| Objective To investigate the expression of Integrinβ1 (ITGB1) protein and unravel its clinical significance in pancreatic carcinoma. And to investigate the inhibitory effect of RNA interference (RNAi) on ITGB1 gene expression and protein level of PANC-1 cell, as well as its effect on cell invasiveness, proliferative ability, apoptosis level, and drug sensitivity of PANC-1 cell. To lay the foundation for further investigation on the invasion and drug resistance mechanisms of pancreatic carcinoma.Methods The immunohistochemical method was used to determine the expression of ITGB1 in 28 paraffin embedded samples of pancreatic carcinoma, compared with that in normal pancreas tissues of 14 cases, then revealed the relationship between ITGB1 expression and clinical features of pancreatic carcinoma. SiRNA for ITGB1 gene was designed and synthesized, then be transfected into pancreatic carcinoma cell line PANC-1 by positive ion liposome Lipofectamine 2000. Transfect effiency was assessed by flow cytometry. Use RQ-PCR (Real-time Quantitative Polymerase Chain Reaction) to detect the expression of ITGB1 and ab1 mRNA, then estimate the interference efficiency, survey how long the ITGB1 siRNA induced RNAi effect can preserve, and screening the most effective siRNA. Expression of ITGB1 protein was detected by western blot. The influence of RNAi on invasion ability of PANC-1 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 pyramidate concentration of 5-fluorouracil (5-Fu), and the method of Annexin V/PI was used to detect cell apoptosis level under the presence and absence of 5-fluorouracil (5-Fu).Results (1) There are more ITGB1 protein expression in pancreatic carcinoma tissues than in normal pancreatic tissues, statistical significance can be seen between the two groups. There are more ITGB1 protein expression in pancreatic carcinoma tissues of these patients who has lymphatic metastasis than those no lymphatic metastasis. The expression levels of ITGB1 were obviously different in clinical stages, but had nothing to do with the differentiation of pancreatic carcinoma. (2) It present better transfection rate when cell concentration is 2.0×105/hole and siRNA concentration is 50 nmol/L. While concentration dependent manner was not detected and the transfection rate tends to decrease when the concentration be further increased. Inhibition effect of siRNA3 on ITGB1 expression is most significant within designed four siRNAs, and the expression of ITGB1 mRNA can be maitain at a very low level at least 96 hours. 48 hours after transfection, the PANC-1 cells express a significant lower ITGB1 protein quantity then origin. (3) After transfection, invasiveness of PANC-1 cell in vitro decreased obviously, and it seemed have no variation on proliferation ability and apoptosis rate of PANC-1 cell, but the cell survival rates of PANC-1 cell under certain concentration of 5-Fu was decreased markedly.Conclusions The expression of ITGB1 is potentialized in pancreatic carcinoma, and it is positively correlated with the clinical stages or lymph node metastasis of pancreatic carcinoma, but have nothing to do with cancer differentiation. The ITGB1 siRNA is successfully constructed in vitro and efficiently transfected to PANC-1 cells. ITGB1 siRNA can effectively suppress ITGB1 mRNA and protein expression in human pancreatic carcinoma cell line PANC-1 for about 96 hours, and at the same time, there are not marked difference on proliferation ability and apoptosis rate of PANC-1 cell, but invasiveness of PANC-1 cell in vitro decreased obviously, and they became sensitive about apoptosis induced by 5-Fu. All these suggested that ITGB1 gene could be a target for gene therapy of pancreatic carcinoma, RNAi is expected to be an effective measure for tumor gene therapy. |
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