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中华细胞与干细胞杂志(电子版) ›› 2019, Vol. 09 ›› Issue (04) : 230 -235. doi: 10.3877/cma.j.issn.2095-1221.2019.04.007

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论著

沉默Gpr48基因表达对皮肤鳞状细胞癌细胞系A431 PKC信号通路活化及细胞侵袭的影响
张瑞华1,(), 杨海平1, 刘彬1   
  1. 1. 210046 南京大学医学院附属泰康仙林鼓楼医院皮肤科
  • 收稿日期:2019-06-19 出版日期:2019-08-01
  • 通信作者: 张瑞华

Effects of silencing Gpr48 gene expression on PKC signaling pathway activation and cell invasion in skin squamous cell carcinoma cell line A431

Ruihua Zhang1,(), Haiping Yang1, Bin Liu1   

  1. 1. Department of Dermatology, Taikang Xianlin Drum Tower Hospital Affiliated to Medical College of Nanjing University, Nanjing 210046, China
  • Received:2019-06-19 Published:2019-08-01
  • Corresponding author: Ruihua Zhang
  • About author:
    Corresponding author:Zhang Ruihua, Email:
引用本文:

张瑞华, 杨海平, 刘彬. 沉默Gpr48基因表达对皮肤鳞状细胞癌细胞系A431 PKC信号通路活化及细胞侵袭的影响[J/OL]. 中华细胞与干细胞杂志(电子版), 2019, 09(04): 230-235.

Ruihua Zhang, Haiping Yang, Bin Liu. Effects of silencing Gpr48 gene expression on PKC signaling pathway activation and cell invasion in skin squamous cell carcinoma cell line A431[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2019, 09(04): 230-235.

目的

分析沉默G蛋白偶联受体48 (Gpr48)基因表达对皮肤鳞状细胞癌(SCC)蛋白激酶C (PKC)信号通路及细胞侵袭能力的影响。

方法

构建ShRNA感染皮肤SCC细胞株A431,设计干扰Gpr48表达shRNA(shRNA-Gpr48组)及阴性对照shRNA-NC组,采用实时定量反转录聚合酶联反应(RT-PCR)检测Gpr48相对表达量;以蛋白印迹法(Western Blot)测定PKC相关抗体蛋白表达情况,进行Transwell小室实验检测细胞侵袭能力,总结Gpr48缺失对三丙胺(TPA)刺激A431细胞株PKC活化及细胞侵袭能力的影响,为SCC靶向治疗提供参照。组间比较采用独立样本t检验。

结果

qRT-PCR结果:shRNA-Gpr48组Gpr48相对表达量(0.27±0.06)低于shRNA-NC组(1.01±0.12),差异具有统计学意义(t?= 17.441,P?< 0.01),shRNA-Gpr48组p-PKCδ (0.463±0.035)低于shRNA-NC组(0.721±0.074),shRNA-?Gpr48组NF-κB p65 mRNA (0.631±0.079)低于shRNA-NC组(0.834±0.102),差异具有统计学意义(t?= 9.966、4.975,P均< 0.01),Notch1 mRNA(0.981±0.037)高于shRNA-NC组(0.562±0.041),差异具有统计学意义(t?= 23.991,P?< 0.01);Western Blot结果:shRNA-Gpr48组p-PKCδ(0.221±0.034)低于shRNA-NC组(0.292±0.046)、NF-κB p65蛋白表达(0.512±0.047)低于shRNA-NC组(0.636±0.051),差异具有统计学意义(t?= 3.925,5.653,P均< 0.01),Notch1(0.524±0.063)高于shRNA-NC组(0.421±0.076),差异具有统计学意义(t?= 3.299,P?< 0.01);shRNA-Gpr48组侵袭细胞率为(35.03±1.54)﹪,低于shRNA-NC组的(51.83±2.76)﹪,差异具有统计学意义(t?= 16.809,P?< 0.01)。

结论

沉默Gpr 48缺失表达可能通过抑制PKC活化,降低p-PKCδ、NF-κB p65表达,上调抑癌基因Notch1表达,抑制癌细胞增殖、侵袭。

Objective

To analyze the effects of silencing G-protein-coupled receptor 48 (Gpr48) gene expression on protein kinase C (PKC) signaling pathway and cell invasion in skin squamous cell carcinoma (SCC) .

Methods

Gpr48 interfering shRNA (shRNA-Gpr48 group) and negative control shRNA were infected into skin SCC cell line A431. And the relative expression of Gpr48 was detected by real-time quantitative reverse transcription polymerase chain reaction (RT-?PCR) . Expressions of PKC-related antibody proteins were determined by Western Blot. Transwell chamber assay was used for cell invasion. Statistical analysis was performed with SPSS 20.0-software. The independent sample t test was used to compare the differences of the above indexes in shRNA-Gpr48 group and shRNA-NC group. The effects of Gpr48 deletion on activation of A431 cell line PKC and and cell invasion stimulated by tripropylamine (TPA) were determined.

Results

qRT-PCR results showed the relative expression of Gpr48 in the shRNA-Gpr48 group was lower than that in the shRNA-NC group (0.27±0.06) vs (1.01±0.12) (t = 17.441, P < 0.001) . The mRNA expression of p-PKCδ (0.463±0.035) vs (0.721±0.074) , and NF-κB p65 was lower in the shRNA-Gpr48 group compared to the shRNA-NC group (0.631±0.079) vs (0.834±0.102) (t?= 9.966, 4.975, all P?< 0.01) while the mRNA expression of Notch1 was higher (0.981±0.037) vs (0.562±0.041) (t?= 23.991, P?< 0.001) in the shRNA-Gpr48 group. Western Blot results showed that the protein expression of p-PKCδ (0.221±0.034) vs (0.292±0.046) , and NF-κB p65 (0.512±0.047) vs (0.636±0.051) (t?=3.925, 5.653, all P?< 0.01) was lower in the shRNA-Gpr48 group compared to the shRNA-NC group while the expression of Notch1 was higher in the shRNA-Gpr48 group (0.524±0.063) vs (0.421±0.076) (t?= 3.299, P?< 0.01). The cell invasion rate in the shRNA-Gpr48 group was lower than that in the shRNA-NC group (35.03±1.54) ﹪ vs (51.83±2.76) ﹪ (t?= 16.809, P?< 0.01) .

Conclusion

Silencing Gpr 48 expression may inhibit cancer cell proliferation and invasion by inhibiting PKC activation, decreasing expressions of p-PKCδ and NF-κB p65 and up-regulating the expression of tumor suppressor gene Notch1.

表1 RT-PCR反应各引物序列
图1 SCC A431细胞稳转株Gpr48基因敲出效率
图2 倒置显微镜下观察两组细胞形态(×400)
表2 Gpr48缺失对SCC A431细胞株各基因表达的影响( ± s
图3 Western Blot测定A431细胞各蛋白表达
表3 Gpr48缺失对SCC A431细胞株各蛋白表达的影响( ± s
图4 Gpr48缺失对SCC A431细胞株侵袭能力的影响
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