RVC does not affect tissue morphology or the expression and distribution of proteins in tissue samples.

<p>(A) Morphological features of murine liver tissue samples stored in different preservative solutions. (B) The abovementioned paraffin-embedded tissue samples were sliced and stained with H&E for the examination of cellular morphological characteristics. RNAlater and 100% ethanol caused significant tissue dehydration, whereas the RVC-based preservative solution did not cause this phenomenon. (C-D) Determination of EGFR and PCNA protein expression and distribution by immunohistochemical staining in tissue samples treated with different preservative solutions. The results from the RVC-based groups were consistent with the data of the control group.</p

Both the RVC-based preservative solution and RNAlater can effectively delay RNA degradation.

<p>(A-B) Quality of RNA in tissue samples (n = 3) preserved with different concentrations of RVC in the preservative solution at 4°C or −80°C for 7 days. (C) Quality of RNA in tissue samples (n = 5) preserved in different preservative solutions at 4°C for 7 days and −80°C for one month (D). NT: stored without any preservative solution; RVC+G: RVC+15% glycerol; RVC+D: RVC+5% DMSO; * p < 0.05, ** p < 0.01, *** p < 0.001 (compared to the NT group).</p

The RVC-based preservative solution has a milder effect on protein activity than 100% ethanol and RNAlater.

<p>(A-B) Determination of the effect of different preservative solutions on intracellular luciferase activity. The lysate of cells expressing luciferase was reacted with different preservative solutions, and luciferase activity was measured after 0, 0.5, 1, and 3 hours (n = 3). *** p < 0.001 compared to the NT group. (C) The effects of various preservative solutions on intracellular GFP activity. The RVC-based preservative solution exerted a significantly milder effect on GFP fluorescent activity than 100% ethanol and RNAlater.</p

Different types of preservative solutions cause changes in the microenvironment of cells and affect gene expression levels.

<p>(A, B) RNA was isolated from tissue samples immersed in different preservative solutions at −80°C for 1 month and subjected to whole-transcriptome analysis. Different preservative solutions affected the gene expression levels within tissue samples to different degrees. The total number of genes affected by the different preservation solutions compared to the control group are shown in (C). (D) The RNA from the abovementioned samples was analyzed for the expression of 12 miRNAs by real-time RT-PCR using <i>snoRNA202</i> as an internal control. Different preservative solutions also affected the expression of miRNA in the tissue samples. * p < 0.05, ** p < 0.01, *** p < 0.001 (compared to the NT group). (E) The genomic DNA was isolated from the samples processed as described above and subjected to PCR for amplification of an <i>IFN-r</i> gene fragment. (F) The PCR-amplified <i>IFN-r</i> gene fragments were subjected to sequence analysis by the Sanger sequencing method. The correct sequences were obtained from the samples stored using all preservation methods.</p

Peritumoral Small EphrinA5 Isoform Level Predicts the Postoperative Survival in Hepatocellular Carcinoma

<div><h3>Background</h3><p>EphrinA5, a member of Eph/Ephrin family, possesses two alternative isoforms, large ephrinA5 isoform (ephrinA5L) and small ephrinA5 isoform (ephrinA5S). EphrinA5L is a putative tumor suppressor in several types of human cancers. However, the role of ephrinA5S in hepato-carcinogenesis remains unclear. In this study, we evaluate the role of ephrinA5 isoforms in human hepatocellular carcinomas (HCC).</p> <h3>Methodology/Principal Findings</h3><p>A total of 142 paired HCCs and peritumoral liver tissue was examined for relative expression of ephrinA5L and ephrinA5S by using quantitative real-time polymerase chain reaction. We analyzed their expression in relation to clinical parameters, disease-free survival and overall survival. Functional assays were performed to dissect the possible underlying mechanisms. Both ephrinA5L and ephrinA5S were significantly downregulated in HCCs, as compared to those in peritumoral tissue (<em>p = </em>0.013 and 0.001). Univariate analysis demonstrated that ephrinA5S was positively correlated with old age and histological grade. In multivariate analysis, high ephrinA5S expression in peritumoral tissue had better disease-free survival (<em>p = </em>0.002) and overall survival (<em>p = </em>0.045) in patients with HCC after surgical resection. Functional analysis in HCC cell lines revealed that ephrinA5S had a more potent suppressive effect than ephrinA5L on cell proliferation (<em>p</em><0.05) and migration (<em>p</em><0.01). Furthermore, forced expression of both ephrinA5 isoforms in HCC cell lines significantly down-regulated epidermal growth factor receptor (EGFR) expression by promoting c-Cbl-mediated EGFR degradation.</p> <h3>Conclusions/Significance</h3><p>EphrinA5S might be a useful prognostic biomarker for HCCs after surgical resection. EphrinA5, especially ephrinA5S, acts as a tumor suppressor in hepatocarcinogenesis. Peritumoral small ephrinA5 isoform level could determine the postoperative survival in hepatocellular carcinoma.</p> </div

Stepwise multivariate Cox proportional hazard model for independent predictors for postoperative survival.

<p>HR: Hazard Ratio, CI: Confidence Interval.</p

Clinical parameters of HCC patients analyzed.

<p>Clinical parameters of HCC patients analyzed.</p

EphrinA5 isoforms suppressed EGFR expression by enhancing c-Cbl-mediated EGFR degradation.

<p>(A) Both ephrinA5L and ephrinA5S reduced EGFR protein expression level in Hep3B cells. Ectopic ephrinA5 reduced endogenous EGFR protein expression (left panel) but had no transcriptional modification of EGFR in RT-PCR (right panel). The differences were statistically significant between the treated group and untreated group. Experiments in each group were performed in triplicate. The level of significance was set at <i>p</i><0.05 (*), <i>p</i><0.01 (**), or <i>p</i><0.001 (***). (B) Ectopic expresison of ephrinA5L and ephrinA5S reduced endogenous EGFR protein expression in Hep3B cells, which was rescued after c-Cbl knockdown by siRNA.</p

Association between ephrinA5 isoforms, clinical parameters and disease-free survival/overall survival.

a<p>Kaplan-Meier analysis with log rank test.</p>b<p>Relative expression of ephrinA5 mRNA assessed by real-time RT-PCR using peritumoral liver tissues.</p><p>CI: Confidence Interval,</p>*<p>P<0.05.</p

Relative expression of ephrinA5L and ephrinA5S and its relation to disease-free survival and overall survival.

<p>(A) RNA of 142 paired human HCC tissues was extracted and subjected into primer-specific real-time PCR to detect the expression of ephrinA5L and ephrinA5S. Both ephrinA5L and ephrinA5S were significantly downregulated in tumor as compared with normal tissues (<i>p = </i>0.013 and 0.001). (B) Kaplan-Meier curve for the disease-free survival and overall survival of HCC patients with high and low ephrinA5S expression. The disease-free survival and overall survival were significantly different in log-rank test with <i>p-</i>values of 0.019 and 0.045, respectively.</p