目的研究SMAD4新突变体对胰腺导管腺癌(PDAC)发生发展的影响。方法 将构建好的空白对照组(NC组)、野生型组(SMAD4 wt组)、突变型组(SMAD4 Y353C组)3组质粒用TransIntro TM EL转染试剂分别导入PDAC细胞PANC-1中,首先进行实时荧光定量聚合酶链反应(RT-qPCR)和Western Blot实验,分析不同质粒对PDAC细胞mRNA和蛋白质表达的影响,然后进行伤口愈合实验和细胞侵袭实验,研究不同质粒对PDAC细胞PANC-1伤口愈合和侵袭能力的影响。结果 RT-qPCR和Western Blot检测结果显示,NC组、SMAD4 wt组、SMAD4 Y353C组PDAC细胞PANC-1 SMAD4 mRNA和蛋白表达量比较差异均有统计学意义( F =695.76、 194.98 , P <0.05),而且SMAD4 Y353C组mRNA和蛋白表达量均低于SMAD4 wt组,高于NC组,差异均有显著性( P <0.05)。体外伤口愈合实验和细胞侵袭实验结果显示,NC组、SMAD4 wt组和SMAD4 Y353C组细胞的迁移面积和侵袭能力比较差异有统计学意义( F =1 741.15、299.12, P <0.05),SMAD4 Y353C组细胞的迁移面积和侵袭能力明显大于SMAD4 wt组细胞,小于NC组细胞,差异均有显著意义( P <0.05)。结论 突变型SMAD4 Y353C能抑制PANC-1细胞 SMAD4 mRNA和蛋白质的表达,促进PANC-1细胞的迁移和侵袭,为PDAC的治疗提供理论依据。
Objective To study the effect of a new SMAD4 mutant on the development and progression of pancreatic ductal adenocarcinoma (PDAC). Methods Three groups of plasmids, i.e., normal control group (NC), wild-type group (SMAD4 wt), and mutant-type group (SMAD4 Y353C) were transfected into PANC-1 PDAC cells with TransIntro TM EL transfection reagent. RT-qPCR and Western Blot were performed to analyze the effects of different plasmids on the mRNA and protein expression of PDAC cells. And wound healing assay and cell invasion assay were then carried out to investigate the effects of these plasmids on wound healing and invasion of PANC-1 PDAC cells. Results The results of RT-qPCR and Western Blot showed that there were significant differences in the mRNA and protein expression of SMAD4 in the PANC-1 PDAC cells between these groups ( F =695.76,194.98, P <0.05). Moreover, the mRNA and protein expression in the mutant-type group was significantly lower than that in the wild-type group but significantly higher than that in the NC group ( P <0.05). Meanwhile, in vitro wound healing assay and cell invasion assay showed that there were significant differences in the cell migration area and invasion ability between these groups ( F =1 741.15,299.12, P <0.05), and the cell migration area and invasion ability in the mutant-type group were significantly higher than those in the wild-type group but smaller than those in the NC group ( P <0.05). Conclusion The SMAD4 Y353C mutant can inhibit the mRNA and protein expression of SMAD4 in PANC-1 cells, and promote the migration and invasion of these cells as well, which provides a theoretical basis for the treatment of PDAC.
Journal of Precision Medicine
Carcinoma, pancreatic ductal