no = 12) demonstrated negative (75%, 9/12) or weak staining (25%, 3/12). Following surgical pathologic staging criteria (established by the International Federation of Gynecology and Obstetrics, 6th Edition, 2002), of the 88 carcinoma samples of tissue microarray (mean age, 53 years; range, 27-84 years), 2 were in stage I, 54 were in stage II, and 32 were in stage III. As shown in Figure 3B, immunoreactivity of MCM2 was weakly positive in 9 patients (10.2%), moderately to highly positive in 70 patients (79.6%), and negative in 9 patients (10.2%). On the contrary, no or weakly positive staining, moderately to highly positive was observed in 68.2%, 27.3% and 4.5% of adjacent tissues, respectively. Furthermore, the immunostaining revealed that the expression of MCM2 was significantly higher in advanced HCC tumors than that in the primary stage. As shown in Figure 3C and 3D, all negative staining of MCM2 were shown in the primary stage I. For stage II and III, over 90% cases exhibited positive staining. Among them, 76.9% cases of stage II were with moderate or strong staining. In 32 stage III specimens, weakly positive staining was detected in 9.4% (3/32), and moderate or strong staining was detected in 90.6% (29/32). Our studies suggested that MCM2 could be developed as an 16


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independent diagnostic factor for HCC.

Figure 3. Overexpression of MCM2 in HCC tumor tissues at protein and mRNA levels. (A) Expression of MCM2 in 12 paraffin-embedded tissues by immunohistochemistry analysis (p < 0.001). The Fisher's exact test was used. (B) MCM2 was overexpressed in tumor tissues in 88 HCC clinic samples by tissue microarray (p < 0.001). (C) MCM2 exhibited higher expression in advanced HCC 17



cases than that in the primary stage. The number of HCC cases in different clinical stages with MCM2 expression was shown in the table, and their percent among the clinical stage was shown in the brackets. The p value was calculated by the chi-square test. (D) The standard grading immunostaining was indicated by “-/+/++/+++”. (E) RNAseq data from TCGA database indicated that the mRNA level of MCM2 in tumor tissues was higher than that in adjacent tissues. ***p< 0.001 (t test). (F) The mRNA expression of MCM2 was gradually increased from stage I to stage III. *p< 0.05, **p< 0.01, ***p< 0.001. Further, a series of validation experiments were carried out to compare its transcripts by analysis of RNAseq data from TCGA database, and the mRNA level of MCM2 was observed in all the carcinoma and adjacent tissues (carcinoma tissues, 373; adjacent tissues, 50). A significant difference in the mRNA level of MCM2 was observed between carcinoma and the corresponding adjacent tissues (p < 0.001) (Figure 3E). As shown in Figure 3F, the mRNA level of MCM2 was progressively increasing from stage I to stage III (one-way ANOVA analysis, p < 0.001). Suppression of MCM2 Decreased Liver Cancer Cell Proliferation. The over-expression of MCM2 was detected in several HCC cell lines including HepG2, HCC 97H and Hep3B compared with the normal liver cell line LO2 (Figure S5). Thus, the dynamics of cell physiology was further explored by treating liver cancer cell lines with MCM2-siRNA. The siRNA oligonucleotides sequence has been used in previous study49 (Figure S6). After transfection MCM2-siRNA for 48 h, mRNA and protein levels of MCM2 were all significantly decreased by quantitative real time PCR and Western blot, respectively (Figure 4A, Figure S7A). The siRNA silencing on the essential roles of MCM2 were in S phase progression of HepG2 cancer cells by flow cytometry analysis and cell proliferation (Figure 4B and Figure 4C). Furthermore, the cell cycle and cell proliferation at S phase in HCC 97H and Hep3B 18


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cells was decreased and the cells were growing at a much lower pace after transfection of MCM2-siRNA (Figure S7). Our data demonstrated that blocking MCM2 influenced the cell proliferation of HepG2, HCC 97H and Hep3B cells.

Figure 4. MCM2 impacted the cell cycle and proliferation of liver cancer cells. (A) Real time PCR and Western blot results suggested that the level of MCM2 was decreased after MCM2-siRNA transfection. (B) The S phase dynamics after MCM2 downregulation. Cell cycle phases including G1, S and G2/M were highlighted. (C) Proliferation curves of HepG2 cells after MCM2-siRNA transfection. The absorbance value at 72 h was significantly down-regulated after MCM2-siRNA transfection. **p < 0.01. For further exploring the plausible mechanism of MCM2 blocking the proliferation of HCC cells, siRNA transfection combined with label-free quantitative proteomics was performed. The nuclear proteins were extracted and analyzed by label-free quantification after siRNA transfection since cell cycle related proteins such as MCM2 were mainly located in the nucleus (Figure 5A). In our proteomic experiment, 1898 proteins were identified, and most proteins were located on the nuclei based on the cellular component of the GO pathway analysis (Figure S8). 19



Among these proteins identified, 158 proteins were differentially expressed including 71 up-regulated and 87 down-regulated proteins (Figure 5B, Table S3). Down-regulated proteins were analyzed through KEGG pathway analysis and enriched in DNA replication, spliceosome, cell cycle and so on which were closely related to the dynamic of cell cycle especially DNA replication and cell cycle pathways (Figure 5C and Figure 5D). Among them, MCM2-7 were both enriched in the DNA replication and cell cycle pathways, and a roughly equal reduction of MCM2-7 was achieved when it was treated with MCM2-siRNA suggesting that individuals of MCM2-7 might form hetero-hexametric channels in HCC (Table S4)49. Recent reports have demonstrated that CDKs can regulate and phosphorylate MCM2-7, and recruite them during DNA synthesis50,51. CDK2/7 proteins also play important roles in cell division and modulate transcription through regulation of the cell cycle52. Particularly, CDK2 combines with cyclin E to form the cyclin E/CDK2 complex for the transition from G1 to S phase of the cell cycle53,54. As shown in Figure 5E and Figure 5F, proteins of the cell cycle pathway and CDK2/7 were significantly decreased based on proteomic data and Western blot analysis after transfection. These data indicated that inhibition of MCM2 expression by siRNA might lead to significant apoptosis induction of HepG2 cells and S arrest in cell cycle distribution in vitro through influencing CDK2/7.

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Figure 5. MCM2 might impact the proliferation of HCC cells through cell cycle related proteins. (A) The workflow of siRNA transfection combined with label-free proteomics analysis. (B) The quantified nuclear proteins. Red plots displayed up-regulated and blue represented down-regulated proteins after siRNA transfection against MCM2. (C) The cluster of down-regulated proteins after siRNA transfection through KEGG pathway analysis. (D) Analysis of down-regulated proteins of the DNA replication and cell cycle pathway through the ClueGO plugin of Cytoscape. (E) Proteins of the cell cycle pathway were down-regulated after MCM2-siRNA transfection. (F) Western blot analysis indicated that MCM3-4, CDK2, CDK7 were reduced after MCM2-siRNA transfection. MCM2-targeted NIR-II Imaging of HCC Tumors. To obtain additional insight 21



into the in vivo effects, we observed the effects and specificity of MCM2 suppression on a tumor xenograft model. Considering the advantages of NIR-II imaging, a novel small-molecule based MCM2-targeted NIR-II probe CH1055-MCM2 was developed and its imaging properties in vivo were investigated (Figure 6A). CH1055-MCM2 was prepared through the direct conjugate addition of one of the maleimide groups of CH1055-PEG4-Su (see Figure S9 and ESI† in supporting information) with a MCM2 targeting antibody. CH1055-MCM2 was purified using HPLC and characterized using MALDI-TOF-MS [calcd. for: 152650, found: m/z 152652.6 (Figure S10)]. The maximum emission wavelength CH1055-MCM2 probe was shown at 1055 nm in PBS buffer (Figure 6B) and the fluorescence quantum yield (QY) of CH1055-MCM2 probe was ~0.2% in water (IR-26 as a reference QY = 0.5%) under 785 nm excitation. The CH1055-MCM2 also exhibited high photostability in PBS, medium and fetal bovine serum (FBS) with negligible decay under continuous excitation for 1 h (Figure 6C). The probe CH1055-MCM2 (50 µg) was injected intravenously through tail vein into nude mice bearing HepG2 xenografts (n = 3). From NIR-II imaging, the blood vessels of the tumor could be clearly visualized from the surrounding background tissue from 6 h to 48 h p.i. (Figure S11). The maximum of the tumor uptake was reached ~12 h (T/N = 4.4) (Figure S12). The targeting specificity of probe CH1055-MCM2 for HCC tumors was confirmed by the blocking experiment for NIR-II imaging (Figure 7). After co-injection of MCM2 antibodies (300 µg), the fluorescence signal of the tumor was particularly weak, indicating the high MCM2 targeting specificity of CH1055-MCM2 probe for HCC tumor tissues (Figure 7A). 22


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The nude mice were sacrificed at 48 h after injection probe to evaluate ex vivo biodistribution of CH1055-MCM2 in major organs (Figure 7B). It was noteworthy that, consistent with the outcomes obtained in vivo results, fluorescence signal was much higher than that in normal organs except liver and kidney. In addition, the fluorescence signal accumulated in kidney and liver suggested that the clearance routes of CH1055-MCM2 were through both hepatobiliary and renal systems. Thus, NIR-II imaging rapidly validated MCM2 as a tumor target that is readily accessible to antibody injected intravenously for tumor targeting and imaging in vivo.

Figure 6. The synthesis and optical properties of CH1055-MCM2. (A) The synthesis of CH1055-MCM2. MCM2 antibody was indicated by the red round. (B) Absorbance and fluorescent emission spectra of CH1055-MCM2 in PBS (105 mW/cm2). (C) Photostability of CH1055-MCM2 in different biological media under continuous 808 nm excitation (82 mW/cm2) for 1 h.

23



Figure 7. MCM2-targeted NIR- II imaging of HCC tumors in vivo and ex vivo. (A) NIR-II images of HepG2 tumor-bearing nude mice including different tumor sizes (6.0 mm and 7.2 mm) and the blocking group at different time points were performed under 808 nm excitation (82 mW/cm2,1000 LP and 120 ms). White arrows and white dotted lines indicated the tumor. (B) The biodistribution of CH1055-MCM2 in HepG2 tumor-bearing nude mice at 48 h under an 808 nm excitation (82 mW/cm2, 1000 LP and 150 ms). White arrows indicated the tumor.

■CONCLUSIONS We describe a new proteomic-based discovery and rapid validation approach in a 24


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noninvasive manner that integrates newly developed, small-molecule based NIR-II imaging and iTRAQ quantitative proteomic techniques to map the proteins overexpressed in vivo in HCC tumors. This approach has demonstrated distinct molecular signatures in adjacent and tumor tissues. This strategy was applied 6 paired tumors and adjacent tissues to uncover, from identified proteins expressed in HCC tissues from 6 patients, approximately 260 differentially expressed proteins, including an evolutionarily conserved group of proteins MCM2-7，DNA replication licensing factors which were significantly enriched in HCC tumor tissues and permitted rapid targeted imaging in vivo. Among them, MCM2 was one of the most significantly altered proteins, and its overexpression was identified and further confirmed by immunostaining and Western blot analysis. After transfection with siRNA-MCM2, our data supported that the MCM2 may impact the proliferation of HepG2 cells at S arrest in cell cycle distribution in vitro through interacting CDK2 and CDK7. Our first MCM2 NIR-II imaging, using intravenously injected CH1055-MCM2 specific to the new protein MCM2 in HCC tumors, visualized targeting and tissue accumulation with high sensitivity and superior resolution. NIR-II imaging thus provided a rigorous and objective validation of accessibility and tissue specificity of the targets and ultimately demonstrated potential utility such as, how rapidly and to what extent the antibody targeted probe targets single organs or solid tumors in vivo. Future studies will focus on the imaging-guided sRNA therapy for HCC and apply this novel integrated analytical strategy broadly applicable to the other malignancy system for imaging and therapy in a noninvasive manner. 25



■ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:10.1021/acs.jproteo-me.XXX Extended methods. Table S2. Upregulated proteins in HCC tumor tissues. Table S3 Downregulated proteins after treated with MCM2 siRNA. ■ AUTHOR INFORMATION Corresponding Authors *Tel: (86)-027-68759887. Fax：(86)-027-68759987. Email: [email protected]. Π

Lead Contact

Dr. Xuechuan Hong ORCID Xuechuan Hong: orcid.org/0000-0001-6834-3278 Author Contributions This manuscript was written through the contributions of all of the authors. All of the authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ※

These authors contributed equally to this work

■ ACKNOWLEDGMENT This work was partially supported by the Chinese National Basic Research Programs 26


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(2017YFC0906600,

2016YFA0501300,

2017YFA0505000,

2017YFD0501500,

2016YFA0100900), NNSCF (81773674, 81573383, 21390402, 31470809, 31670834, 31700723, 81761148029, 2178812, 81425015), ICP (2014DFB30020), IFM (2017CXJJ19, BTPLTS&T 2016ACA126),

16CXZ027&

BWS14J052),

(Z161100004916024), ABRPSTCS

NNSFB

NSFHP

(SYG201521),

(Grant

(2017CFA024, NSFJP

No.

5152008),

2017CFB711,

(BK20160387),

SSTRG

(JCYJ20170303170809222), NSFTAG (2016ZR-15-9), FUYTTAR (QCZ2016-10), FSKLP (SKLP-Y201501), the Fundamental Research Funds for the Central Universities and the Open Research Fund Program of the Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals.

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For TOC only:

A new proteomic-based discovery and rapid validation approach in a noninvasive manner that integrates newly developed, small-molecule based NIR-II imaging and iTRAQ quantitative proteomic techniques to map MCM2 overexpressed in vivo in HCC tumors. This analytical strategy has demonstrated distinct molecular signatures in adjacent and tumor tissues and can uncover novel accessible targets of HCC useful for imaging and therapy.

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Proteomic Analysis and NIR-II Imaging of MCM2 ... - ACS Publications

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