Neuromedin B receptor mediates neuromedin B-induced COX-2 and IL-6 expression in human primary myometrial cells
Neuromedin B receptor mediates neuromedin B-induced COX-2 and IL-6 expression in human primary myometrial cells
复制标题
DOI：
10.1136/jim-2020-001412
复制
发表时间：
2020-07
影响因子：
2.6
通讯作者：
Jingfei Chen;Yun Shi;Jingrui Huang;Jiefeng Luo;Weishe Zhang
中科院分区：
文献类型：
--
作者：
Jingfei Chen;Yun Shi;Jingrui Huang;Jiefeng Luo;Weishe Zhang
导致分娩的确切机制尚不清楚。在我们最初的互补DNA（cDNA）微阵列实验中，我们发现，神经介肽B受体（NMBR）在人类子宫肌层中的差异表达在自发或催产素诱导的劳动。我们以前的研究表明，神经介肽B（NMB）可以通过核因子κ B（NF-κB B）转录因子p65（p65）和Jun原癌基因激活蛋白1（AP-1）转录因子亚基（c-Jun）诱导原代人子宫肌细胞表达白细胞介素6（IL-6）和2型环氧合酶（考克斯-2）。本研究旨在探讨NMB诱导的效应是否需要NMBR。本研究建立了子宫肌层细胞原代培养模型，为研究肌松在分娩启动中的作用机制提供了一个合适的模型。进行免疫化学染色以验证NMBR在原代子宫肌层细胞中的表达。采用真实的定量PCR（RT-qPCR）和Western blotting检测NMBR、p65、c-Jun、考克斯-2和IL-6的mRNA和蛋白表达。将含有靶向NMBR的shRNA或含有NMBR cDNA序列的慢病毒转染至原代子宫肌层细胞以敲低或过表达NMBR。通过膜联蛋白V和碘化丙啶染色测定细胞死亡，并通过流式细胞术分析。NMB处理后，NMBR的敲低显著减弱了考克斯-2和IL-6的上调，p65和c-Jun的磷酸化，而过表达的NMBR则增强了p65和c-Jun的磷酸化，总p65和c-Jun无显著变化。总之，本研究表明，NMBR介导的NMB诱导NF-κB和AP-1活化，进而诱导IL-6和考克斯-2在原代子宫肌层细胞中的表达。
The precise mechanisms that lead to parturition remain unclear. In our initial complementary DNA (cDNA) microarray experiment, we found that the neuromedin B receptor (NMBR) was differentially expressed in the human myometrium during spontaneous or oxytocin-induced labor. We have previously shown that neuromedin B (NMB) could induce interleukin 6 (IL-6) and type 2 cyclo-oxygenase enzyme (COX-2) expression in the primary human myometrial cells via nuclear factor kappa B (NF-κB) transcription factor p65 (p65) and Jun proto-oncogene, activator protein 1 (AP-1) transcription factor subunit (c-Jun). This study is aimed to investigate whether NMBR is required for NMB-induced effect. Primary myometrial cell culture was established to provide a suitable model to investigate the mechanism of NMB in labor initiation. Immunochemical staining was conducted to validate the NMBR expression in primary myometrial cells. The mRNA and protein expression of NMBR, p65, c-Jun, COX-2 and IL-6 were assessed by Quantitative Real Time PCR (RT-qPCR) and western blotting. Lentiviruses with shRNAs targeting NMBR or containing cDNA sequence of NMBR were transfected to primary myometrial cells to knockdown or overexpress NMBR. Cell death was determined by annexin V and propidium iodide staining and analyzed by flow cytometry. The upregulation of COX-2 and IL-6 and phosphorylation of p65 and c-Jun were significantly attenuated by knockdown of NMBR and enhanced by overexpressed NMBR following NMB treatment, with no significant change in total p65 and c-Jun. In summary, this study showed that NMBR-mediated NMB-induced NF-κB and AP-1 activation, which in turn, induce expression of IL-6 and COX-2 in primary myometrial cells.