Effects Suppression of Bcl-xl Expression by RNAi on Proliferation and Apoptosis of Human Pancreatic Cancer Cells and Chemosensitivity to Gemcitabine
|Keywords||pancreatic cancer Bcl-xl RNAi gemcitabine chemosensitivity|
Objective Bcl-xl is members of Bcl-2 family plays inhibiting cell apoptosis role function protein, it expressed in a variety of tumor tissues, and in pancreatic cancer tissue also presents the high expression, contribut to tumor cell proliferation and apoptosis and chemoradiation tolerance impact. RNA interference (RNAi) can highly specific to downgrade gene expression, this study aims at carrying RNAi technique to pancreatic cancer cell line PANC1 inhibit Bcl-xl gene expression, to observe its impact to pancreatic cancer cell proliferation and apoptosis, invasion ability and the gemcitabine chemotherapy sensitivity, discussed the influence of Bcl-xl genes in pancreatic cancer occurrence, development, and the correlation with pancreatic cancer drug resistance produces, for pancreatic cancer gene therapy explore a new avenue.Methods Design and preparation for Bcl-xl gene siRNA (small interfering RNA), with cationic liposomes lipofectamineTM2000 as the carrier to transfect human pancreatic cancer cell line PANC1, application of flow cytometric analysis, determine the optimal carrying conditions. Using RT-PCR to detect the Bcl-xl mRNA expression of PANC1 cells after transfection,filtrate the most effective siRNA. The influence of RNAi on invasion ability of PANC1 cells was measured by transwell chamber experiment, MTT assay was used to detect cell proliferation ability as well as the cell survival rates under the use of gemcitabine, and the method of Annexin V-FITC /PI was used to detect cell apoptosis level under the presence and absence of gemcitabine.Results①SiRNA was successfully transfected into human pancreatic cancer cell line PANC1,it present better transfection rate when cell concentration is 5×104/hole , cell rendezvous degrees about 70% and siRNA concentration is 60nmol/L in 24 hole boards,the maximum efficiency up to 79.6%, continue to improve concentration can not obviously improve the efficiency.②SiRNA induce Bcl-xl mRNA expression obviously down-regulated, siRNA1 group is the most significant reduced.③Transwell chamber experiment Transwell small room experiments, the relative inhibition rate of the siRNA group, the negative-control group,the blank control group for 45.6%, 3.2%, 0% . Respectively, compared with control group, PANC1 cells in vitro carrying group significantly weakened; Compared with carrying group and chemotherapy group, joint group cell proliferation more slowly, growth curve more gently; AnnexinV- FITC/PI detection, the early apoptosis rate of joint group is 39.3%, carrying group and chemotherapy group’s early apoptosis rate 15.9% and 17.8%, respectively combined treatment group compared with carrying group and chemotherapy group was statistically significant difference (p < 0.05).Conclusions Bcl-xl protein high expression in pancreatic cancer cells, through siRNA successfully transfected pancreatic cancer cells,the expression of Bcl-xl gene suppressed, PANC1 cell proliferation is restrained,and inducing apoptosis, weakened the invasion ability, strengthened the proliferation suppression and apoptosis-inducing role to gemcitabine. It’s suggests that RNAi can effectively silence PANC1 cells Bcl-xl gene expression, still can increase the chemotherapy sensitivity of pancreatic cancer to gemcitabine. Bcl-xl could be a potential pancreatic cancer gene therapy targets, targeted Bcl-xl gene silencing maybe an effective method to overcome cancer chemotherapy drug resistance .