MPB83蛋白诱导NF-κBp65核转位抑制肿瘤细胞凋亡过程中miR-130b作用机制的研究

Intravesical Bacille Calmette-Guerin (BCG) therapy has been proved to be a useful way in treating cancer in clinic. However, recent research showed that tumor cells infected by BCG exhibit resistant to apoptosis induced by TNF-αreagent, but which bacterial component is involved and how it works in this procedure are still unknown. The data acquired from our previous research showed that the incubation of cervical cancer cells with the MPB83 protein, a secreted protein derived from Bacille Calmette-Guerin, could decrease the apoptosis ratio of tumor cells induced by TNF-α relying on suppressed expression of PTEN protein which was caused by the enhancement of NF-κBp65 nuclear translocation. On the basis of bioinformatics analysis, we considered that miR-130b not only participated in the down-regulation of PTEN protein expression caused by the enhanced NF-κBp65 nuclear translocation but also resulted in the enhancement of NF-κBp65 nuclear translocation. Our study aims to confirm the conclusion reached according to the bioinformatics data analysis using the experimental methods such as luciferase reporter assays. And then we can reveal the mechanisms by which the miR-130b mediated the down-regulated expression of PTEN protein caused by the enhanced NF-κBp65 nuclear translocation and by which the increased expression of miR-130b gene enhanced the NF-κBp65 nuclear translocation based on the experimental data. The uncovered mechanism will be beneficial to understand how the MPB83 protein inhibits the apoptosis of tumor cells and to offer an evidence to support the hypothesis that there may be a potential danger in treating cancer using BCG therapy in clinic.

卡介苗接种是临床上治疗癌症的一种方法。但近期研究人员却发现，卡介苗感染肿瘤细胞后，能使细胞对肿瘤坏死因子-α(TNF-α)诱导的凋亡作用产生抗性。而参与这一过程的细菌组分及其作用机制还有待研究。本项目前期实验结果显示卡介苗的外分泌蛋白MPB83与宫颈癌细胞共孵育后，可以通过增强NF-κBp65核转位抑制PTEN蛋白表达，进而抑制TNF-α诱导的细胞凋亡。同时前期生物信息学预测结果显示，miR-130b基因表达不仅可以介导NF-κBp65核转位对PTEN蛋白表达的抑制作用，而且还能反馈调控NF-κBp65核转位。本项目拟用荧光素酶报告基因检测系统等实验手段证实信息学的预测结果，并以此揭示miR-130b基因表达介导NF-κBp65核转位抑制PTEN蛋白表达以及调控NF-κBp65核转位的机制，解释MPB83蛋白抑制宫颈癌细胞凋亡的现象，最终为临床上应用卡介苗治疗肿瘤存在风险的推测提供依据。

报道称，卡介苗感染肿瘤细胞后，能使细胞对肿瘤坏死因子-α(TNF-α)诱导的凋亡作用产生抗性。本项目研究显示，MPB83与宫颈癌HeLa和Siha细胞孵育会导致NF-κBp65核定位现象的出现和miR-130b表达水平的上升；siRNA-p65转染靶细胞抑制NF-κBp65表达能抑制细胞内NF-κBp65在靶细胞系中表达，干扰MPB83诱导性NF-κBp65核定位和miR-130b水平升高现象；基因PCR扩增联合测序技术测定结果验证了miR-130b编码基因的转录起始位点；荧光素酶报告基因检测系统测试结果证实了转录起始位点附近NF-κBp65激活miR-130b基因表达的位点，上述结果说明，NF-κBp65核转位介导MPB83对miR-130b表达的激活作用。生物信息学预测联合荧光素酶报告基因检测系统测试结果显示，PTEN、TOLLIP编码mRNA3’UTR存在miR-130b的靶定位点；mimics和inhibitors转染靶细胞分别降低和升高两种靶基因mRNA及蛋白表达水平；与NC相比，miR-130b-3p转染靶细胞能降低细胞的凋亡水平，促进细胞增殖导致细胞活性上升；与pcDNA3.1相比，miR-130b-3p与pcDNA3.1::PTEN共转染靶细胞能增加细胞凋亡水平，抑制细胞增殖导致细胞活性下降；与NC相比，miR-130b-5p转染靶细胞能增强NF-κBp65核转位；与pcDNA3.1相比，miR-130b-5p与pcDNA3.1::TOLLIP共转染靶细胞干扰细胞NF-κBp65核转位，上述结果说明，miR-130b-3p通过抑制PTEN蛋白表达可以降低细胞凋亡水平，促进细胞增殖导致细胞活性上升，miR-130b-5p通过抑制TOLLIP蛋白可以增强NF-κBp65核转位。综上所述，本研究证实MPB83诱导性miR-130b基因表达介导了MPB83对宫颈癌细胞活性的增强作用，应用卡介苗治疗肿瘤存在风险。