Smad4静默对K-ras突变小鼠胰腺PanIN细胞增殖及转移能力的影响

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背景与目的:由己建立的小鼠基因打靶模型证实,K-ras突变启动了胰腺癌前病变—胰腺上皮内瘤变(pancreatic intraepithelial neoplasia,PanIN),p53、p16失活均可单独促进小鼠PanIN发展为浸润性胰腺癌。作为人胰腺癌中另一失活频率高发的抑癌基因Smad4,本课题组前期研究提示,应用RNA干扰技术沉默PanIN细胞株中内源性Smad4表达,促使PanIN细胞恶性转化。基于此目的,本研究进一步探讨siRNA干扰Smad 4基因对PanIN细胞体内外增殖及转移能力的影响,从而有助于阐明PanIN恶性转化的新机制。方法:构建和筛选能特异性静默PanIN细胞中Smad4表达的最佳siRNA稳定表达质粒,稳定转染最佳siRNA表达质粒进入PanIN细胞,用Zeocin筛选稳定转染克隆,挑选阳性克隆、扩增,稳定转染后细胞命名为PanIN-S。本研究运用细胞计数、克隆形成实验等分别比较两组在活细胞率和体外增殖能力的影响,同时为进一步验证Smad 4基因静默对PanIN细胞体外迁移、侵袭能力的影响,本研究采用Transwell和Mitigel assay检测其干扰前后体外运动、侵袭能力。动物实验中,建立PanIN及PanIN-S细胞组裸鼠移植瘤模型,并应用免疫组化方法检测并比较两组PCNA、VEGF和MMP-9的表达及其差异。结果:成功构建了Smad4基因siRNA体系;体外实验中与PanIN组相比,PanIN-S组细胞增殖、侵袭能力明显增强,差异有统计学意义(P<0.05);动物模型免疫组化结果显示,与PanIN组相比,PanIN-S组PCNA、VEGF和MMP-9表达显著增高(P<0.05)。结论:K-ras突变基础上小鼠PanIN细胞Smad4基因静默促使PanIN细胞的恶性转化;PanIN基础上Smad4静默促使小鼠PanIN细胞体内外增殖及转移能力的增强,其增殖及转移可能与PCNA、VEGF和MMP-9高表达有关。 BACKGROUND & AIM: The established murine gene targeting model confirmed that K-ras mutation initiates pancreatic precancerous lesions, pancreatic intraepithelial neoplasia (PanIN), and p53 and p16 inactivation can both promote PanIN Development of invasive pancreatic cancer. As another tumor suppressor gene, Smad4, which is frequently inactivated in human pancreatic cancer, our previous study suggested that RNA interference could silence the expression of endogenous Smad4 in PanIN cells and promote the malignant transformation of PanIN cells. For this purpose, this study further explored the impact of Smad 4 siRNA on the proliferation and metastasis of PanIN cells in vitro and in vivo, which may help to clarify the new mechanism of PanIN malignant transformation. METHODS: The optimal siRNA expression plasmid for silencing Smad4 in PanIN cells was constructed and screened. The optimal siRNA expression plasmid was stably transfected into PanIN cells. The stable clones were screened by Zeocin and the positive clones were selected and amplified. The cells were named PanIN-S after transfection. In this study, the effects of Smad 4 gene silencing on the migration and invasion ability of PanIN cells in vitro were compared between the two groups by cell counting and clonogenic assay. In this study, Transwell and Mitigel assay to detect its interference before and after in vitro exercise, invasive ability. Animal experiments, the PanIN and PanIN-S cell transplantation group was established nude mice model, and the immunohistochemical method was used to detect and compare the two groups of PCNA, VEGF and MMP-9 expression and differences. Results: The Smad4 gene siRNA system was successfully constructed. Compared with PanIN group, the proliferation and invasion ability of PanIN-S group were significantly increased in vitro (P <0.05). The results of immunohistochemistry in animal model showed that, Compared with PanIN group, the expression of PCNA, VEGF and MMP-9 in PanIN-S group was significantly increased (P <0.05). Conclusion: The Smad4 gene silencing in PanIN cells induced the malignant transformation of PanIN cells based on the K-ras mutation. The silencing of SmaIN4 by PanIN enhanced the proliferation and metastasis ability of PanIN cells in vitro and in vivo, And MMP-9 high expression.
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