The Effects Of Silencing K-ras In Pancreatic Cancer Cells On Cell Proliferation, Apoptosis And Cell Signaling

BACKGROUND:Pancreatic Cancer is one of the most aggressive human solid tumors, with rapid growth, hidden symptoms and early metastatic, as well as resistance to chemotherapeutic drugs. The only effective therapy known is surgery. While it is difficult to discover this tumor in its early stage, few patients have the chance of surgery resection. The5-year survival rate among patients with pancreatic cancer, even after surgery, is about12-20%. The pancreatic cancer currently rank fourth among cancer-related deaths in western countries.The causes of pancreatic cancer remain unknown. Several environmental factors and multiple genes abnormally expresstion have been implicated. Activation of oncogenes and inactivation of tumor suppressor genes are believed to be basic moleculer events. K-ras oncogene plays a vital role in pancreatic cancer’s growth and maintenance. A K-ras mutation is present in up to80～95%of sporadic pancreatic cancers and is reported to be an early genetic event. K-ras encodes a small molecular GTP/GDP binding protein, when binds to GTP, it is in the active state and activates proteins necessary for the growth and proliferation of cells, and other cell signal factors. The protein product of the normal K-ras gene performs an essential function in normal tissue signaling. While mutated, this transformed protein is trapped in the activated state enables downstream signaling pathways without the activation of growth factors upstreams, which finally leads to unlimited cell proliferation and results in the invasive carcinoma.Point mutation is the most common format in K-ras mutation, especially in the codon12. As the earliest and most common genetic mutation in pancreas cell transformation and tumor progression, as well as the central importance of ras signaling for cell function and survival, highlight that mutant K-ras is a new attractive molecular marker for the early diagnosis and target for the treatment of pancreatic cancer. The extraordinary sequence specificity of RNA interference (RNAi) offers a hope for cancer therapy that may differentiate the small difference between mutant and normal K-ras genes.ARHI is a human suppressor gene that belongs to the Ras superfamily. Unlike Ras, It exerts opposite functions against most of its family member, which attracted us in its function in pancreatic cancer. It has been proved to be inactive in most pancreatic cancers. While reexpression, ARHI can inhibits the proliferation of PC cell lines, induces apoptosis, leads to cell cycle arrest and blocks the downstream signaling pathways of Ras. It even reduces the expression of Ras. The interaction between ARHI and Ras remains to be studied.OBJECTS:Specific knockdown of activated K-ras via RNA interference in pancreatic cancer cell lines, then examine the proliferation, apoptosis and cell cycle and corresponding molecular consequences of K-ras cell signaling pathways. Determine whether the expression of ARHI is increased.METHODS:Two human pancreatic carcinoma lines with activated K-ras (PANC-1and MiaPaCa-2) of point mutation in codon12were used. K-ras inhibition was accomplished by small interfering RNA (siRNA) knockdown of K-ras expression via transfected using lipofectmine2000. Tranfection effects were examined by fluorescence microscope and flow cytometry. K-ras mRNA and protein expression were analyzed by RT-PCR and western blot respectively. Cell proliferations were determined by CCK-8method. Apoptosis were analyzed by flow cytometry, as well as Hoechst staining and Acridine orange staining in morphology. Real-time PCR was used to analyzing the relative quantity of cyclin A and E,as well as ARHI, and the cell signaling associated proteins as p-ERK, p-AKT, pan-AKT and the tumor suppressor gene p53and ARHI were detected by western blot method.RESULTS:The transfection effects using lipofectmine2000can be higher than90%in both celss lines. K-ras gene in both cell lines reduced the expression of K-ras mRNA and protein, which were significant24h or48h after transfection. The effect of reduction was strongest when using1OOnM siRNA concentration. The reduction was specific as oligonucleotides with a single bp alteration, while the oligonucleotides specific for the other cell line were ineffective. MiaPaCa-2cells showed significant proliferation after K-ras RNAi, p<0.05, and both PANC-1and MiaPaca-2cells showed increased apoptosis, p<0.05. Both cell lines showed alteration in the cell cycle, a reduction of S phase from32.4%to21.8%in PANC-1,26.5%to21.7%in MiaPaCa-2, and cellular arrest in the G1phase,50.4%to65.1%in PANC-1and58.7%to67.4%in MiaPaCa-2, statistically significant (p<0.05). mRNA levels of cyclin A and E tent to be dereaesed, but the realative quantities were higher than0.5, while the alteration of mRNA level of ARHI remained unclear. The levels of p-ERK, p-AKT protein expression decreased significantly while which of AKT remains. Furthermore, the tumor suppressor gene p53showed a increased level of protein expression, and ARHI protein band was not found after RNAi.SUMMARY:Specific silencing mutant K-ras in human pancreatic cancer cell lines through RNAi by transient transfection can knockdown the specific mutant K-ras sequences, which results in reduced proliferation in MiaPaCa-2, increased apoptosis in both cell lines and cell cycle arrest. The protein levels of crucial downstream cell signaling associated proteins were reduced thus inhibit the signal transduction. The interaction between ARHI and Ras needs futher research.