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

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

SETD7, H3K4me2, ZBTB20, and CDKN2D level in HCC was determined by TMA IHC.

<p>The two rows on the left indicate HE staining for HCC tumor tissues and paired ANLTs, whereas the two rows on the right indicate IHC staining for SETD7, H3K4me2, ZBTB20, and CDKN2D protein in HCC tumor tissues and paired ANLTs (<i>n</i> = 225). (Original magnified 100×; Inserted figures magnified 400×).</p

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

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

Correlation between the SETD7 and clinicopatologic variables in HCC.

<p>Correlation between the SETD7 and clinicopatologic variables in HCC.</p

<i>SETD7</i> expression changes liver cancer cell proliferation by regulating the cell cycle.

<p>(A) Western blot analysis was performed to detect the interference efficiency of siRNA after transfected with si-<i>SETD7</i>-1, si-<i>SETD7</i>-2, si-<i>SETD7</i>-3, or scramble RNA (NC) in SMMC-7721. (B) Western blot analysis was performed to detect <i>SETD7</i> expression in HepG2 transfected with pGV141-<i>SETD7</i> (OV-<i>SETD7</i>) or pGV141 plasmid (Control). (C, D) CCK8 array was used to assess proliferation in <i>SETD7</i> knockdown in SMMC-7721 cells or <i>SETD7</i> overexpression in HepG2 cells. *, P<0.05; **, P<0.01 by student’s t test. (E, F) Cell cycle was assessed in <i>SETD7</i> knockdown in SMMC-7721 cells or <i>SETD7</i> overexpression in HepG2 cells by flow cytometry assay.</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

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

Expression of <i>SETD7</i> in HCC tumor tissues, paired ANLTs and cell lines.

<p>(A) qRT-PCR analysis was performed to analyze <i>SETD7</i> expression in 20 pairs of HCC tumor tissues and ANLTs, the data shown are the mean of –ΔCT, and the expression of <i>SETD7</i> in HCC is significantly higher than that in ANLTs (P<0.05); (B) Western blot assay was performed to detect <i>SETD7</i> expression in 20 pairs of HCC tumor tissues and ANLTs; (C) Gray value of Western blot result (P<0.05); (D) Western blot assay was performed to analyze <i>SETD7</i> expression in normal hepatocyte cell line (HL-7702) and liver cancer cell lines (HepG2, SMMC-7721, QGY-7703, HCC-0010, and Bel-7404); (E). Typical IHC staining of SETD7 in HCC tumor tissues and ANLTs (with figures on the left magnified 100× and figures on the right magnified 400×); (F) Box plots indicate the IHC scores of SETD7 in HCC tumor tissues and ANLTs [mean, 3.204 (SD, 0.092) vs 1.427 (SD, 0.083), P<0.01].</p