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中华细胞与干细胞杂志(电子版) ›› 2017, Vol. 07 ›› Issue (05) : 265 -270. doi: 10.3877/cma.j.issn.2095-1221.2017.05.003

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论著

基质交联分子1表达下调对人胰腺癌SW1990细胞周期及细胞凋亡的影响
黎美琳1, 顾鹏2,()   
  1. 1. 214000 无锡市第五人民医院(无锡市传染病医院)肝病科
    2. 214000 无锡市锡山人民医院泌尿外科
  • 收稿日期:2016-11-03 出版日期:2017-10-01
  • 通信作者: 顾鹏

Effects of down-regulation of stormal interaction molecule 1 on cell cycle and apoptosis of pancreatic cancer cell line SW1990

Meilin Li1, Peng Gu2,()   

  1. 1. Department of Hepatology, Wuxi No.5 People's Hospital, Wuxi 214000, China
    2. Department of Urology, Xishan People's Hospital of Wuxi, Wuxi 214000, China
  • Received:2016-11-03 Published:2017-10-01
  • Corresponding author: Peng Gu
  • About author:
    Corresponding author: Gu Peng, Email:
引用本文:

黎美琳, 顾鹏. 基质交联分子1表达下调对人胰腺癌SW1990细胞周期及细胞凋亡的影响[J/OL]. 中华细胞与干细胞杂志(电子版), 2017, 07(05): 265-270.

Meilin Li, Peng Gu. Effects of down-regulation of stormal interaction molecule 1 on cell cycle and apoptosis of pancreatic cancer cell line SW1990[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2017, 07(05): 265-270.

目的

探讨基质交联分子1(STIM1)表达下调对人胰腺癌细胞株SW1990细胞周期和细胞凋亡的影响,并研究其分子作用机制。

方法

构建携带STIM1基因siRNA的慢病毒载体转染SW1990细胞,分为对照组、空载体组和STIM1组。采用半定量逆转录聚合酶链反应(RT-PCR)和Western blot验证STIM1组SW1990细胞STIM1表达下调。通过MTT增殖实验和流式细胞术检测STIM1表达下调对细胞增殖、周期和细胞凋亡的影响,采用RT-PCR和Western blot法检测细胞周期和细胞凋亡相关分子表达的变化。两组间比较采用t检验。

结果

STIM1组SW1990细胞中STIM1表达mRNA(0.261±0.029)、蛋白(0.120±0.032)低于空载体组mRNA(1.002±0.091)、蛋白(0.996±0.053),t =20.74、26.89,P均< 0.01。SW1990细胞24、48、72 h的增殖水平STIM1组分别为(0.122±0.008)、(0.252±0.031)、(0.373±0.028),相比空载体组(0.223±0.035)、(0.618±0.017)、(0.924±0.140),t = 6.48、16.90、23.99,P < 0.01,受到抑制。STIM1组中SW1990细胞G2/M期细胞比例(41.47±0.66)﹪高于空载体组(10.30?±?2.24)﹪,t = 23.14,P < 0.01。STIM1组中SW1990细胞凋亡率(25.21±1.96)﹪高于空载体组(3.71±1.23)﹪,t =16.03,P < 0.01。半定量RT-PCR和Western blot提示,STIM1组细胞周期蛋白B1(cyclin B1)表达mRNA(0.344±0.031)、蛋白(0.776±0.042)相比空载体组mRNA(1.011±0.060)、蛋白(1.034±0.036),t = 40.06、8.51,P均< 0.01下调;STIM1组p21表达mRNA(1.970±0.107)、蛋白(1.315±0.093)相比空载体组mRNA(1.025±0.044)、蛋白(0.998±0.036),t =17.10、9.52,P均< 0.01上调;STIM1组Bcl-2表达mRNA(0.156±0.025)、蛋白(0.381±0.028)相比空载体组mRNA(1.010±0.072)、蛋白(0.980±0.057),t =15.46、14.63,P均< 0.01下调;STIM1组survivin表达mRNA(0.188±0.022)、蛋白(0.022±0.019)相比空载体组mRNA(1.016±0.090)、蛋白(0.994±0.047)t = 58.08、442.58,P均< 0.01下调;STMI1组procaspase-3表达蛋白(0.389±0.030)相比空载体组蛋白(1.008±0.040)下调,差异有统计学意义(t = 19.22,P?< 0.01)。

结论

在胰腺癌SW1990细胞中,沉默STM1可阻滞细胞于G2/M期,抑制细胞增殖,诱导细胞凋亡,有望成为胰腺癌治疗的分子靶点。

Objective

To investigate effects of down-regulation of STIM1 on cell cycle and apoptosis of pancreatic cancer cell line SW1990 and explore its possible molecular mechanism.

Methods

Lentiviral vectors, which carrying siRNA of STIM1 gene, were contructed. Then lentiviral vectors were used to transfect the SW1990 cells and were divided into three groups:an untreated group, an empty vector group and an STIM1 group. The effects of down-regulation of STIM1 expression on cell proliferation, cell cycle and apoptosis were assessed by MTT assay and flow cytometry, respectively. Further expression changes of genes related to cell cycle and apoptosis were detected by semi-quantitative RT-PCR and Western blot. All values present the mean±SD.ttest was used between the two groups.

Results

The STIM1 expression of STIM1 group mRNA(0.261?±?0.029), protein(0.120?±?0.032)was down-regulated compared with the empty vector group[mRNA(1.002?±?0.091) t= 20.74,P< 0.01],[protein(0.996?±?0.053) t?= 26.89,P< 0.01]in the SW1990 cells. Cell proliferation of SW1990 in the STIM1 group in 24?h, 48 h, 72 h(0.122?±?0.008),(0.252?±?0.031),(0.373±0.028)was significantly inhibited compared with the empty vector group[(0.223±?0.035) t= 6.48,P< 0.01],[(0.618±0.017) t= 16.90,P< 0.01],[(0.924?±?0.140) t= 23.99,P?< 0.01]. The results of flow cytometry revealed that the percentage of cells at G2/M phase in the STIM1 group was(41.47±0.66)﹪, significantly higher than that in the empty vector group[(10.30±2.24)﹪,t= 23.14,P< 0.01]. In addition, the percentage of apoptotic cells in the STIM1 group was(25.21±1.96)﹪, significantly higher than that in the empty vector group[(3.71?±1.23)﹪,t= 16.03,P< 0.01]. Moreover, the results of semi-quantitative RT-PCR and Western blot showed that Cyclin B1 expression of the STIM1 group[mRNA(0.344±0.031)],[protein(0.776±0.042)]was down regulated compared with the empty vector group[mRNA(1.011±0.060)t= 40.06,P= 0.000][protein(1.034±0.036)t= 8.51,P< 0.01], P21 expression of STIM1 group[mRNA(1.970±0.107)],[protein(1.315±0.093)]was regulated compared with the empty vector group[mRNA(1.025?±0.044)t= 17.10,P< 0.01],[protein(0.998±0.036)t?= 9.52,P< 0.01], Bcl-2 expression of STIM1 group[mRNA(0.156±0.025)],[protein(0.381±0.028)]was down regulated compared with the empty vector group[mRNA(1.010±0.072)t= 15.46,P< 0.01],[protein(0.980±0.057) t= 14.63,P< 0.01]. Survivin expression of STIM1 group[mRNA(0.188±0.022)],[protein(0.022±0.019)]was down regulated compared with the empty vector group[mRNA(1.016±0.090)t= 58.08,P?< 0.01],[protein(0.994±0.047)t= 442.58,P< 0.01]. Procaspase-3 expression of STIM1 group[protein(0.389±0.030)]was down regulated compared with the empty vector group[protein(1.008±?0.040)t= 19.22,P< 0.01].

Conclusion

STIM1 may play a pivotal role in the proliferation and apoptosis of pancreatic cancer cell line SW1990, which could become a molecular target for therapy of pancreatic carcinoma.

表1 RT-PCR引物序列
表2 STIM1 siRNA靶向干扰SW1990细胞后,STIM1 mRNA和蛋白相对表达量(±s)
图1 倒置荧光显微镜下观察慢病毒载体转染SW1990细胞72 h (×100)
图2 STIM1?siRNA靶向干扰SW1990细胞后STIM1的表达变化
表3 抑制STIM1表达对SW1990细胞增殖的影响(±s)
表4 抑制STIM1表达对SW1990细胞周期影响(﹪, ± s)
表5 抑制STIM1表达对SW1990细胞周期与凋亡相关基因mRNA和蛋白表达的变化(﹪, ± s)
图3 抑制STIM1表达对SW1990细胞周期与细胞凋亡相关分子变化
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