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    SMAD4 基因缺失在胰腺癌细胞对吉西他滨耐药中的作用研究

    Study on the Role of SMAD4 Gene Deletion in Gemcitabine Resistance in Pancreatic Cancer Cells

      摘要
    • 摘要:
      目的 探究 SMAD4 基因与胰腺癌细胞对吉西他滨耐药性的关系，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Western blotting试验检测 SMAD4 基因的 mRNA 和蛋白水平的表达，划痕试验和 Transwell试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 blotting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，能增强吉西他滨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。

       

      Abstract:
      OBJECTIVE  To explore the relationship between SMAD4 gene and gemcitabine resistance in pancreatic cancer, and provide new research ideas for individualized treatment of pancreatic cancer.
      METHODS  Bioinformatics analysis was used to analysis the SMAD4 gene expression in pancreatic cancer cells, high throughput sequencing was used to detect AsPC-1 and MIA-PaCa2 genes; CCK8 method and Trypan blue staining assay were used to detect the toxicity of gemcitabine at different concentrations on pancreatic cancer cells, and then the optimal concentration and time were selected. Real time fluorescent quantitative PCR and Western blotting were used to detect the mRNA and protein expression of SMAD4 gene. Scratch test and Transwell test were used to detect the effect of silenced or overexpressed SMAD4 gene on cell migration and invasion under the combined action of gemcitabine.
      RESULTS  According to cBioPortal database, 33% of pancreatic cancer patients had SMAD4 gene mutations, and most of the mutations were deletion, missense and truncation mutations; High throughput sequencing analysis, real-time fluorescence quantitative PCR, and Western blotting confirmed that the expression levels of SMAD4 gene at mRNA and protein levels in MIA PaCa-2 cells were significantly higher than those in AsPC-1 cells. The optimal concentration of gemcitabine for AsPC-1 cells was 10−5 mol·L−1, and the optimal treatment time was 48 h. The optimal concentration of gemcitabine for MIA PaCa-2 cells was 10−7 mol·L −1, and the optimal treatment time was 48 h. Compared with the control group, silencing the SMAD4 gene significantly increased the survival rate of MIA PaCa-2 cells treated with gemcitabine. Compared with the PBS group, the number of cells migrating and invading in the drug treated group was significantly reduced, but the number of cells migrating and invading in the transfected group was significantly upregulated compared to the non transfected group. After overexpression of the SMAD4 gene, the survival rate of the transfected group cells treated with gemcitabine was significantly lower than that of the control group, and the scratch area of the transfected plasmid drug group decreased the slowest, with a significant reduction in the number of invasive cells passing through the chamber.
      CONCLUSION  There are significant differences in the SMAD4 gene between AsPC-1 cells and MIA PaCa-2 cells. SMAD4 gene deletion is associated with gemcitabine resistance in pancreatic cancer cells. SMAD4 gene, as a tumor suppressor gene, can enhance the ability of gemcitabine to inhibit the proliferation, migration and invasion of pancreatic cancer cells, and is expected to become a new target to solve the problem of drug resistance in pancreatic cancer

       

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