miR-429靶向调控PKP1在慢性非细菌性前列腺炎发病中的作用及机制研究

Chronic Nonbacterial Prostatitis (CNP) affects the quiality of life of the patients severely and the pathogenesis of CNP is largely unexplored. It is reported that inflammatory cytokines play a prominent role in pathogenesis of CNP. Plakophilin1 (PKP1) is a type of cell-junctional protein, whose role in CNP is unexplored. Our previous study revealed PKP1 is highly expressed in normal prostate while downregulated in prostatitis tissue; Knockdown of PKP1 in prostatic cells leads to significant upregulation of inflammatory cytokines, increased proliferation and reduced apoptosis. Preliminary data revealed that miR-429 regulated the expression of PKP1 and knockdown of PKP1 led to activation of NF-kB pathway. Therefore, we hypothesize that miR-429 may target PKP1 and activate downstream NF-kB pathway, thereby upregulating the expression of inflammatory cytokines and play an important role in the pathogenesis of CNP. This project plans to detect the regulation of PKP1 and inflammatory cytokines by miR-429 and proliferation, apoptosis, adhesion of the prostatic cells via luciferase assay and other experiments; Explore the activation of NF-kB pathway by immunofluorescene experiments, pathway inhibitors and other methods; Establish CNP rat model to validate in vitro results, which may provide a new theoretical basis and targets for prevention and treatment of CNP.

慢性非细菌性前列腺炎（CNP）严重影响生活质量，其发病机制尚未明确。炎性细胞因子在其发病过程中发挥重要作用。斑菲素蛋白1（PKP1）是细胞连接蛋白，未见其在CNP中的作用报道。我们前期研究发现，CNP组织中PKP1表达较正常组织显著降低；在正常前列腺上皮细胞中敲低PKP1可激活NF-kB通路，促使多种炎性细胞因子上调，进而促进增殖和抑制凋亡；生物信息学及分子生物学实验初步表明PKP1受到miR-429调控。因此，我们推测miR-429可能靶向调控PKP1而激活NF-kB通路，促进下游炎性细胞因子表达，从而参与CNP的发生、发展。本课题拟通过荧光素酶报告基因等技术验证miR-429对PKP1、炎性细胞因子的表达调控及对细胞增殖、凋亡、黏附的影响；采用通路阻滞剂、免疫荧光等方法检测PKP1对NF-kB通路的激活及下游效应；构建CNP大鼠模型验证体外实验结果，为其防治提供新的理论依据与作用靶点。

慢性非细菌性前列腺炎（CNP）严重影响生活质量，其发病机制尚未明确。炎性细胞因子在其发病过程中发挥重要作用。斑菲素蛋白1（PKP1）是细胞连接蛋白，未见其在CNP中的作用报道。我们前期研究发现，CNP组织中PKP1表达较正常组织显著降低；在正常前列腺上皮细胞中敲低PKP1可激活NF-kB通路，促使多种炎性细胞因子上调，进而促进增殖和抑制凋亡；生物信息学及分子生物学实验初步表明PKP1受到miR-429调控。因此，我们推测miR-429可能靶向调控PKP1而激活NF-kB通路，促进下游炎性细胞因子表达，从而参与CNP的发生、发展。本课题通过荧光素酶报告基因等技术验证了miR-429对PKP1的靶向调控，构建了miR-429过表达/低表达体系，并测定了miR-429调控PKP1对炎性细胞因子表达及对细胞增殖、凋亡、黏附的影响，发现miR-429过表达时细胞中PKP1表达量显著下调，而IL6、IL8等炎症因子的表达量显著上调，同时细胞增殖显著上调、凋亡下调、细胞粘附增加。同时利用BPH-1-shPKP1细胞株，测定了NF-kB经典及非经典信号途径的激活情况及使用通路抑制剂后的细胞表型，发现NF-kB通路依靠经典及非经典信号途径的激活。顺利建立了CNP大鼠及小鼠模型，发现毛萼乙素可以缓解CNP小鼠模型中前列腺炎症并缓解盆腔疼痛的严重程度。