The Loss of miR-26a-Mediated Post-Transcriptional Regulation of Cyclin E2 in Pancreatic Cancer Cell Proliferation and Decreased Patient Survival

<div><p>Background</p><p>miR-26a plays a critical role in tumorigenesis, either as a tumor suppressor or as an oncogenic miRNA, depending on different tumor types. However, the function of miR-26a in pancreatic cancer has not been clearly elucidated. The present study was designed to determine the roles of miR-26a in pancreatic cancer and its association with the survival of patients with pancreatic cancer.</p><p>Methods</p><p>The expression of miR-26a was examined in 15 pairs of pancreatic duct adenocarcinoma (PDAC) and their adjacent benign pancreatic tissues (ABPT), by qRT-PCR. The results were confirmed by <i>in situ</i> hybridization using two panels of 106 PDACs and their ABPT microarray. The association of miR-26a expression with overall survival was determined. The proliferation and cell cycle distribution of Capan-2, SW-1990, and Panc-1 cells, transfected with miR-26a mimics or a miR-26a inhibitor, were assessed using the Cell Counting Kit-8 assay and flow cytometry, respectively. The cell tumorigenicity was evaluated via murine xenograft experiments. Cyclin D2, E2, EZH2, and PCNA levels were analyzed by Western blot and immunohistochemistry.</p><p>Results</p><p>miR-26a was expressed in the cytoplasm of pancreatic ductal epithelial cells, whereas its expression was significantly downregulated in PDAC tissues compared with that of ABPT. Patients with low miR-26a expression had a significantly shorter survival than those with high miR-26a expression. The <i>in vitro</i> and <i>in vivo</i> assays showed that overexpression of miR-26a resulted in cell cycle arrest, inhibited cell proliferation, and decreased tumor growth, which was associated with cyclin E2 downregulation.</p><p>Conclusions</p><p>miR-26a is an important suppressor of pancreatic ductal carcinoma, and can prove to be a novel prognostic factor and therapeutic target for pancreatic cancer treatment.</p></div

Representation of cell cycle distribution in Capan-2 (A,B,C), SW-1990 (E,F,G), and Panc-1 (I,J,K) cells.

<p>Cells were either transfected with miR-26a mimics or inhibitors. The controls were left untreated. D, H, and L indicate the statistical cell cycle distribution for Capan-2, SW-1990, and Panc-1, respectively.</p

miR-26a expression in pancreatic cancer specimens and overall survival.

<p>(A) Average expression level of miR-26a in human PDAC specimens (<i>n</i> = 15) and normal pancreatic tissues (<i>n</i> = 15). miRNA abundance was assessed by qRT-PCR and normalized to U6 RNA. Values are presented as the mean ± S.D. (B) Overall survival following resection of pancreatic cancer with the miR-26a-negative versus miR-26a-positive groups. The miR-26a-negative group had significantly shorter survival than the miR-26a-positive group (<i>P</i> = 0.029). (C,D) <i>In situ</i> hybridization for miR-26a in pancreatic lesions. <i>In situ</i> hybridization showed much lower miR-26a expression in PDAC tissues (C) than in ABPT (D). The inset shows the negative control (scrambled sequence probe). Cytoplasmic staining in the ductal epithelial cells stands in contrast with the negative staining with the scrambled probe. (E,F) <i>In situ</i> hybridization for miR-21 (positive control) in pancreatic lesions. <i>In situ</i> hybridization showed much stronger miR-21 expression in PDAC tissues (E) than in ABPT (F). The inset shows the negative control (scrambled sequence probe). Cytoplasmic staining in tumor cells stands in contrast with the negative staining of the scrambled probe. Original magnification, 100×.</p

Clinicopathological characteristics of 106 cases of pancreatic cancer.

<p>Clinicopathological characteristics of 106 cases of pancreatic cancer.</p

miR-26a overexpression inhibited pancreatic cancer cell growth by the downregulation of cyclin E2 expression.

<p>The qRT-PCR analysis demonstrated the transcription of miR-26a in mimics, inhibitor, and control groups (A,B,C), and the expression of cyclin E2 in cyclin E2 siRNA, control siRNA, and control groups (D,E,F). The proliferation of PDAC cell lines transiently transfected with miR-26a mimics, miR26a inhibitor or cyclin E2 siRNA was analyzed by the CCK-8 proliferation assay (G,H,I). The regulation of cyclin E2 or EZH2 expression by miR-26a was analyzed by Western blot (J,K,L), and Western blot analysis also confirmed the expected efficiency of cyclin E2 siRNA in three PDAC cell lines (M,N,O).</p

Immunohistochemical expression of cyclin D2, cyclin E2, and PCNA in PDAC tissues as well as overall survival.

<p>The PDAC tumor tissues (PDAC) were negative for cyclin D2 (A) and strongly positive for cyclin E2 (B), with a high PCNA proliferative index (C). The adjacent benign pancreatic tissues (ABPT) were negative for cyclin D2 was (D), and positive for cyclin E2, as observed in the ductal epithelial cells of ABPT (E). PCNA was very low in normal pancreatic tissues (F). The insets of A, B, and C show the negative controls. The inset in D indicates that cyclin D2 is a positive control of lung adenocarcinoma. Original magnification, 200×. Overall survival after resection of pancreatic cancer with the cyclinE2-positive versus cyclinE2-negative groups was not significant (<i>P</i> = 0.676) (G), whereas overall survival after resection of pancreatic cancer with the PCNA high proliferative index versus PCNA low proliferative index groups had a significantly shorter survival (<i>P</i> = 0.007) (H).</p

The relationship between miR-26a expression and clinicopathological features in pancreatic cancer.

*<p>Chi-square test.</p>**<p>Mann–Whitney test.</p

Ectopic expression of miR-26a inhibits pancreatic cancer growth in nude mice.

<p>(A) Nude mice were subcutaneously inoculated with SW-1990 cells transfected with pTM (pTM: hTERT–miR-26a plasmid) or pT (pT: hTERT control plasmid), in their flanks. The image is representative of tumors formed in 8 mice. (B) Growth curves of tumor volumes. The graph is representative of tumor growth, 5 weeks after inoculation. Tumor volume was calculated and all data are shown as the mean ± S.D. (<i>n</i> = 8). (C–H) Expression of cyclin D2, cyclin E2, and PCNA was measured by immunohistochemistry in the tissues of mice inoculated with pTM- transfected SW-1990 cells or the control cells. The figure insets in C, E, and G indicate a negative control field. Cells colored brown indicate positive staining. Original magnification, 200×.</p

Puerarin Suppresses Proliferation of Endometriotic Stromal Cells Partly via the MAPK Signaling Pathway Induced by 17ß-estradiol-BSA

<div><h3>Background</h3><p>Puerarin is a major isoflavonoid compound extracted from <em>Radix puerariae</em>. It has a weak estrogenic action by binding to estrogen receptors (ERs). In our early clinical practice to treat endometriosis, a better therapeutic effect was achieved if the formula of traditional Chinese medicine included <em>Radix puerariae</em>. The genomic and non-genomic effects of puerarin were studied in our Lab. This study aims to investigate the ability of puerarin to bind competitively to ERs in human endometriotic stromal cells (ESCs), determine whether and how puerarin may influence phosphorylation of the non-genomic signaling pathway induced by 17ß-estradiol conjugated to BSA (E₂-BSA).</p> <h3>Methodology</h3><p>ESCs were successfully established. Binding of puerarin to ERs was assessed by a radioactive competitive binding assay in ESCs. Activation of the signaling pathway was screened by human phospho-kinase array, and was further confirmed by western blot. Cell proliferation was analyzed according to the protocol of CCK-8. The mRNA and protein levels of cyclin D1, Cox-2 and Cyp19 were determined by real-time PCR and western blotting. Inhibitor of MEK1/2 or ER antagonist was used to confirm the involved signal pathway.</p> <h3>Principal Findings</h3><p>Our data demonstrated that the total binding ability of puerarin to ERs on viable cells is around 1/3 that of 17ß-estradiol (E₂). E₂-BSA was able to trigger a rapid, non-genomic, membrane-mediated activation of ERK1/2 in ESCs and this phenomenon was associated with an increased proliferation of ESCs. Treating ESCs with puerarin abrogated the phosphorylation of ERK and significantly decreased cell proliferation, as well as related gene expression levels enhanced by E₂-BSA.</p> <h3>Conclusions/Significance</h3><p>Puerarin suppresses proliferation of ESCs induced by E₂-BSA partly via impeding a rapid, non-genomic, membrane-initiated ERK pathway, and down-regulation of Cyclin D1, Cox-2 and Cyp19 are involved in the process. Our data further show that puerarin may be a new candidate to treat endometriosis.</p> </div

Effect of E₂-BSA and puerarin on cell proliferation in ESCs.

<p>(A) ESCs were treated with various concentrations (10⁻¹⁰ to 10⁻⁶ mol/L) of E₂-BSA for 4 d. (B) Growth curves of ESCs incubated with E₂-BSA (10⁻⁸ mol/L) and/or puerarin (10⁻⁹ mol/L) for 0, 1, 2, 4 d. (C) Cells were treated for 4 d with E₂-BSA (10⁻⁸ mol/L) in the absence or presence of puerarin (10⁻⁹ mol/L) and/or pretreated with U0126 (20 µmol/L) for 60 min. The figures represent data obtained from three experiments; each experiment was performed in triplicate, and data are represented as the mean ± SD. DMSO treatment was used as the vehicle control. <i>Vs. control</i>,**, <i>P</i><<i>0.01,</i> *, <i>P</i><<i>0.05; vs. E₂-BSA, ▴, P</i><<i>0.05.</i></p