Journal: Nature Communications

Article Title: Amphiphilic nanocarrier-induced modulation of PLK1 and miR-34a leads to improved therapeutic response in pancreatic cancer

doi: 10.1038/s41467-017-02283-9

Figure Lengend Snippet: High miR-34a/low PLK1 levels are associated with prolonged survival of PDAC patients. a Kaplan–Meier curves representing the percent OS in PDAC patients based on combined miR-34a and PLK1 expression levels in the TCGA data set ( n = 180). Statistical significance between miRNA/mRNA expression and OS was determined by the Log-rank test ( P < 0.05). b High miR-34a levels in FFPE specimens of PDAC patients exhibiting long-term survival (LTS, n = 7) compared to patients exhibiting short-term survival (STS, n = 3) analyzed by qRT-PCR. c Representative images of PLK1 immunostaining from the same PDAC patients showing low PLK1 levels in LTS compared to STS. Scale bar, 100 μm. d Quantification of PLK1 immunostaining presented in c showing negative correlation to miR-34a levels shown in b in PDAC patients. Data represent mean ± SD. (Student’s t -test)

Article Snippet: Sections were also stained with rabbit anti-PLK1 (1:50, cat# bs-3535R, Bioss Antibodies), rabbit anti-MYC (1:100, cat# 10828-1-AP, Proteintech) and mouse anti-αSMA (1:300, cat# A2547, Sigma - Aldrich).

Techniques: Expressing, Quantitative RT-PCR, Immunostaining

Journal: Nature Communications

Article Title: Amphiphilic nanocarrier-induced modulation of PLK1 and miR-34a leads to improved therapeutic response in pancreatic cancer

doi: 10.1038/s41467-017-02283-9

Figure Lengend Snippet: Physicochemical characterization of APA-miRNA–siRNA polyplexes. a Schematic illustration of APA nanocarrier complexed with small RNAs (polyplex) and chemical structure of APA polymeric nanocarrier. b Polyplex formation of APA with miR-34a and PLK1-siRNA (total of 50 pmol oligonucleotides, miRNA/siRNA ratio of 1:1) showing the optimal Nitrogen/Phosphate (N/P) ratio using EMSA. c Hydrodynamic diameter and surface charge of the polyplex at N/P ratio of 2, measured by particle size analyzer and Zetasizer, respectively. d Representative TEM images of the polyplex. e miR-34a release from the polyplex obtained in vitro by the polyanion heparin displacement assay. f miR-34a release from the polyplex by cathepsin B (2 units per mg polymer) cleavage of the PGA backbone. g Direct labeling of active cathespins in PDAC tumor xenograft and in normal adjacent tissues. Frozen sections were fixed on slides, incubated with 0.25 µM Cy5-labeled cathepsin activity-based probe (in red), stained with 4′,6-diamidino-2-phenylindole (DAPI, in blue) and imaged with fluorescent microscope. For specificity of staining, additional slides were treated with a non-labeled cathepsin inhibitor (GB111, 5 µM) prior to incubation with the Cy5-labeled cathepsin activity-based probe (right image). Scale bar, 10 µm

Article Snippet: Sections were also stained with rabbit anti-PLK1 (1:50, cat# bs-3535R, Bioss Antibodies), rabbit anti-MYC (1:100, cat# 10828-1-AP, Proteintech) and mouse anti-αSMA (1:300, cat# A2547, Sigma - Aldrich).

Techniques: In Vitro, Labeling, Incubation, Activity Assay, Staining, Microscopy

Journal: Nature Communications

Article Title: Amphiphilic nanocarrier-induced modulation of PLK1 and miR-34a leads to improved therapeutic response in pancreatic cancer

doi: 10.1038/s41467-017-02283-9

Figure Lengend Snippet: Proliferation, migration and survival inhibition of MiaPaCa2 cells following treatment with APA nanocarrier containing miRNA–siRNA combination. a miR-34a levels in MiaPaCa2 cells following treatment with APA polyplexes containing miR-34a or NC-miR, quantified relative to U6 RNA using qRT-PCR, showing in vitro delivery efficacy by APA nanocarrier. Untreated cells served as control. b miR-34a’s target genes (CDK6, MET, Notch, and Bcl2) levels 48 h following the same treatment as in a . c PLK1 mRNA levels following treatment with APA polyplexes containing PLK1-siRNA or NC-siRNA for 24 h, quantified relative to GAPDH mRNA using qRT-PCR. d PLK1 protein levels following the same treatment as in c . Densitometric analysis of western blot is presented as percentage of band intensity compared to untreated cells (unit). e – g Proliferation of MiaPaCa2 cells following treatment with APA polyplexes containing different concentrations of miR-34a or NC-miR e , PLK1-siRNA or NC-siRNA f , or miR-34a (100 nM) and PLK1-siRNA (50 nM) combination g . Statistical significance is shown for the comparison between miR-34a and NC-miR in e and between PLK1-siRNA and NC-siRNA in f ( n = 3). h , i Migration of MiaPaCa2 cells 48 h following incubation with the same treatments as in g . Representative images of the cells from 0 to 48 h time points h quantified as wound confluence (percent out of initial wound at time 0) using the IncuCyte software i . (2 biological repeats were done in triplicates). j Representative images of cell survival via colony formation assay for 11 days. k Quantification of colonies from 3 biological repeats as their total area relative to untreated cells (control) using ImageJ software (experiments were done in triplicates). Data represent mean ± SD. (Student’s t -test, NS; not significant for P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001.)

Article Snippet: Sections were also stained with rabbit anti-PLK1 (1:50, cat# bs-3535R, Bioss Antibodies), rabbit anti-MYC (1:100, cat# 10828-1-AP, Proteintech) and mouse anti-αSMA (1:300, cat# A2547, Sigma - Aldrich).

Techniques: Migration, Inhibition, Quantitative RT-PCR, In Vitro, Western Blot, Incubation, Software, Colony Assay

Journal: Nature Communications

Article Title: Amphiphilic nanocarrier-induced modulation of PLK1 and miR-34a leads to improved therapeutic response in pancreatic cancer

doi: 10.1038/s41467-017-02283-9

Figure Lengend Snippet: Biocompatibility of APA-siRNA polyplex. a APA alone or complexed with siRNA (50 and 200 nM) was added to freshly isolated human PBMCs that were seeded in 12-well plates. PBMCs medium and LPS (2 µg ml −1 ) were used as negative and positive controls, respectively. Culture supernatants were collected after 24 h and assayed for human IL-6 and TNF-α cytokines by ELISA. b miR (35 μM) alone or complexed with APA was incubated in FBS for several time points (0, 1, 3, 6 and 12 h) at 37 °C and was run on an electrophoresis agarose gel. c Red blood cells lysis assay following 1 h incubation with APA-miRNA polyplexes. Results are presented as percentage of hemoglobin released by 1 wt %/vol solution of Triton X-100 (100% lysis). Sodium dodecyl sulfate (SDS) and dextran were used as positive and negative controls, respectively. d H&E staining of normal pancreas following 3 sequential i.v. injections of PBS or APA-miR-34a-PLK1-siRNA polyplex (2 mg kg −1 oligonucleotide dose). Scale bar, 10 μm. e Blood glucose levels of normal mice treated as in d . Blood was withdrawn at days 0, 2, 7, and 9 from treatment initiation ( n = 2/3). Data represent mean ± SD

Article Snippet: Sections were also stained with rabbit anti-PLK1 (1:50, cat# bs-3535R, Bioss Antibodies), rabbit anti-MYC (1:100, cat# 10828-1-AP, Proteintech) and mouse anti-αSMA (1:300, cat# A2547, Sigma - Aldrich).

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Incubation, Electrophoresis, Agarose Gel Electrophoresis, Lysis, Staining

Journal: Nature Communications

Article Title: Amphiphilic nanocarrier-induced modulation of PLK1 and miR-34a leads to improved therapeutic response in pancreatic cancer

doi: 10.1038/s41467-017-02283-9

Figure Lengend Snippet: In vivo antitumor effect of miRNA–siRNA combination. a Trial design for testing miRNA–siRNA combination efficacy in the orthotopic PDAC model. b Tumor growth curves from biweekly fluorescent measurements of tumor-bearing mice treated with APA complexed with miR-34a/PLK1-siRNA, miR-34a/NC-siRNA, PLK1-siRNA/NC-miR, NC-miR/NC-siRNA or PBS (treatments are marked with arrows). ( n = 6, 7). Data represent mean ± SEM. One way ANOVA. c In vivo toxicity via mouse body weight evaluation. Data represent mean ± SEM. d An image of a representative mouse from each treatment group 33 days post tumor inoculation showing the difference in tumor fluorescent signal. e Kaplan–Meier survival graph. Log-Rank test, P < 0.05 for the combination miR-34a/PLK1-siRNA compared to all other treatment groups. f Effect of miR-34a-PLK1-siRNA combined treatment on proliferation, apoptosis and angiogenesis in MiaPaCa2 orthotopic xenograft tumors. Representative images of H&E, Ki67, cleaved caspase 3 and CD31 immunostaining of tumors from the different treatments, following 45 days from tumor inoculation, are shown (8–10 fields per slide). Scale bars, 200 µm. g Quantification of immunostaining that were shown in f . MVD, microvessel density. Data represent mean ± SD. (Student’s t -test, * P < 0.05; ** P < 0.01; *** P < 0.001)

Article Snippet: Sections were also stained with rabbit anti-PLK1 (1:50, cat# bs-3535R, Bioss Antibodies), rabbit anti-MYC (1:100, cat# 10828-1-AP, Proteintech) and mouse anti-αSMA (1:300, cat# A2547, Sigma - Aldrich).

Techniques: In Vivo, Immunostaining

Journal: Nature Communications

Article Title: Amphiphilic nanocarrier-induced modulation of PLK1 and miR-34a leads to improved therapeutic response in pancreatic cancer

doi: 10.1038/s41467-017-02283-9

Figure Lengend Snippet: Synergistic anticancer effect by the combination of the restoration of miR-34a and silencing of PLK1 is via myc. a miR-34a binding site within MYC 3′-UTR. b PLK1 and MYC protein levels in MiaPaCa2 cells transfected with miRNA and siRNA monotherapies and their combination. (Representative blot out of 3 biological repeats is shown). c MYC immunostaining of tumors from the different treatments of the in vivo experiment shown in Fig. 8. d MYC immunostaining of short-term and long-term PDAC FFPE specimens. Representative images are shown. e Quantification of MYC immunostaining based on histology scores (0–3: 0- none, 1- weak, 2- moderate, 3- high). f Cell viability of cMYC overexpressed-MiaPaCa2 cells (transiently transfected with MYC ORF-containing plasmid 24 h prior to treatments) and naive cells following treatment with the combination for 48 h. Immunoblotting of MYC is depicted beneath the graph ( n = 3 biological repeats). g Proposed model of synergism via MYC as a common target for miR-34a and PLK1. STS; short-term survivors, LTS; long-term survivors. cMYC OE; cMYC overexpression. Data represent mean ± SD. (Student’s t -test, NS; not significant for P > 0.05, * P < 0.05, ** P < 0.01)

Article Snippet: Sections were also stained with rabbit anti-PLK1 (1:50, cat# bs-3535R, Bioss Antibodies), rabbit anti-MYC (1:100, cat# 10828-1-AP, Proteintech) and mouse anti-αSMA (1:300, cat# A2547, Sigma - Aldrich).

Techniques: Binding Assay, Transfection, Immunostaining, In Vivo, Plasmid Preparation, Western Blot, Over Expression

Journal: Cancer research

Article Title: Polo-like Kinase 1 Is Involved in Invasion through Extracellular Matrix

doi: 10.1158/0008-5472.CAN-07-2348

Figure Lengend Snippet: PLK1 expression is necessary but not sufficient for invasion. A, schematic presentation of the HMT-3522 model of metaplastic breast cancer progression (13). B, RT-PCR analysis for mRNA and Western blot for protein levels of PLK1. S1 cells in two-dimensional or three-dimensional lrECM were grown in the absence of epidermal growth factor (EGF) to completely growth arrest cells as a negative control for PLK1 signal. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. C, Western blot for PLK1 in cells transfected with siRNA to PLK1 versus scrambled control siRNA (Scr.); 44% and 46% reduction in T4-2 and S3-C cells, respectively. Invasion assay for T4-2 or S3-C cells (induced by T4-2 conditioned medium) after cells were transfected with PLK1 or scrambled control siRNA; three experiments, duplicate samples. D, the percentage of Ki67-positive T4-2 cells transfected with PLK1 (◆) or scrambled control (■) siRNAs at indicated time points after release from synchrony; invasion assay for these T4-2 cells. E, the percentage of TUNEL-positive T4-2 cells after transfection with the PLK1 or scrambled control siRNAs; two experiments, duplicate samples.

Article Snippet: Tissues were blocked in 1.5% normal horse serum in PBS and incubated with 10 μg/mL of PLK1 antibody [anti-PLK1, human (rabbit); Calbiochem].

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Negative Control, Transfection, Control, Invasion Assay, TUNEL Assay

Journal: Cancer research

Article Title: Polo-like Kinase 1 Is Involved in Invasion through Extracellular Matrix

doi: 10.1158/0008-5472.CAN-07-2348

Figure Lengend Snippet: Vimentin (VIM) and β1 integrin are necessary for PLK1-mediated invasion. A, the mechanism proposed. B, Western blot for vimentin in T4-2 cells transfected with the indicated siRNAs. Vim2 used for subsequent experiments. Invasion assay for T4-2 cells transfected with the indicated siRNAs. Four experiments, duplicate samples. C, cell surface expression of β1 integrin. Four experiments. Values normalized to S1. P < 0.05, between T4-2 and all other cell types. Invasion assay for T4-2 cells treated with the indicated amounts of β1 integrin blocking antibody A2BII. P < 0.05, compared with untreated control, six experiments. D, invasion assay for T4-2 cells treated with siRNAs against PLK1 or vimentin, or β1 blocking antibody, in combinations indicated; four experiments, duplicate samples. P < 0.05, compared with scrambled control siRNA.

Article Snippet: Tissues were blocked in 1.5% normal horse serum in PBS and incubated with 10 μg/mL of PLK1 antibody [anti-PLK1, human (rabbit); Calbiochem].

Techniques: Western Blot, Transfection, Invasion Assay, Expressing, Blocking Assay, Control

Journal: Cancer research

Article Title: Polo-like Kinase 1 Is Involved in Invasion through Extracellular Matrix

doi: 10.1158/0008-5472.CAN-07-2348

Figure Lengend Snippet: PLK1 affects invasion via phosphorylating vimentin and down-regulating cell surface β1 integrin. A, Western blot for Ser82 phosphorylated vimentin in T4-2 cells treated with the indicated siRNAs. B, Western blot for Ser82 phosphorylated vimentin in T4-2 cells infected with lentivirus expressing WT vimentin pVIM (WT) or mutated vimentin pVIM (S82A). Invasion assay for T4-2 samples infected with lentivirus expressing a WT vimentin pVIM (WT) or mutated vimentin pVIM (S82A). Normalized to WT values. P = 0.036, three experiments, triplicate samples. C, cell surface expression of β1 integrin on T4-2 cells treated with the indicated siRNAs. P = 0.0007 (PLK1-scrambled control siRNA) and 0.003 (vimentin-scrambled control siRNA); four experiments. D, cell surface β1 integrin levels (totaland active, as indicated), normalized to T4-2 cells expressing WT vimentin; four experiments.

Article Snippet: Tissues were blocked in 1.5% normal horse serum in PBS and incubated with 10 μg/mL of PLK1 antibody [anti-PLK1, human (rabbit); Calbiochem].

Techniques: Western Blot, Infection, Expressing, Invasion Assay, Control

Journal: Cancer research

Article Title: Polo-like Kinase 1 Is Involved in Invasion through Extracellular Matrix

doi: 10.1158/0008-5472.CAN-07-2348

Figure Lengend Snippet: PLK1 in vivo. A, frequency of tumor formation and mean tumor volumes in fat pad. T4-2 transfected with siRNA against PLK1 (n = 10) versus scrambled control siRNA (n = 5). B, control experiment for immunohistochemical detection of PLK1, comparing mock antibody with PLK1 antibody-treated samples. Bar, 100 µm. C, example; PLK1 immunohistochemical signal in normal, in situ, and invasive samples from the same patient. Bar, 100 µm. D, PLK1 signal intensity. Two normal (N) and two invasive (I) cases as controls, compared with eight cases each containing areas of normal as well as in situ/preinvasive (P) and invasive carcinomas.

Article Snippet: Tissues were blocked in 1.5% normal horse serum in PBS and incubated with 10 μg/mL of PLK1 antibody [anti-PLK1, human (rabbit); Calbiochem].

Techniques: In Vivo, Transfection, Control, Immunohistochemical staining, In Situ