The Study Of Related Gene Mutation Condition And Abnormal Expression Of RASA1in Pancreatic Cancer

Background:It is well-known that pancreatic cancer is one of the most malignant tumor. Due to its insidious onset and rapid progression, patients would see the doctor until they have advanced lesion or with distant metastasis.Its overall mortality is high, and the average survival time is6months after diagnosis.Due to the limited role of traditional treatment in pancreatic cancer, targeted therapies to pancreatic cancer has applied more and more to improve efficacy and reduce drug toxicity,.The EGFR signaling pathway plays an important role in the process of cell proliferation and differentiation. Its signaling molecule mutations is of great significance in the tumor development process. EGFR, KRAS, and BRAF are important molecules in the signaling pathway, their gene mutations and signaling pathway activation, tumor development are closely related.RAS-GAPs, by increasing RAS proteins intrinsic GTPase activity, hydrolysis RAS-GTP to RAS-GDP a non-active form, to close the RAS signaling pathway to implement negative regulation to the pathway. RASA1is one the RAS-GAPs, a key regulator of the RAS signaling pathway molecules. About the role and significance of RASA1in pancreatic cancer is still unknown.Aim:This study through the detection of EGFR-the KRAS-ERK signaling pathway critical molecular mutation status and RASA1expression in pancreatic cancer cells and tissues, explores the mutations and RASA1possible role in pancreatic cancer and its clinical significance. It will provide a theoretical basis for further elucidation of the molecular mechanisms of pancreatic cancer development process and a new target of pancreatic cancer diagnosis and treatment.Method:We collected32cases of paraffin-embedded specimens of removal pancreatic cancer from2000to2009in Guangdong Province People’s Hospital, with5cases of pancreatic cysts,5cases of chronic pancreatitis tissue samples as a control.Genomic DNA was extracted using QIAamp DNA FFPE Tissue kit, amplificated the EGFR, KRAS, the BRAF common mutations point by PCR with Premix Ex TaqTM Hot Start Version kit, the progress is95℃5min—(95℃45s—58℃45s—72℃1min)×35cycles—72℃5min,12℃for ever.Product was rinsed, pre-denatured and transferred to96well plates, and direct sequencd it using ABI3100Genetic Analyzer.Total RNA was isolated with Trizol (Life Technologies).Quantitative real-time PCR (qRT-PCR) was performed using an ABI PRISM7500Quantitative PCR system (Applied Biosystems, Foster City, CA), for95℃10min—(95℃30s—58℃30s—72℃30s)×50cycles. The primers used were designed using Primer Premier5.0software. Each sample was examined in triplicate, and the amount of product was normalized in relation to GAPDH.The product was recovered and agarose gel electrophoresis was performed.2×106cells were lysated in400μl1×SDS lysate, boiled for10mins and centrifuged4℃20,000×g for10mins. Supernatant concentration was measured by BCA and adjusted to1μg/μl. Twenty micrograms of the total lysate protein were run for SDS—PAGE electrophoresis. Proteins were blotted onto PVDF membranes and immunostaining was done using polyclonal anti-RASA1antibody (1:1000), monoclonal anti-GAPDH antibody (1:2000),for4℃overnight. Membranes was labeled using HRP conjugated goat anti-rabbit secondary antibody, and colored by ECL. Grayscale of bands was analyzed by BandScan5.0.Briefly, deparaffinized sections were subjected to heat-induced epitope retrieval. After blocking by3%H2O2, tissues were incubated with anti-RASA1antibody (1:50),followed by HRP-conjugated En Vision goat anti-rabbit.3,3’-Diaminobenzidine was used as a substrate (Dako). All sections were counterstained with hematoxylin. Human ovarian cancer specimens were used as the positive control, and PBS, substituting the primary antibody, as the negative control. Positive cells must have clear cell structure, accurate position of positive granules, distinct contrast to the backdrop. The staining intensity was seored as follows:O(no staining),1(weak staining, light yellow or yellow) and2(moderate staining, yellowish brown or brown).All statistical analyses were performed using the SPSS13.0statistical software package. The Chi-square test and Fisher’s exact test were used to assess the difference of KRAS mutation rate and RASA1protein expression level between pancreatic cancer tissues and pancreatic benign lesion tissues. The relation between the RASA1protein expression and clinicopathologic parameters was respectively analyzed by χ2test, two independent samples Mest and Wilcoxon U test, according to the different data types. The differences of RASA1mRNA and protein level between the pancreatic cells were assessed by the one way ANOVA and the differences between groups were assessed using the SNK test.a=0.05(two side)as the difference level, and P<0.05was considered indicative significance.Result:Conclusion:KRAS mutations plays an important role in the abnormal activation of EGFR/RAS/MEK signaling pathway in pancreatic cancer.RASA1plays an important role in the pancreatic cancer development as a potential tumor suppressor gene.