miR-21 induced the activation of AKT and ERK1/2, and the expression of HIF-1α and VEGF.

<p>(A) DU145 cells were seeded into 60 mm dishes. When cells were 60% confluence, cells were transfected with pre-miRNA negative control or pre-miR-21 as described above. Total proteins were extracted 36 h after the transfection, and analyzed for the expression of phospho-AKT (p-AKT), total AKT, phospho-ERK1/2 (p-ERK), and ERK2 by immunoblotting. (B) DU145 cells were transfected with pre-miR-21 precursor as described above. After the transfection for 36 h, the cells were treated with solvent alone, 10 µM of LY294002, or 10 µM of U0126 for 6 h. The expression levels of p-AKT, total AKT, p-ERK, ERK2, HIF-1α and HIF-1β were analyzed by immunoblotting. (C-D) After the transfection of cells with pre-miR-21, the cells were cultured for 36 h, then treated with solvent alone, 10 µM of LY294002, or 10 µM of U0126 for 12 h. VEGF mRNA expression was analyzed by RT-PCR (C), and by real-time RT-PCR (D). ##, indicates the significant decrease when compared to that of the control (<i>p</i><0.01).</p

PTEN was the downstream target of miR-21 in inducing angiogenesis.

<p>(A) DU145 cells were infected with adenovirus carrying GFP (Ad-GFP) or PTEN (Ad-PTEN). After 24 h, cells were transfected with pre-miR-21 or scrambled control. The next day, cells were trypsinized and angiogenesis assay was performed as above. The representative plugs from treatments of Ad-GFP plus scrambled control, Ad-GFP plus pre-miR-21, and Ad-PTEN plus pre-miR-21 were shown in the picture. <i>Scale</i>, 2 mm. (B) The relative angiogenesis was showed in the bar graph. Results from each group are presented as mean ±SD of 6 samples. * indicates the significant increase when compared to the Ad-GFP plus scrambled control group (<i>p</i><0.05). ^#, indicates the significant decrease when compared to Ad-GFP plus miR-21 group (<i>p</i><0.05).</p

MicroRNA miR-21 overexpression increased HIF-1α and VEGF expression and induced tumor angiogenesis.

<p>(A) Human prostate cancer cells DU145 were transfected with pre-miRNA negative control or pre-miR-21 at 25 nM. After the transfection for 36 h, cells were collected and subjected to qRT-PCR for miR-21 expression. (B) DU145 cells were treated as in <i>A</i>, the total protein was extracted and subjected to Western blotting assay. (C–D) DU145 cells were collected after the transfection for 36 h. Total RNAs were extracted and analyzed for VEGF and GAPDH mRNA expression by RT-PCR analysis (C), and by real-time RT-PCR (D). (E) DU145 cells were transfected as above. After 24 h, cells (2×10⁶cells, 15 µl) were mixed with 15 µl Matrigel. The cell mixture was implanted onto the CAM of a 9-day old chicken embryo. After 4 days of implantation, the CAM was cut off, and the amount of blood vessels on the CAM induced by the DU145 plugs was determined from eight different replicated experiments. Representative plugs from negative control and miR-21 treatment groups were shown. <i>Scale</i>, 2 mm. (F) The number of blood vessels were counted from replicate experiments, and normalized to that of the negative control group as relative angiogenesis. The data are mean±SD from 6 replicates. ** indicates the significant increase when compared to the pre-miRNA negative control (<i>p</i><0.01).</p

The inhibitor of miR-21 inhibited miR-21-inducing expression of HIF-1α and VEGF, activation of AKT and ERK1/2, and angiogenesis.

<p>(A) The total proteins were prepared 36 h after transfection, and analyzed by immunoblotting for the expression of PTEN, β-actin, p-AKT, AKT, p-ERK1/2, ERK2, HIF-1α, and HIF-1β. (B–C) The expression levels of VEGF in the cells described as above were analyzed by RT-PCR (B), and by real-time RT-PCR (C). (D) The cells above were used for angiogenesis assay. Representative plugs from the negative control and miR-21 inhibitor treated groups were shown. <i>Scale</i>, 2 mm. (E) The number of blood vessels were counted from replicate experiments, and normalized to that of the control group as relative angiogenesis. The data are the mean ±SD from 6 replicate experiments. ##, indicates the significant decrease when compared to that of the control (<i>p</i><0.01).</p

miR-21 induced angiogenesis through mediating AKT and ERK1/2 activation.

<p>(A) DU145 cells were transfected with pre-miR-21. After transfection for 36 h, the cells were trypsinized, mixed with 15 µl Matrigel in the presence or absence of 10 µM of LY294002 or U0126. Representative plugs from DMSO control, LY294002, and U0126 treatment groups were shown. <i>Scale,</i> 2 mm. (B) The number of blood vessels were counted from 8 replicate experiments, and normalized to that of the pre-miR-21 group as relative angiogenesis. ^##, indicates the significant decrease when compared to that of pre-miR-21 treatment group (<i>p</i><0.01).</p

HIF-1α expression is required in miR-21 inducing VEGF expression and tumor angiogenesis.

<p>(A) DU145 cells were transfected with pre-miR-21. After 24 h incubation, cells were infected with adenovirus carrying GFP (Ad-SCR) or siRNA against HIF-1α (Ad-siHIF-1α) at 20 MOI for 24 h. The total proteins were analyzed by immunoblotting. (B–C) The expression levels of VEGF and GAPDH mRNA were analyzed by RT-PCR (B), and by real-time RT-PCR (C). (D) After DU145 cells were transfected with pre-miR-21 for 24 h, the cells were infected with Ad-GFP or Ad-siHIF-1α at 20 MOI for 24 h. Then the cells were used to perform angiogenesis assay as described above. The picture showed the representative plugs from Ad-GFP and Ad-siHIF-1α treatment groups. <i>Scale</i>, 2 mm. (E) The number of blood vessels were counted from replicate experiments, and normalized to that of the Ad-siSCR group as relative angiogenesis. The data are mean ± SD from 6 replicate experiments. ##, indicates the significant difference when compared to that of the Ad-siSCR group (<i>p</i><0.01).</p

Decreased Expression of Beclin 1 Correlates Closely with Bcl-xL Expression and Poor Prognosis of Ovarian Carcinoma

<div><p>Background</p><p>It has been suggested that autophagy-related Beclin 1 plays a critical role in the regulation of tumor development and/or progression, but its prognostic significance and relationship with Bcl-xL expression in ovarian carcinoma are unclear.</p><p>Methodology/Principal Findings</p><p>In the present study, the methods of Western blotting and immunohistochemistry (IHC) were utilized to investigate the expression status of Beclin 1 and Bcl-xL in fresh ovarian tissues and paraffin-embedded epithelial ovarian tumor tissues. Decreased expression of Beclin 1 was examined by IHC in 8.3% of normal ovaries, in 15.4% of cystadenomas, in 20.0% of borderline tumors, and in 55.6% of ovarian carcinomas, respectively. In ovarian carcinomas, decreased expression of Beclin 1 was correlated closely with ascending histological grade, later pT/pN/pM status and/or advanced clinical stage (<i>P</i><0.05). In univariate survival analysis, a highly significant association between low-expressed Beclin 1 and shortened patient survival was evaluated in ovarian carcinoma patients (<i>P</i><0.01), and Beclin 1 expression was an independent prognostic factor as evidenced by multivariate analysis (<i>P</i> = 0.013). In addition, decreased expression of Beclin 1 was inversely correlated with altered expression of Bcl-xL in ovarian carcinoma cohort, and combined analysis further showed that the low Beclin 1/high Bcl-xL group had the lowest survival rate.</p><p>Conclusions/Significance</p><p>Our findings suggest that Beclin 1 expression, as examined by IHC, could be served as an additional tool in identifying ovarian carcinoma patients at risk of tumor progression, and predicting patient survival in ovarian carcinomas with increased expression of Bcl-xL.</p></div

Receive operating characteristic curve analysis for several clinicopathological features and Beclin 1 expression was used to evaluate the survival status.

<p>(A) Age [area under curve (AUC) = 0.487, <i>P</i> = 0.769], histological grade (AUC = 0.588, <i>P</i> = 0.054), FIGO stage (AUC = 0.750, <i>P</i><0.0001), pT status (AUC = 0.638, <i>P</i> = 0.003), pN status (AUC = 0.599, <i>P</i> = 0.031), pM status (AUC = 0.614, <i>P</i> = 0.012), histological type (AUC = 0.446, <i>P</i> = 0.242),Beclin 1 expression (AUC = 0.673, <i>P</i><0.0001) and Bcl-xL (AUC = 0.588, <i>P</i> = 0.056) implied statistical associations with the survival in whole study population. (B) Age (AUC = 0.504, <i>P</i> = 0.943), histological grade (AUC = 0.609, <i>P</i> = 0.044), FIGO stage (AUC = 0.767, <i>P</i><0.0001), pT status (AUC = 0.617, <i>P</i> = 0.030), pN status (AUC = 0.625, <i>P</i> = 0.020), pM status (AUC = 0.631, <i>P</i> = 0.015), histological type (AUC = 0.472, <i>P</i> = 0.598),and Beclin 1 expression (AUC = 0.675, <i>P</i><0.0001) were applied to test the survival status in highly-expressed Bcl-xL population. (C) Age (AUC = 0.449, <i>P</i> = 0.583), histological grade (AUC = 0.511, <i>P</i> = 0.907), FIGO stage (AUC = 0.714, <i>P</i> = 0.022), pT status (AUC = 0.744, <i>P</i> = 0.009), pN status (AUC = 0.551, <i>P</i> = 0.583), pM status (AUC = 0.551, <i>P</i> = 0.583), histological type (AUC = 0.427, <i>P</i> = 0.434),and Beclin 1 expression (AUC = 0.603, <i>P</i> = 0.272) were employed to evaluate the survival in low-expressed Bcl-xL population.</p

Association of Beclin 1 Expression with Patients' Clinicopathologic Features in Ovarian Carcinomas.

*<p>Chi-square test; ^†Mean age; FIGO indicates International Federation of Gynecology and Obstetrics.</p

The expression patterns of Beclin 1and Bcl-xL in ovarian carcinoma and Kaplan-Meier survival analysis.

<p>Consecutive sections were used for immunohistochemical study for Beclin 1 and Bcl-xL. (A) High Beclin 1/high Bcl-xL case (Case 9). (B) High Beclin 1/low Bcl-xL case (Case 17). (C) Low Beclin 1/high Bcl-xL (Case 42). (D) Low Beclin 1/low Bcl-xL case (Case 63, Magnification, ×100). (E) Kaplan-Meier analysis of overall survival (OS) for Beclin 1 expression in Bcl-xL–high population. (F) Kaplan-Meier analysis of OS for Beclin 1 expression in Bcl-xL–negative population. (G) Combined analysis of Beclin 1 and Bcl-xL expression in the prognostic value of patients with ovarian cancer (Log-rank test).</p