Rab14 Was Upregulated In Pancreatic Cancer And Regulated Cell Proliferation, Invasion, Migration And Chemoresistance Through YAP/Bcl-2 Signaling Pathway

1 ObjectiveThe pancreatic cancer has a high malignancy,poor prognosis and death to early metastasis[1].Although there has been a further study of the changes in the genetic aspect and apparent aspect of pancreatic cancer,the pathogenesis is still unclear,and this experiment is intended to study further[2-6].The Rab GTPase family protein Rab14 belongs to RAS oncogene superfamily of small G-protein which regulate membrane vesicle trafficking,signal transduction,and recycling of various membrane receptors.Rab family proteins play various roles during development and progression of human cancers.Rab14 has been implicated in cancer development,including ovarian cancer[11],oral squamous cell carcinoma[7],gastric cancer[8],renal cancer[9].However,its clinical significance in pancreatic cancers and its biological effects have not been examined.In this research,expression pattern and clinical significance of Rab14 in pancreatic cancer was explored.In addition,the effect of Rab14 on the biological behavior of pancreatic cancer and the potential mechanism was analyzed.2 Materials and methods2.1 Surgical specimens103 cases of pancreatic cancer specimens were collected from pancreatic cancer patients who received radical operation without preoperative therapy in the First Affiliated Hospital of China Medical University.Patients didn't undergo chemoradiotherapy before surgery.Histological grade and differentiation degree were determined according to WHO classification criterion.2.2 Immunohistochemistry analysisTissues of formalin-fixed paraffin-embedded surgical specimens were constructed.The sections were examined by two professional pathologists individually.2.3 Cell cultureBxpc3 and capan2 cells were purchased from American Type Culture Collection?Manassas,VA,USA?.They were cultured in RPMI 1640 supplemented with 10%heat-inactivated fetal bovine serum?FBS?in a humidified cell incubator at 37?with an atmosphere of 5%CO₂.2.4 Cell transfection and interferenceThe plasmid of pCMV6-Rab14 was purchased from Origene and transfected into cells using lipofect3000.SMARTpool siRNA for Rab14 and Non-targeting siRNA were purchased from Dharmacon.siRNA transfection was performed using Dharmafect1 reagent.2.5 Western blotThe total protein was collected from pancreatic cancer cells treated with Rab14plasmid and siRNA.The protein concentration was measured through Bradford method.The proteins were electrophoresed and electrotransfered.2.6 Real-time Quantitative polymerase chain reactionTotal RNA was extracted from cell lines using Trizol following the manufactory instructions.Real-time PCR was performed using SYBR Green master mix?TAKARA,Dalian,China?.PCR was performed using 7500 Real-Time PCR System?Applied Biosystems?.?-actin was used as the reference gene.The relative expression of target genes was calculated using the 2^-??Ct??Ct method.Experiments were repeated in triplicate.2.7 Cell proliferation assayFor MTT assay,cells were plated in 96-well plates and cultured overnight.MTT solution was added to each well and incubated for 4 h at 37°C,then the supernatant was removed from each well.DMSO was added to dissolve the formazan crystals.Absorbance was measured at 490 nm.2.8 Transwell assayCells in serum free medium were added to the upper chamber,and 15%FBS-containing medium was used as chemoattractant in the lower chamber.ECMatrix^TMwas pre-coated to the upper chamber for invasion assay.And cells on the bottom of the membrane were stained and checked.2.9 Flow cytometryCells were collected and washed twice with PBS,followed by being resuspended in 250?l of binding buffer.Cells were fixed in 1%paraformaldehyde and then stained with 5 mg/ml propidium iodide alone or together with Annexin V/FITC for cell cycle or apoptosis analysis,respectively.After incubation in the dark for 15 min,cells were analyzed by FACS Calibur flow cytometer.2.10 Statistical analysisSPSS version 13 for Windows was used for all statistical analyses.A?2 test was usedtoexaminepossibleassociationsbetweenRab14expressionand clinicopathological factors.The Student's t-test was used to compare differences between control and treatment groups.P<0.05 was considered to indicate statistical significance.3 Results3.1 Expression pattern of Rab14 in pancreatic cancerExpression of Rab14 in pancreatic cancer tissue was determined using immunohistochemistry.Rab14 was located in cytoplasm.Rab14 was overexpression in pancreatic cancers.3.2 Clinical significance of Rab14 in pancreatic cancerRab14 overexpression in pancreatic cancers was significantly correlated with advanced TNM stage,histological grade and lymph node metastasis.3.3 The expression of Rab14 in pancreatic cancer cell linesBxpc3 cells and Capan2 cells were chosen for Rab14 plasmid and siRNA transfection.Western blot was employed to estimate the transfection efficiency.The results showed that Rab14 protein expression was upregulated in Bxpc3 cells while downregulated in Capan2 cells.3.4 Rab14 facilitates proliferation of pancreatic cancer cellsMTT assay showed that the proliferation rate of pancreatic cancer cells increased significantly after Rab14 overexpression while Rab14 depletion inhibited proliferation of pancreatic cancer cells.Colony formation assay showed that Rab14 overexpression upregulated colony number while Rab14 depletion facilitated colony formation ability in pancreatic cancer cells.3.5 Rab14 promotes invasion of pancreatic cancer cellsTranswell test results showed that the numbere of invading cells increased in Bxpc3 cells after Rab14 overexpression,while decreased in Capan2 cells after Rab14depleption.3.6 Rab14 promotes chemoresistance in pancreatic cancer cellsAnnexin V/PI analysis was used to detect the level of apoptosis after treatment ofgemcitabine.The results showed that Rab14 significantly suppressed the rate of apoptosis while its depletion upregulated gemcitabine induced apoptosis in pancreatic cancer cells.3.7 Rab14 regulates expression of cell cycle,invasion and apoptosis related proteinExpression of related proteins was examined using western blot.The results showed that cyclinA,cyclinD1,cyclinE,p-Rb,bcl-2 levels increased while p21 levels decreased in pancreatic cancer cell with Rab14 overexpression.On the contrary,expression of cyclinA,cyclinD1,cyclinE,p-Rb,bcl-2 was downregulated while p21was upregulated in decreased in pancreatic cancer cell with after Rab14 depletion.3.8 Rab14 regulates cell apoptosis through YAP/bcl-2 signalingRab14 overexpression upregulated YAP level in pancreatic cancer cell line Bxpc3 while Rab14 depletion downregulated YAP level in Capan2 cells.Rab14overexpression inhibited pancreatic cancer cell apoptosis through YAP/bcl-2signaling.4 ConclusionIt is identified that Rab14 is overexpression in pancreatic cancer and was significantly correlated with advanced TNM stage,histological grade and lymph node metastasis.Rab14 promoted pancreatic cancer cell proliferation,invasion,cell cycle transitionand chemoresistance to gemcitabine.Rab14 activatedYAP/bcl-2 signaling in pancreatic cancer cells.