miR-1224-5p通过靶基因SIN3A特异性调控NF-kB信号通路在幽门螺杆菌致病中的作用机制

We found from a genome-wide microRNA screen that, three days post transfection of miR-1224-5p into AGS cells，the NF-κB activation induced by H.pylori infection was abrogated significantly. SIN3A is predicted to be a potential target gene of miR-1224-5p.RNAi silencing of SIN3A could specifically inhibit the NF-κB signal pathway activated by H.pylori infection, instead of induction by TNFα or IL1β. It was shown that SIN3A is a key regulator of deacetylation of H3K27 and plays important roles in gene regulation，and the expression level of miR-1224-5p and its target gene SIN3A in various cancer cell lines is significantly high and low, respectively.However, there is no evidence about the functions miR-1224-5p and its target gene SIN3A play during the NF-κB signal pathway activated by H.pylori infection and the progression of related gastric diseases.Therefore, from different levels of cell cultures, molecular mechanisms, clinic tissue samples and animal model, we proposed here by applying diverse in vitro and in vivo experimental techniques, to investigate the dynamic expression level of miR-1224-5p and its target gene SIN3A in gastric epithelial cell lines and clinic tissue samples in different phases of H.pylori infection related diseases, to reveal how they are involved in the NF-κB signal pathway activated by H.pylori infection，and to study the molecular mechanisms on how miR-1224-5p and SIN3A play specific roles in NF-κB activation by affecting H3K27Ac and subsequently the activity of the promoters of the target genes of SIN3A. The completion of this project will aid in understanding the initiation and development of H.pylori infection related gastric diseases, pave the way for better diagnostics and treatment, and provide theoretic basis for searching for novel marker and effective targets.

全基因组miRNA筛选发现，AGS细胞转染miR-1224-5p后，由Hp感染诱发的NF-κB通路下调;SIN3A是miR-1224-5p的重要靶基因之一。RNAi沉默SIN3A可特异性抑制由Hp激活的NF-κB通路。SIN3A对H3K27去酰基化和基因转录起重要作用，且SIN3A及miR-1224-5p在多种肿瘤细胞系中呈差异表达。但miR-1224-5p及SIN3A对Hp感染诱导NF-κB信号通路激活及其疾病相关性尚未见报道。本研究拟借助细胞、分子、组织样本及动物模型，分析miR-1224-5p及SIN3A在胃上皮细胞和Hp感染不同病变胃粘膜中的动态表达，研究其与NF-κB激活的关系，探讨miR-1224-5p及SIN3A在Hp感染起始阶段如何通过影响H3K27Ac及其靶基因启动子的活性来激活NF-κB，将为Hp感染相关疾病的诊断与防治寻找新标志物与有效靶点提供理论依据。

SIN3A是一种转录抑制因子，可以拮抗MYC的原癌活性，也可以与MXD1-MAX 异源二聚体相互作用而抑制转录，对组蛋白H3K27Ac进行负调控，并通过序列特异性与染色质上的特定DNA结合，调节基因转录。现有研究表明，SIN3A在不同疾病中发挥着不同的角色，其在胃相关疾病中的作用及调控机制尚不清楚。本研究主要研究内容为:1.细胞水平：通过体外细胞实验研究H.p与miR-1224-5p/SIN3A的调控作用，以及对胃癌细胞生物学行为的影响和机制。2.组织水平：研究miR-1224-5p/SIN3A在胃癌及癌旁正常组织中的表达情况及与H.p感染、临床病理参数的关系。 3.基因组水平：通过RNA-seq实验对SIN3A相关基因表达谱的变化进行分析；同时利用co-IP技术，筛选能够与SIN3A直接结合的蛋白；利用生物信息学技术，预测在胃癌中有差异表达且可能受组蛋白去乙酰化调控的基因，并进一步构建ceRNA网络。4、双荧光素酶基因报告：采用双荧光素酶基因报告系统确证SIN3A是miR-1224-5p的靶基因。本研究表明，miR-1224-5p在胃癌细胞和胃癌组织中低表达，能够抑制胃癌细胞增殖和转移；SIN3A在胃癌细胞和胃癌组织中高表达，能够促进胃癌细胞增殖和侵袭转移，抑制凋亡。SIN3A表达与细胞周期无关，差异无统计学意义（p＞0.05）。在胃癌细胞中，miR-1224-5p靶向结合SIN3A，从而调控EMT相关指标Vimentin、Ecadherin、snail的表达。. 结论：1.miR-1224-5p/SIN3A可通过促进胃癌细胞EMT以及激活NF-κB信号转导通路促进胃癌的侵袭和迁移。2.miR-1224-5p/SIN3A能通过调控胃癌细胞组蛋白去乙酰化，从而调控肿瘤相关基因表达。

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