| 引用本文: | 吴博雅,安倩,曹睿,陈静.SMAD4 基因缺失在胰腺癌细胞对吉西他滨耐药中的作用研究[J].中国现代应用药学,2025,42(6):22-32. |
| wuboya,anqian,caorui,chenjing.Role of SMAD4 Gene Deletion in Gemcitabine Resistance in Pancreatic Cancer Cells[J].Chin J Mod Appl Pharm(中国现代应用药学),2025,42(6):22-32. |
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| 摘要: |
| 目的 探究 SMAD4 基因与胰腺癌对于吉西他滨的耐药性的关系，为胰腺癌的个体化医疗提供新的研究思路。方法 生物信息学分析 SMAD4 基因在胰腺癌细胞中的表达水平，高通量测序检测Aspc-1与MIA-paca2基因序列， CCK8 法与台盼蓝染色实验检测不同浓度吉西他滨对胰腺癌细胞的毒性作用，进而选取最佳作用浓度与时间， 实时荧光定量 PCR 和蛋白免疫印迹实验检测 SMAD4 基因的 mRNA 和蛋白水平的表达，划痕实验和 Transwell 实验检测沉默或过表达 SMAD4 基因在吉西他滨共同作用下对细胞迁移、侵袭能力的影响。结果 cBioPortal 数据库分析有33%胰腺癌患者发生 SMAD4 基因突变，且突变多为缺失、错义突变和截断突变；高通量测序分析、实时荧光定量PCR及Western blot证明 SMAD4 基因在 MIA PaCa-2 细胞的mRNA与蛋白水平的表达量均明显高于AsPC-1 细胞，吉西他滨作用于 AsPC-1 细胞的最佳浓度为 10 -5 mol·L?1、最佳作用时间是 48 h，吉西他滨作用 MIA PaCa-2 细胞的最佳浓度为 10 -7 mol·L?1、最佳作用时间是 48 h。 与对照组相比，沉默 SMAD4 基因后吉西他滨作用 MIA PaCa-2 细胞的存活率明显上升；与 PBS 组相比，加药组细胞迁移和侵袭的细胞数明显降低，但转染组相较于无转染组的迁移侵袭的细胞数明显上调。 过表达 SMAD4 基因后，吉西他滨作用于转染组细胞的存活率明显低于对照组，且转染质粒加药组的细胞划痕面积减少最慢，通过小室的侵袭细胞数明显减少。结论 SMAD4 基因在 AsPC-1 细胞和 MIA PaCa-2 细胞中存在明显差异。 SMAD4 基因缺失与胰腺癌细胞对吉西他滨耐药性相关。SMAD4 基因作为一种抑癌基因，能增强 GEM 抑制胰腺癌细胞增殖、迁移和侵袭的能力，有望成为解决胰腺癌耐药性问题的新靶点。 |
| 关键词: 胰腺癌 SMAD4 吉西他滨 耐药性 |
| DOI： |
| 分类号:R284.1；R917.101 |
| 基金项目:Mfn2介导的线粒体自噬与力达霉素抗肿瘤作用机制研究 |
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| Role of SMAD4 Gene Deletion in Gemcitabine Resistance in Pancreatic Cancer Cells |
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wuboya, anqian, caorui, chenjing
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North China University of Science and Technology
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| Abstract: |
| ABSTRACT: OBJECTIVE To detect the expression level of SMAD4 gene in pancreatic cancer AsPC-1 and MIA PaCa-2 cells, and to explore the specific relationship between SMAD4 gene and pancreatic cancer resistance to gemcitabine, in an attempt to solve the clinical problem of ineffectiveness for drug-resistant pancreatic cancer. In order to provide new research ideas for the personalized medicine of pancreatic cancer. METHODS 1 Bioinformatics analysis of SMAD4 gene expression in pancreatic cancer . 2 CCK8 assay to detect the toxic effects of different concentrations of gemcitabine on pancreatic cancer AsPC-1 cells and MIA PaCa-2 cells . 3 Trypan blue assay to detect the effect of gemcitabine on the survival rate of pancreatic cancer AsPC-1 cells and MIA PaCa-2 cells. 4 Quantitative PCR and Western blotting to detect the mRNA and protein levels of SMAD4 gene. 5 Transfection of plasmid or siRNA to overexpress or silence SMAD4 gene in pancreatic cancer cells. 6 Detecting the effect of gene and drug on cell migration by scratch test. 7 Transwell experiments were used to observe the effects of genes and drugs on cell migration and invasion. RESULTS 1 The mutation rate of SMAD4 gene in pancreatic cancer was 33%. High-throughput sequencing analysis showed that the expression of SMAD4 gene in AsPC-1 cells and MIA PaCa-2 cells was significantly different. 2 he mRNA and protein levels of SMAD4 gene in AsPC-1 cells were significantly lower than those in MIA PaCa-2 cells, and the sensitivity of AsPC-1 cells to gemcitabine was significantly lower than that in MIA PaCa-2 cells. 3 Compared with the control group, the survival rate of gemcitabine-treated MIA PaCa-2 cells increased after the knockout of SMAD4 gene, and the number of cells transfected with siRNA in the Transwell chamber was significantly higher than the number of cells without siRNA transfection. The number of cells passing through the chamber in the dye-added drug group was higher than that in the single drug-added group. 4 After overexpression of SMAD4 gene, the survival rate of gemcitabine-treated cells in the transfection plasmid group was significantly lower than that in the control group, and the cell scratch area in the transfection plasmid plus drug group decreased the slowest, and the number of cells passing through the Transwell chamber decreased significantly. CONCLUSION The expression of SMAD4 gene in AsPC-1 cells and MIA PaCa-2 cells was significantly different. Through CCK8 and trypan blue experiments, it was concluded that the best concentration of gemcitabine on AsPC-1 cells was 10-5M, and the best time was 48h; the best concentration of gemcitabine on MIA PaCa-2 cells was 10-7M, and the best time was 48h.Gemcitabine can inhibit the migration and invasion of pancreatic cancer cells, and the SMAD4 gene knockout significantly increases the migration and invasion ability of pancreatic cancer cells, and can partially inhibit the killing effect of gemcitabine on pancreatic cancer cells after knockout. |
| Key words: pancreatic cancer SMAD4 gemcitabine resistance |
