shRNA-mediated GSTP1 gene silencing enhances androgen-independent cell line DU145 chemosensitivity

Purpose

Design short hairpin RNA (shRNA) interference sequence to silence glutathione S-transferase P1 (GSTP1) gene of androgen-independent prostate cancer cell line DU145, and then to explore its effect on sensitivity to chemotherapeutics.

Methods

Target sequence was picked up to form the shRNA. DU145 cell was divided into five groups according to the shRNA added for transfection: shRNA255, shRNA554, shRNA593, negative-shRNA and blank group. Fluorescence microscope was used to pick up the shRNA with the highest transfection ratio. Western blotting and RT-PCR were taken to pick up the shRNA with the best gene silencing result. 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay and terminal de-oxynucleotidyl transferase-mediated dUTP nick end-labeling assay were used to detect survival ratio and apoptosis ratio of DU145 administered of fluorouracil (5-FU) or paclitaxel (PA) at different concentrations before and after shRNA transfection.

Results

Three different shRNA oligonucleotides (shRNA255; shRNA554; shRNA593) targeting the coding sequence of GSTP1 mRNA and one negative control shRNA were constructed. The transfection ratio of shRNA554 (76.2 ± 0.68 %) was higher than that of shRNA255 (63.3 ± 1.04 %) (P < 0.01) or shRNA593 (72.7 ± 0.33 %) (P < 0.01). After transfection of shRNA554, the mRNA and protein of level were the lowest, P < 0.01. The survival ratio of DU145 administered with 5-FU of different concentrations (30, 60, 120, 240 μg/ml) declined after transfection (P < 0.01). Besides, the apoptosis ratio increased after transfection (P < 0.01). Similarly the survival ratio of DU145 administered with PA of different concentrations (0.2, 2, 10, 20 μg/ml) declined (P < 0.01) and the apoptosis ratio increased (P < 0.01) after transfection.

Conclusions

The gene GSTP1 silence via shRNA transfection to androgen-independent prostate cancer cell line DU145 enhances the sensitivity to chemotherapeutics.     

Proteomic analysis of cerebrospinal fluid: toward the identification of biomarkers for gliomas

Gliomas are the most common primary brain tumors in adults and, despite advances in the understandings of glioma pathogenesis in the genetic era, they are still ineradicable, justifying the need to develop more reliable diagnostic and prognostic biomarkers for this malignancy. Because changes in cerebrospinal fluid (CSF) are suggested to be capable of sensitively reflecting pathological processes, e.g., neoplastic conditions, in the central nervous system, CSF has been deemed a valuable source for potential biomarkers screening in this era of proteomics. This systematic review focused on the proteomic analysis of glioma CSF that has been published to date and identified a total of 19 differentially expressed proteins. Further functional and protein-protein interaction assessments were performed by using Protein Analysis Through Evolutionary Relationships (PANTHER) website and Ingenuity Pathway Analysis (IPA) software, which revealed several important protein networks (e.g., IL-6/STAT-3) and four novel focus proteins (IL-6, galanin (GAL), HSPA5, and WNT4) that might be involved in glioma pathogenesis. The concentrations of these focus proteins were subsequently determined by enzyme-linked immunosorbent assay (ELISA) in an independent set of CSF and tumor cyst fluid (CF) samples. Specifically, glioblastoma (GBM) CF had significantly lower GAL, HSPA5, and WNT4 levels than CSF from different grades of glioma. In contrast, IL-6 level was significantly higher in GBM CF when compared with CSF and, among different CSF groups, was highest in GBM CSF. Therefore, these candidate protein biomarkers, identified from both the literatures and in silico analysis, may have potentials in clinical diagnosis, prognosis evaluation, treatment response monitoring, and novel therapeutic targets identification for patients with glioma.     

Laparoscopic Spleen-Preserving Distal Pancreatectomy: Comparative Study of Spleen Preservation with Splenic Vessel Resection and Splenic Vessel Preservation

Background

Spleen-preserving laparoscopic distal pancreatectomy (SPLDP) can be performed with splenic vessel resection (SVR) or splenic vessel preservation (SVP). The purpose of this comparative study was to evaluate the clinical outcomes of patients who underwent SPLDP with SVR or SVP at a single institution.

Methods

We retrospectively reviewed the records of 246 patients who underwent SPLDP at Asan Medical Center, Seoul, Korea, for benign or low-grade malignant tumors found in the body or tail of the pancreas between November 2005 and November 2011.

Results

In total, 206 patients (83.7 %) were managed by SVP. SVR was performed in the remaining 40 (16.3 %) cases. There were no significant differences between the SVP and SVR groups in terms of intraoperative blood loss (378 ± 240 vs. 328 ± 204 ml, respectively; P = 0.240) and operating time (193.4 ± 59.1 vs. 204.4 ± 51.8 min, respectively; P = 0.492). Sixty-seven (32.5 %) and 10 patients (25 %) had complications in the SVP and SVR groups, respectively (P = 0.347). At 3 days after surgery, the rates of splenic infarction were 16.0 % (33/206) in the SVP group and 52.5 % (21/40) in the SVR group, but all recovered within 12 months on postoperative computed tomography. The time of recovery from splenic infarction was 3.6 ± 3.1 and 4.7 ± 3.7 months in the SVP and SVR groups, respectively. At 6 months, the rates of gastric varices were 1.9 % in the SVP group and 35 % in the SVR group (P < 0.001) with no progression at 12 months. No gastrointestinal bleeding occurred at a median follow-up of 34 months (range = 12–84).

Conclusions

SPLDP with SVR can be used for patients with large and benign or low-grade malignant tumors that distort and compress vessel course, as the higher rate of early splenic ischemia and perigastric varices is acceptable.     

脑卒中后遗症病人社区康复和预防的研究现状

从转变康复观念、矫正不良行为、调整影响因素、提供康复服务方面，综述了脑卒中后遗症病人社区康复的现状，并对脑卒中的预防进行了分析。     

Mid to Distal Small Bowel Resection with the Preservation of the Terminal Ileum Improves Glucose Homeostasis in Diabetic Rats by Activating the Hindgut-Dependent Mechanism

Background

The aim of this study was to develop a novel surgical model to test the “hindgut hypothesis” and thereby study the role of the gut in glucose homeostasis and the mechanism of action of bariatric surgery.

Method

Sprague-Dawley rats were given a high-fat and high-sugar diet and treated with 25 mg/kg streptozotocin (STZ). The fat-sugar-fed/STZ-treated rats were randomized into mid to distal small bowel resection with the preservation of the terminal ileum (DBRPI) and sham operation (which had a formal celiotomy with bowel manipulation only) groups. Rats were observed for 12 weeks after the operation. The main outcome measures were weight, food intake, non-fasting glucose, an oral glucose tolerance test (OGTT), an insulin tolerance test (ITT), the levels of fasting and glucose-induced insulin, glucagon-like peptide-1 (GLP-1), peptide YY (PYY), serum bile acids, and lipid profile.

Result

The DBRPI and sham groups exhibited no difference in weight and food intake after surgery. When compared to the sham controls, the DBRPI group displayed an improvement in non-fasting glucose, oral glucose tolerance, and insulin tolerance at 4 and 12 weeks postresection. DBRPI elicited an increased serum insulin, PYY and GLP-1 levels at 12 weeks postoperation; furthermore, DBRPI resulted in higher serum levels of triglyceride, total bile acids, total bilirubin, and direct bilirubin levels and lower free fatty acid level at 12 weeks.

Conclusions

This study provides strong evidences for the key role of hindgut in the amelioration of diabetes after bariatric surgery. Moreover, these findings confirm that DBRPI is a simple and effective surgical model for testing the “hindgut hypothesis” and focused study of biliary enterohepatic recycling in the context of bariatric operations.     

EZH2, a Potential Regulator of Dental Pulp Inflammation and Regeneration

Introduction

Dental pulp has limited capability to regenerate, which happens in the early stage of pulpitis. An ambiguous relationship exists; inflammation may impair or support pulp regeneration. Epigenetics, which is involved in cell proliferation and inflammation, could regulate human dental pulp cell (HDPCs) regeneration. The aim of this study was to determine the role of the epigenetic mark, enhancer of zeste homolog 2 (EZH2), in the inflammation, proliferation, and regeneration of dental pulp. We used trimethylated histone H3 lysine 27(H3K27me3) and its lysine demethylase 6B (KDM6B) to monitor functional effects of altered EZH2 levels.

Methods

We detected epigenetic marks (EZH2, H3K27me3, and KDM6B) in pulp tissue by immunohistochemistry and immunofluorescence. EZH2 levels in HDPCs in inflammatory responses or differentiation were analyzed by quantitative polymerase chain reaction and Western blot. Quantitative polymerase chain reaction was used to assess the effects of EZH2 inhibition on interleukins in HDPCs upon tumor necrosis factor alpha stimulation. Cell proliferation was tested by cell counting kit-8, cell cycle, and apoptosis analysis. HDPC differentiation was investigated by quantitative polymerase chain reaction, alkaline phosphatase activity, and oil red O staining.

Results

EZH2 and H3K27me3 were decreased, whereas KDM6B was increased in infected pulp tissue and cells, which were similar to HDPC differentiation. EZH2 inhibition suppressed IL-1b, IL-6, and IL-8 messenger RNA (mRNA) in HDPCs upon inflammatory stimuli and impeded HDPC proliferation by decreasing cell number, arresting cell cycle, and increasing apoptosis. Suppressed EZH2 impaired adipogenesis, peroxisome proliferator-activated receptor r (PPAR-r), and CCAAT-enhancer binding protein a (CEBP/a) mRNA in adipogenic induction while enhancing alkaline phosphatase activity, Osx, and bone sialoprotein (BSP) mRNA in mineralization induction of HDPCs.

Conclusions

EZH2 inhibited HDPC osteogenic differentiation while enhancing inflammatory response and proliferation, suggesting its role in pulp inflammation, proliferation, and regeneration.