The Effect Of Pancreatic Cancer Cells On Fibroblasts

BackgroundRecent studies have shown that the microenvironment of cancers,which include stroma cells,cytokines,chemotactic factors and so on,plays important roles in tumour initiation and progression.Among the content of microenvironment,cancer-associated fibroblasts(CAFs) were thought of more and more important.While pancreatic tumor cells are surrounded by extensive stroma,especially the CAFs.Many researches have shown that CAFs can accelerate tumor growth and migration in pancreatic cancer,but the studies of how the CAFs were generated,and the role of pancreatic cancer plays in the process were rare.Objectiv1. To investigate whether the pancreatic cancer cells BXPC-3,SW1990 and the MVs derived from them can activate normal fibroblasts(NFs) into cancer-associatedfibroblasts(CAFs),and the effect into the progression of fibroblasts.2. To find the possible molecular mechanism that the pancreatic cancer cells activate the NFs into CAFs.Methods1. NFs were isolated from mouse pancreas tissues of wild C57,then NFs were cultured with BXPC-3 or SW1990 cells by indirect co-cultures, and detected the content of the specific proteins α-SMA,FAP in fibroblasts by western assay,the progression of fibroblasts by edu assay.2.Microvesicles(MVs) isolated from BXPC-3,SW1990 cells by differential centrifugation were used to stimulate NFs and calculate the content of the α-SMA,FAP protein and the progression of fibroblasts3. Total RNA was extracted from the fibroblasts using Trizol reagent,then 12 different kinds of mi RNAs in fibroblasts and MVs were detected by q RT-PCR.4. NFs were transfected with pre-mi R-155 according to the previous results, then the content of the α-SMA,FAP protein and the progression of fibroblasts were detected.5. MVs that collected from the BXPC-3 and SW1990 cells transfected with anti-mi R-155 were used to stimulate the NFs, then the content of the α-SMA,FAP protein and the progression of fibroblasts were detected similarly.6. TP53inp1 was selected as the target gene of mi R-155,and the content of TP53inp1 were detected in fibroblasts which were co-cultured with cancer cells,or stimulated with MVs,or transfected with pre-mi R-155,or stimulated with MVs which had been knocked down mi R-155.7. NFs was transfected with TP53inp1 si RNA,then the content of the α-SMA,FAP protein and the progression of fibroblasts were detected.Results1.After co-cultured with BXPC-3 or SW1990 cells, the α-SMA protein level in fibroblasts increased( 84.70 ± 0.36) %,( 69.11 ± 0.26) %( P<0.05) respectively,while the FAP protein level increased(59.82±0.49)%,(42.17 ±0.11)%(P<0.05)respectively,and the progression of fibroblasts increased (57.33±0.08)%,(48±0.12)%(P<0.05)respectively.2.After the stimulation of the MVs derived from BXPC-3 or SW1990 cells, the α-SMA protein level in fibroblasts increased(61.06±0.12)%,(54.47±0.20)% (P<0.05)respectively,while the FAP protein level increased(85.46±0.67)%, (49±0.24)%(P<0.05)respectively,and the progression of fibroblasts increased(3.47±0.44)folds,(4.65±0.60)folds(P<0.05)respectively.3. Among the selected 12 kinds of mi RNAs, the content of mi R-155 increased (1.50±0.45)folds,(1.95±0.65)folds(P<0.05)respectively after co-cultured with BXPC-3 or SW1990 cells.Similarly, the content of mi R-155 increased (1.11±0.35)folds,(87.90±0.27)%(P<0.05)respectively after the stimulation of the MVs derived from BXPC-3or SW1990 cells.In BXPC-3 or SW1990 cells, the content of mi R-155 in MVs were(7.37±0.66)times or(7.85±0.62)times (P<0.05)respectively compared with those in MV-free.4.After transfected with pre-mi R-155,the α-SMA,FAP protein level in fibroblasts increased(1.52±0.11)folds,(1.71±0.67)folds respectively(P<0.05), the progression of fibroblasts increased(94.11±0.17)%(P<0.05).5. After the stimulation of MVs which knocked down mi R-155,the α-SMA,FAP protein level in fibroblasts had no significantly change,and the progression of fibroblasts also had no significantly change.6. After co-cultured with BXPC-3 or SW1990 cells,the content of TP53inp1 protein level decreased(49.15±0.09)%,(27.54±0.10)%(P<0.05)respectively. After the stimulation of the MVs derived from BXPC-3 or SW1990 cells, the content of TP53inp1 protein level decreased(78.80±0.12)%,(67.95±0.14)% ( P<0.05) respectively.After transfected with pre-mi R-155, the content of TP53inp1 protein level decreased(35.82±0.02)%(P<0.05).But there was no significantly change of TP53inp1 protein level in fibroblasts after the stimulation of MVs which knocked down mi R-155.7. After transfected with TP53inp1 si RNA, the α-SMA,FAP protein level in fibroblasts increased( 90.28 ± 0.11) %,( 94.64 ± 0.29) %( P<0.05) respectively,and the progression of fibroblasts increased( 92.27 ± 0.30) % (P<0.05).Conclusions1. Pancreatic cancer cells BXPC-3,SW1990 and MVs derived from the cells can motivate the activation of NFs into CAFs,and the progression of CAFs increased markly.2. Mi R-155,targeting TP53inp1, might be secreted from pancreatic cancer cells,packed by MVs, transfered into NFs and then motivate them into CAFs.