基于荧光共振能量转移技术的STAT3二聚化抑制剂筛选模型的建立

目的%20构建STAT3二聚化抑制剂筛选模型,为STAT3抑制剂筛选提供实验方法。方法%20分别构建pECFP-N1-STAT3和pEYFP-N1-STAT3的荧光报告载体,利用脂质体转染技术将二者共转入HEK-293T细胞,利用ECFP和EYFP两种荧光蛋白之间能量共振转移,检测磷酸化STAT3分子的二聚化水平,并检测Stattic对二聚化的影响。结果%20成功构建了pECFP-N1-STAT3和pEYFP-N1-STAT3的荧光报告载体。将两种荧光报告载体共转染HEK-293T细胞后,Western%20Blot%20检测结果显示STAT3以及p-STAT3的表达水平明显增加。以458nm波长激发ECFP,其发射波长可激发EYFP。加入STAT3二聚化抑制剂Stattic后,荧光强度降低,且呈现一定的剂量依赖性。结论%20基于荧光共振能量转移技术的STAT3二聚化抑制剂筛选模型构建成功。     

Inhibition of STAT3 signaling targets both tumor-initiating and differentiated cell populations in prostate cancer

Despite of tremendous research efforts to profile prostate cancer, the genetic alterations and biological processes that correlate with disease progression remain partially elusive. In this study we show that the STAT3 small molecule inhibitor Stattic caused S-phase accumulation at low-dose levels and led to massive apoptosis at a relatively high-dose level in prostate cancer cells. STAT3 knockdown led to the disruption of the microvascular niche which tumor-initiating cells (TICs) and non-tumor initiating cells (non-TICs)depend on. Primary human prostate cancer cells and prostate cancer cell line contained high aldehyde dehydrogenase activity (ALDH^high) subpopulations with stem cell-like characteristics, which expressed higher levels of the active phosphorylated form of STAT3 (pSTAT3) than that of non-ALDH^high subpopulations. Stattic could singnificantly decreas the population of ALDH^high prostate cancer cells even at low-dose levels. IL-6 can convert non-ALDH^high cells to ALDH^high cells in prostate cancer cell line as well as from cells derived from human prostate tumors, the conversion mediated by IL-6 was abrogated in the presence of STAT3 inhibitor or upon STAT3 knockdown. STAT3 knockdown significantly impaired the ability of prostate cancer cells to initiate development of prostate adenocarcinoma. Moreover, blockade of STAT3 signaling was significantly effective in eradicating the tumor-initiating and bulk tumor cancer cell populations in both prostate cancer cell-line xenograft model and patient-derived tumor xenograft (PDTX) models. This data suggests that targeting both tumor initiating and differentiated cell populations by STAT3 inhibition is predicted to have greater efficacy for prostate cancer treatment.     

银屑病调节性T细胞的功能异常及STAT3通路调控机制研究

目的 研究银屑病患者外周血调节性T细胞（Treg）的功能,探讨与功能异常相关的STAT3信号通路机制。 方法 寻常性银屑病患者81例,银屑病面积和严重度指数（PASI）评分10 ～ 30,均为慢性斑块状。对照组46例,为健康献血者。采用流式细胞仪检测外周血中Treg细胞的比例,用体外淋巴细胞混合培养方法检测银屑病患者和健康人外周血中Treg细胞的增殖活性及对效应性T细胞（Tresp）的抑制功能,用流式细胞仪及qRT-PCR检测Treg细胞中磷酸化STAT3的比例及分泌促炎因子干扰素γ（IFN-γ）、肿瘤坏死因子α（TNF-α）、白细胞介素17（IL-17）的水平。最后,用STAT3通路抑制剂Stattic V处理银屑病患者Treg细胞,观察其增殖及抑制功能的恢复及分泌促炎因子的变化。 结果 银屑病患者组外周血Treg细胞数量（6.437% ± 0.186%）与对照组（6.812% ± 0.241%）比较差异无统计学意义（t = 1.224,P ＞ 0.05）,但银屑病患者组外周血Treg细胞增殖活性及对Tresp细胞的抑制功能明显降低,磷酸化STAT3表达水平显著升高,分泌促炎因子IFN-γ、TNF-α、IL-17的水平显著升高（均P ＜ 0.05）。经50 μg/L Stattic V作用后,银屑病患者Treg细胞对Tresp抑制率为61.670% ± 4.640%,未处理组为28.820% ± 11.490%,两组差异有统计学意义（P ＜ 0.05）;50 μg/L Stattic V作用后,促炎因子IFN-γ、TNF-α、IL-17 mRNA表达量（2-△△Ct）分别为1.654 ± 0.879、0.850 ± 0.705、0.572 ± 0.135,均显著低于未处理组（分别为23.350 ± 6.721、4.847 ± 1.525、3.095 ± 0.650）, 差异均有统计学意义（P ＜ 0.05）。 结论 银屑病患者Treg细胞对Tresp细胞的负向调控功能降低,其机制与STAT3信号通路异常活化有关,抑制STAT3通路的活化有可能一定程度地恢复Treg细胞功能。     

Abrogation of constitutive Stat3 activity circumvents cisplatin resistant ovarian cancer

The aim of the present study was to investigate the role of Stat3 in cisplatin resistant ovarian cancer. It was first demonstrated that higher activated Stat3 was detected in cisplatin-resistant ovarian cancer cell lines. To provide evidence that supported the hypothesis that phosphorylated-Stat3 expression may promote cisplatin resistance, ectopic Stat3 was expressed by IL-6 stimulation that partially abrogates Stat3, as opposed to the knock-down of Stat3 by specific siRNA that restores cisplatin sensitivity against ovarian cancer cells. This hypothesis was further confirmed by clinical tumor specimens of ovarian cancer obtained from patients with cisplatin-resistance. Based on these premises, Stattic [1], an effective small molecular inhibitor of Stat3, was used to inhibit Stat3 activation. The data presented here show that Stattic restored the sensitivity to cisplatin in chemoresistant ovarian cancer by significant reductions in the expression of the anti-apoptosis protein Bcl-2, Bcl-X_L, Survivin protein and phosphorylated-Akt levels. Consistent with these observations, this experiment demonstrated the first evidence of Stattic circumvented cisplatin resistance of orthotopic xenograft ovarian cancer in vivo. Altogether, these findings emphasize the importance of Stat3 in cisplatin resistance in ovarian cancer and provide a further impetus to clinically evaluate biological modifiers that may circumvent cisplatin resistance in patients with chemoresistant ovarian cancer.     

基于miR-370-3p与JAK2/STAT3通路相关性探讨活血荣络方促缺血性脑卒中后血管新生的机制

目的基于miR-370-3p与JAK2/STAT3通路相关性探讨活血荣络方促缺血性脑卒中后血管新生的机制。方法将大鼠随机分为6组,MCAO/R法造模,灌胃给药7 d后免疫荧光染色观察脑组织CD31、vWF及血管内皮生长因子(vascular endothelial growth factor,VEGF)表达;Western blot法检测脑组织JAK2、p-JAK2、STAT3、p-STAT3蛋白的表达;Real-time PCR(RT-PCR)法检测脑组织JAK2、STAT3 mRNA及miR-370-3p的表达;Pearson相关性分析脑组织miR-370-3p与JAK2/STAT3通路的相关性;培养大鼠脑血管平滑肌细胞,实时荧光定量PCR(RT-qPCR)检测LncRNA-H19和miR-370-3p表达;荧光素酶报告实验检测LncRNA-H19和miR-370-3p的靶向关系。结果活血荣络方能增加缺血区微血管密度及VEGF平均荧光强度,上调JAK2、STAT3 mRNA,下调miR-370-3p表达,促进JAK2、p-JAK2、STAT3、p-STAT3表达,且miR-370-3p分别与JAK2、STAT3 mRNA呈高度负相关,此过程能被STAT3 SH2结构域抑制剂Stattic逆转。结论活血荣络方可能通过下调miR-370-3p的表达、激活JAK2/STAT3通路、促进下游VEGF的表达而刺激缺血性脑卒中后血管新生,从而改善神经功能缺损症状。