RETRACTED ARTICLE: Quercetin-induced miR-200b-3p regulates the mode of self-renewing divisions in pancreatic cancer

Pancreatic cancer established cell lines; AsPC1 and PANC1 were obtained from the American Type Culture Collection (Manassas, VA, USA) and recently authenticated by a commercial service (Multiplexion, Heidelberg, Germany). The human, primary pancreatic cancer cell line ASANPaCa was generously provided by Dr. Nathalia Giese and is described [26]. Frozen stocks were prepared from initial stocks, and every 3 months a new frozen stock was used for experiments in order to maintain the authenticity of the cell lines. Monthly examination ensured mycoplasma negative cultures. Cells were cultured in DMEM (PAA, Pasching, Austria) supplemented with 10% heat-inactivated FCS (Sigma, Deisenhoffen, Germany) and 25 mmol/L HEPES (PAA, Pasching, Austria).

All cells were synchronized prior to any experiment by serum starvation as previously described [27, 28]. The cells were seeded with 10% FCS culture medium on the first day. On the second day, the medium was removed and the cells synchronized with 0.1% FCS culture medium for 24 h.

PDA cells were resuspended in tumorsphere DMEM/F12 medium (Thermo Fisher Scientific Inc., Dreieich, Germany) supplemented with B27 supplement (50×) (Thermo Fisher), 20 ng/mL rEGF (Thermo Fisher), 20 ng/mL bFGF (Thermo Fisher), 5 μg/mL I nsulin (Sigma, Deisenhoffen, Germany) and 5 mL Glutamax (Thermo Fisher). To avoid false positives as a result of cell aggregation, 1000 cells in 50 μL tumorsphere medium mixed with 50 μL matrigel were cultured per well of a 24-well plate. The mixture was allowed to polymerize for 1 h at 37 °C and 5% CO₂. Subsequently, 1 mL of the tumorsphere medium was added per well, either alone or supplemented with 50 nM mirVana™ mimics or 50 μM quercetin. Seventy-two hours later, the spheres were collected by gentle centrifugation, dissociated into single cells, and cultured for the formation of next generation spheres. Tumor spheres were counted using an inverted microscope (Nikon Eclipse TS100; Nikon GmbH, Düsseldorf, Germany). To quantify cell numbers, the tumor spheres were collected and disassociated with Trypsin-EDTA (Thermo Fisher) to make a single cell suspension. The viable cells were then counted using the Z™ Series COULTER COUNTER® Cell and Particle Counter (Beckman Coulter, Indianapolis, USA).

Cells were plated at low density in μ-Dish 35 mm (Ibidi, Germany) and placed in the Nikon BioStation IM-Q (Nikon Instruments Europe) compact cell incubator and monitoring system. Cells were photographed every 30 min for about 80 h. The microscope was equipped with an incubator that maintains the temperature at 37 °C, a humidifier that maintains a humidity level of 95% and a stable CO₂ flow of 5%. Time-lapse sequences were combined using FIJI software.

Cells were seeded at a density of 4.5 × 10⁴ cells/well in 6-well cell culture plates. At about 90% cell density, cells were left untransfected, treated with 50 μM quercetin or were transfected with 50 nM mirVana™ mimics of miR-200b-3p or a miR-NC (Thermo Fischer Scientific, Waltham, MA USA) using lipofectamine RNAiMax reagent (Life Technologies, Darmstadt, Germany) for every 72 h for a period of 12 days. The cells were washed with PBS, fixed with methanol for 5 min at room temperature and incubated with Oil Red O staining reagent for 20–30 min on a plate shaker at room temperature. Afterwards, the cells were washed with deionized water. Heterogeneous adipocytes are stained red by the Oil Red O staining solution and visualized with a 10× magnification using an inverted microscope (Nikon Eclipse TS100; Nikon GmbH, Düsseldorf, Germany).

Cells were seeded and treated as described above. The cells were washed with PBS, fixed with 10% neutral buffered formalin for 60 s and incubated in the dark with FAST BCIP/NBT (Sigma) for 10–20 min. Afterwards, cells were washed with deionized water. Heterogeneous alkaline phosphatase expressed by osteoblasts develops the NBT substrate into deep bluish purple color.

Cells were seeded and treated as described above. The cells were washed with PBS and fixed with 10% neutral buffered formalin for 60 min at room temperature. After two rounds of washing with distilled water, the cells were incubated overnight in the dark at room temperature with Alcian blue staining solution (Sigma-Aldrich, St. Louis, Missouri, USA). Heterogeneous chondrogenic differentiation was identified as intense dark-blue coloration due to the formation of aggrecan.

Western blot analyses were done as formerly described [29]. Antibodies used include: rabbit antibodies against human Notch (Cell Signaling Technology, Danvers, USA), Numb (Cell Signaling Technology, Danvers, USA) (Cell Signaling Technology, Danvers, USA), and β-actin (Sigma-Aldrich, St. Louis, Missouri, USA).

Patient materials were obtained under the approval of the ethical committee of the University of Heidelberg after receiving written informed consent from the patients. The diagnoses were established by conventional clinical and histological criteria according to the World Health Organization (WHO). All surgical resections were indicated by the principles and practice of oncological therapy.

Staining was done on 6-μm frozen tissue sections or in established cell lines as previously described [29, 30].

MirVana™ mimics (miR-200b-3p and miR-NC) (Thermo Fisher Scientific Inc., Dreieich, Germany), 50 nM each, were transfected into the cells using Lipofectamine 2000 (Thermo Fisher) or Lipofectamine RNAiMAX transfection reagent (Thermo Fisher) with the reverse transfection method as described in the manufacturer’s instructions.

Cells were seeded at a density of 1 × 10⁴ cells per well of a 96-well plate and co-transfected with 50 nM miR mimics, 25 ng/well of firefly luciferase plasmid and 50 ng/well renilla luciferase reporter construct expressing Notch1 3‘UTR (BioCat, Heidelberg, Germany). The cells were lysed in Passive Lysis Buffer of the Dual Luciferase® Assay System (Promega, Mannheim, Germany) at 48 h post-transfection. Renilla and firefly luciferase (Promega) activities were measured on the FLUOstar OPTIMA instrument (BMG Labtech, Ortenberg, Germany).

This was performed using the Agilent Human miRNA Microarray (Release 19.0) as previously described [24].

The miRNA gene targets’ predictions based on miRanda and TargetScan and PicTar were downloaded from http://​www.​microrna.​org [31], http://​www.​targetscan.​org [32] respectively. “Good” mirSVR score refers to miRNA targets with < −0.1 score, and “non-good” mirSVR score refers to targets with > −0.1 score obtained from the support vector regression algorithm mirSVR, available with miRanda predictions from http://​www.​microrna.​org.

This was done as previously described [24]. cDNA was prepared from 500 ng total RNA using the Taqman® Reverse Transcription Reagents (Thermo Fisher Scientific, Dreieich, Germany) in accordance to the manufacturers’ instructions. Quantitative levels of hsa-miR-200b-3p were measured from 1 μl of synthesized cDNA using their respective primer assays and RNU44 as endogenous control (Applied Biosystems, Darmstadt, Germany). Notch1 and Numbl mRNA levels were quantified from 2 μl of synthesized cDNA using the TaqMan Gene Expression Assay and primers for Notch1, Numbl and GAPDH (Thermo Fisher Scientific). StepOne Real-Time PCR System (Applied Biosystems) was used for the PCR. Changes in the relative concentration were calculated with the second derivative maximum method 2^−ΔCT. ΔCT was calculated by subtracting the CT of the housekeeping gene from the CT of the gene of interest. The fold change was obtained by the equation 2^−ΔΔCT.

The quantitative data are presented as the mean ± SD. The data were analyzed using Student’s t-test for statistical significance. P < 0.05 was deemed to be statistically significant.

It is an established fact that CSCs divide by both symmetric and asymmetric cell divisions [5] with asymmetric division maintaining suitable numbers of cell offspring [6] and symmetric division, imperative for growth and regenerative competences [7]. In addition, Notch and Numb genes are crucial to self-renewing divisions as markers for symmetric and asymmetric division markers respectively [5] (Fig. 1a). Hence, we set out to identify the mode of CSCs divisions occurring in PDA. To this end, firstly, we performed immunofluorescence analysis using PDA patient samples, staining for Notch and Numb. The expression of Notch was observed to be more than Numb. Notch expressions were found mostly at the pancreatic ductal glands, while Numb was mostly expressed at the periphery (Fig. 1b). This observation was verified by double immunofluorescence staining of the ductal marker, cytokeratin with notch; cytokeratin with numb and the human proliferation marker, Ki67 with Notch (Additional file 1: Figure S1). Secondly, we performed time-lapse microscopy, monitoring synchronized single PDA cells from the established and CSC-enriched cell lines AsPC1 and PANC1 [33] for a period of 3 consecutive divisions. Studies show that a cell divides symmetrically, if its first three divisions results to 2, 4 and 8 cells respectively, and asymmetrically if it is 2, 3 and 5 cells instead [7, 34] (Fig. 1c). Our results show that the cells were mostly undergoing symmetric division (Fig. 1d, Additional file 2: Movie S1). The monitored AsPC1 cells had 70% symmetric, 25% asymmetric and 5% ambiguous (Data not shown). The monitored PANC1 cells had 80% symmetric, 10% asymmetric and 10% ambiguous (Data not shown). These observations suggest that both symmetric and asymmetric cell division transpire in PDA with symmetric division being more widespread.

To study a putative effect of quercetin, known to target pancreatic CSCs [35, 36], miR signaling and the mode of CSCs divisions, we performed a miR expression profiling of synchronized AsPC1 cells before and after treatment with quercetin for 12 h. The microarray data with the accession number E-MTAB-4718, was uploaded to Array Express and 105 miRs were found to be differentially expressed. Of these, 80 miRs were upregulated and 25 miRs were down-regulated (Fig. 2a). On the grounds that Notch signaling is implicated in CSCs’ fate determination, we performed in silico analyses, with interest to only the miRs out of the 105 differentially regulated that are involved in Notch signaling. This resulted to only 11 miRs namely; let-7c, miR-200a-3p, miR-200b-3p, miR-103a-3p, miR-1202, miR-125b-5p, miR-1268a, miR-6088, miR-3665, miR-4651 and miR-96-5p (Fig. 2a). The microarray data was validated by qRT-PCR analysis using six out of the 11 miRs (Fig. 2b).

To select the most important candidate miR from the 11, we performed a further in silico studies using the TargetScan® database to evaluate which of these miRs was predicted to target Notch and the human segregating determinants; Numb, Prox1 and Trim (Fig. 2c). MiR-200b-3p was identified as the only candidate (Fig. 2d, Additional file 3: Data 1), thus suggesting that this miR could be a cell fate determinant in PDA.

Considering that the segregating determinants, Numb, Prox1 and Trim directly or indirectly inhibits Notch, we opted to validate Notch1 as a direct target of miR-200b-3p. Whence, we performed a dual luciferase reporter assay using a commercially available renilla luciferase reporter construct expressing the Notch1 3’UTR. AsPC-1, AsanPaCa and PANC-1 cells were co-transfected with a reporter construct of the Notch1 3’ UTR or with control firefly plasmid together with either miR-200b-3p mimic or a miR non-coding control (miR-NC). Forty-eight hours after transfection, the luciferase activity was quantified in a luminescence microplate reader. We found a significantly reduced luciferase activity of the Notch1 reporter in the presence of miR-200b-3p, whereas no reduction of luciferase activity occurred with the empty vector control or in the presence of miR-NC (Fig. 3a). These results fitly show that Notch1 is a direct target gene for miR-200b-3p.

To establish the correlation between the observed down-regulation of miR-200b-3p and upregulation of Notch1 in control PDA cells, we quantified the mRNA and protein expressions of Notch1 and Numb. Our results show upregulation of Notch1 and down-regulation of Numb in control groups but a complete reversal of this phenomenon in miR-200b-3p and quercetin treated groups (Fig. 3b, c), indicating a possible switch from symmetric to asymmetric division. To validate this assumption, we performed a time-lapse microscopy, monitoring synchronized miR-NC and miR-200b-3p transfected AsPC1 cells for 3 division phases over 80 h. We observed that 75% of the control cells divided symmetrically, 20% asymmetrically and 5% ambiguously, while 60% of the miR-200b-3p-transfected cells divided asymmetrically, 30% symmetrically and 10% ambiguously (Fig. 3d). In toto, these results imply that miR-200b-3p switches the mode of CSCs division from symmetric to asymmetric.

Next, we questioned if the switch from symmetric to asymmetric division is associated with CSCs self-renewal and proliferation. Hypothetically, symmetric division would result to more self-renewal and proliferation than ACD by inhibiting Notch signaling (Fig. 4a). Then, we performed serial tumorsphere propagation assays to evaluate the impact of miR-200b-3p on self-renewal and proliferation. Resuspended cells in tumorsphere medium were mixed in a ratio of 1:1 with matrigel and plated in a 24-well plate to avoid false positives and ensure that tumorspheres resulted from single CSCs and not from the aggregation of cells. Upon polymerization, the mixture was covered with unadorned tumorsphere medium (CO) or transfection mixture of the tumorsphere medium with 50nM of miR-NC/miR-200b-3p or 50 μM of quercetin in the tumorsphere medium. Subsequently, the resultant tumorspheres were passaged for three generations. MiR-200b-3p and quercetin significantly decreased tumorsphere formation (Fig. 4b). After being passaged for three generations, the ability to form new tumorspheres was practically lost in cells from the miR-200b-3p and quercetin treated groups whereas cells from the control groups (CO and miR-NC) significantly continued to form tumorspheres (Figs. 4b and 5a). These observations are in coherence with the switch from symmetric to asymmetric division by miR-200b-3p. Next, we evaluated the influence on proliferation by dissociating the tumorspheres from each group to single cells using trypsin-EDTA. The viable cells were then counted using the automated Z™ Series COULTER COUNTER® Cell and Particle Counter. Likewise, miR-200b-3p and quercetin tumorspheres had significantly lower cell numbers than the tumorspheres from the control groups (Fig. 5b). This therefore indicates that miR-200b-3p inhibits proliferation and it is consistent with the inhibition of self-renewal and hints of likely differentiation.

To substantiate the likelihood of differentiation, we cultured the dissociated tumorsphere cells in regular medium (DMEM, 10% FCS, 1% HEPES). The cells were left untransfected or treated with 50 μM quercetin or 50nM mirVana™ mimics of miR-200b-3p or a miR-NC for every 72 h for a period of 12 days and subsequently evaluated for osteogenic, adipogenic and chrongenic differentiations. High heterogeneous expressions of osteoblasts, adipocytes and chondrocytes were detected in the miR-200b-3p and quercetin treated groups while little or no differentiation activity was detected in the control groups (Fig. 6a, b and c). These observations suggest that the differentiation is as a result of the inhibition of self-renewal (Additional file 4: Figure S2).