Annals of RSCB
Vol. XVI, Issue 1
EXPRESSION PROFILE OF VEGF AND EGFR mRNA IN ESOPHAGEAL AND GASTRIC CANCERS Cristina Angelescu, F. Burada, M. Ioana, R. Angelescu, Anca Riza, F. Mixich, Doina Voinescu, M. Cruce, A. Săftoiu UNIVERSITY OF MEDICINE AND PHARMACY OF CRAIOVA
Summary Gastric cancer is one of the most commonly diagnosed malignancies worldwide and an important public health problem. The local growth, invasion and distant metastasis of tumors are highly dependent on the development of new blood vessels. We aimed to evaluate the gene expression profiles of VEGF and EGFR in human esophageal and gastric cancer and to determine the correlations between the expression levels and clinico-pathological parameters of the tumor. 33 paired samples (tumor and non-malignant adjacent tissue) were collected from patients investigated by upper gastrointestinal endoscopy at the Research Center in Gastroenterology and Hepatology Craiova. Total RNA was isolated from the paired specimens and reverse-transcribed into the complementary DNA. Quantitative Real-Time PCR with TaqMan specific probes for VEGF-A and EGFR was performed. The studied genes are expressed both in tumor and peritumoral tissue, with a tendency of VEGF overexpression in tumor. We have also found that VEGF or EGFR mRNA relative expression correlates with several clinico-pathological characteristics. Further studies on higher number of patients are required to determine the utility of angiogenesis gene expression as progression and prognostic markers in esophageal and gastric carcinomas. Key words: qRT-PCR, gastric cancer, angiogenesis, VEGF, EGFR
[email protected] phase, in order to grow further, the tumor must develop new blood vessels, phenomenon termed as angiogenic switch (Folkman, 1990). The neo-angiogenesis is a multistep process, tightly regulated by certain angiogenic and anti-angiogenic factors released by tumor and host cells into the tumor microenvironment. The most important angiogenic factors in gastric carcinogenesis include vascular endothelial growth factor (VEGF) family members, fibroblast growth factor (FGF), interleukin8 (IL-8), and angiogenin. The most powerful angiogenic factor is VEGF-A, also termed VEGF. VEGF is produced by tumor cells, but fibroblasts and inflammatory cells also release VEGF (Fukumura et al., 1998). VEGF mediates the process of angiogenesis by stimulating endothelial cell survival, proliferation, migration and increasing microvascular permeability. Over-expression of VEGF
Introduction Despite the decline in mortality, gastric cancer remains an important health problem, rating as the fourth cause of cancer and the second cause of cancerrelated deaths worldwide. Prognosis of gastric cancer is poor mainly because patients presents with advanced stages of the disease (Parkin et al., 2005). Gastric cancer develops from normal tissue through intermediate premalignant changes of chronic gastritis, gastric atrophy, intestinal metaplasia and dysplasia, following Helicobacter pylori (H. pylori) infection in genetically susceptible individuals (Coreea, 1992). The local growth, invasion and distant metastasis of a solid tumor are highly dependent on the development of new blood vessels. In the absence of proper vascularization, a solid tumor can grow up only to maximum 1-2 mm in diameter, because of hypoxia. After this avascular 228
Annals of RSCB
Vol. XVI, Issue 1
protein has been reported in both early and advanced human gastric adenocarcinoma, but the correlations with clinicopathological parameters and prognosis are not completely elucidated (Kolev et al., 2007; Lee et al., 2010). Epidermal growth factor receptor (EGFR) is a trans-membrane tyrosine kinase receptor belonging to the erbB family. Activation of EGFR initiates intracellular signal transduction leading to cell migration, growth, morphological alterations and also angiogenesis through activation of VEGF. Expression of EGFR at elevated levels have been observed in gastric cancer (Lang et al., 2007; Lieto et al., 2008). Expression of VEGF or EGFR have been evaluated in gastric cancer both at mRNA and protein level by several methods. However, the results vary between studies probably because of various methods used, different number of patients included in the study and genetic polymorphisms. The aim of our study was to evaluate the gene expression profiles of VEGF and EGFR in human esophageal and gastric cancer and to determine the correlations between the expression levels and clinicopathological parameters of the tumor.
RNA extraction Total RNA was purified using SV Total RNA Isolation System (Promega) and kept at -80oC until used. The quality of RNA was evaluated by denaturing agarose gel electrophoresis and the concentration and purity were measured spectrophotometrically (Eppendorf Biophotometer). qRT-PCR Two step quantitative Real-Time PCR was performed. In the first step 1μg RNA was reverse transcribed into complementary single stranded DNA (cDNA) using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to manufacturer’s instructions. In the second step, quantitative Real-Time PCR was performed using TaqMan® Gene Expression Master Mix (Applied Biosystems) with specific primers and probes for target genes and for endogenous control gene (VEGF - Hs00900054_m1, EGFR - Hs01076092_m1 and GAPDH Hs99999905_m1). The amplifications were carried out in 20 μl volume, in triplicate, on a Mastercycler®ep realplex (Eppendorf). The cycling parameters were: hold 50°C for 2-minute, hold 95°C for 10 minutes, followed by 50 cycles of PCR at 95°C for 15 seconds and 60°C for 1 minute. Fold changes between tumor and peritumoral mucosa were calculated using the Pfaffl method. The differences between the paired samples were considered significant when the mRNA level varied more than 1.8 folds. Statistics In order to determine whether the variables followed a gaussian distribution, Shapiro-Wilk and Anderson-Darling tests were performed. Because the data did not followed a normal distribution, nonparametric test were performed. Friedman’s and Kruskal-Wallis tests were performed for statistical analysis of target genes expression in paired samples and Mann-Whitney and Kruskal-Wallis test for associations between total VEGF and
Material and methods Patients and samples A total of 33 patients who had undergone upper gastrointestinal endoscopy and endoscopic ultrasonography at the Research Centre in Gastroenterology and Hepatology of Craiova between 2008 and 2010 were included in the study. Both tumor and peritumoral corresponding esophageal or gastric mucosa were biopsied during endoscopy for all the patients. The samples were collected in RNAlater solution, then stored at -80oC until RNA isolation. Other specimens were collected in formalin for histological diagnosis. The informed con-sent for genetic studies was obtained from all the patients. 229
Annals of RSCB
Vol. XVI, Issue 1
EGFR expression in tumor and pathological characteristics. Two-sided p values
