Hp感染是胃癌的重要病因，但全球近半数人感染 Hp,其中仅有约1%的人发展为胃癌。导致Hp感染后不同结局的原因尚不清楚，可能与Hp毒力、宿主和环境因素有关。因此，探寻细菌和宿主与胃癌相关的关键分子，有助于胃癌高危人群的筛查及胃癌的靶向治疗，对于胃癌的早期防治具有重大意义。为此，本研究聚焦Hp的二个重要的毒力因子CagA和OipA,研究了它们的毒力调控网络，筛选出它们对胃上皮细胞癌性转化起作用及其调控的关键信号和分子，在临床、动物和细胞水平验证这些关键信号通路和调控分子，并进一步研究的它们作用机制。本研究集成蛋白质组学、转录组分析技术、生物信息学技术、基因编辑等技术，从分子、细胞和整体不同层次研究Hp致病致癌机制。构建了Hp关键毒力因子(OipA和CagA)的调控网络，发现Hp的毒力因子不是独立存在的，它们之间可相互调控，共同作用导致疾病的发生。发现了多个Hp及其毒力因子OipA和CagA诱导胃上皮细胞癌变的关键信号通路和调控分子，并重点研究了VASN和PMEPA1在Hp致癌中的作用及机制，发现它们涉及细胞分化、增殖、凋亡和迁移等多种细胞生长和死亡相关生物过程，以及炎症免疫、信号传导相关生物过程，并影响多条肿瘤和炎症免疫相关信号通路。发现了Hp及其毒力因子CagA引起细胞自噬丧失和自噬底物p62积累，进而导致Rad51泛素化，从而抑制了DNA损伤修复的能力，引起基因组不稳定性，最终促进胃癌发生。以上发现揭示Hp致胃癌新的分子机制，为Hp相关胃癌的靶向治疗提供了新的理论依据。Helicobacter pylori(Hp)is the main cause of gastric cancer(GC),but nearly half the world’s population is infected with Hp,of which only about 1%develop GC.The cause of different Hp infection outcomes remains unknown,which may be related to Hp virulence,host and environmental factors.Therefore,the exploration of the key molecules related to GC in Hp and host is significant for the screening of high-risk groups for GC and targeted treatment of GC,which is conducive to the early prevention and treatment of GC.This study focused on CagA and OipA,two vital virulence factors of Hp,studied their virulence regulatory networks,and screened out their key signals and regulatory molecules that play a role in the cancerous transformation.These key signal pathways and molecules were validated in clinical specimens,animals and cells,and their mechanisms were further studied to elucidate the effect and mechanism of Hp and its virulence factors on carcinomatous transformation of gastric epithelial cells.The research integrated proteomics,transcriptomics,bioinformatics,gene editing and other technologies to study the pathogenic and carcinogenic mechanism of Hp from different levels.The regulatory network of the two virulence factors of Hp was constructed,and it was found that the virulence factors were not independent,but they could regulate each other and jointly lead to the disease.Several key signal pathways and regulatory molecules(VASN,PMEPA1,MSN,KRT81,NGFR,PPPIR3C,etc.)of carcinogenesis induced by Hp and its virulence factors were discovered,and the role and mechanism of VASN and PMEPA1 in Hp carcinogenesis were mainly studied.They were found to be involved in a variety of biological processes related to cell growth and death,such as cell differentiation,proliferation,apoptosis and migration,as well as biological processes related to inflammatory and signal transduction,and affected multiple tumor and inflammatory-related signaling pathways(PI3K/AKT,JAK-STAT,IL-17,Wnt,Hippo,AMPK,Hedgehog,etc.).It was also found that Hp and its virulence factor CagA caused autophagy loss and p62 accumulation,leading to Rad51 ubiquitination,which inhibited the ability to repair DNA damage,causing genomic instability,and ultimately promoted GC.The above findings reveal the new molecular mechanism of Hp-induced GC and provide a new theoretical basis for the targeted therapy of Hp-related GC.

我们在课题研究中发现了胃癌微环境中有大量中性粒细胞浸润，其表现为激活/抑制双重表型。鉴定出胃癌微环境能够延长中性粒细胞的寿命；胃癌细胞来源的GM-CSF通过JAK-STAT3信号通路激活中性粒细胞并上调其表面PD-L1的表达。阐明了胃癌激活的中性粒细胞通过PD-L1-PD-1通路抑制T细胞的功能，从而抑制胃癌免疫并促进胃癌进展。总结出中性粒细胞百分比和数量以及PD-L1+中性粒细胞的百分比和数量与胃癌进展密切相关，并且可以预测病人的总体生存率。本研究还发现，肿瘤浸润的单核细胞/巨噬细胞的百分比与IFNγ+和TNFα+NK细胞百分比成负相关。体外研究表明，与胃癌相关的单核细胞/巨噬细胞会损害NK细胞表达IFNγ+和TNFα+和Ki-67。而阻断TGFβ1可减弱单核细胞/巨噬细胞介导的NK细胞功能损伤。我们首次证明了Hp感染中MMP-10的前定植和促炎作用。MMP-10在胃粘膜中表达升高，由Hp和IL-22通过ERK途径协同诱导产生，其与Hp的定植和胃炎的严重程度相关，非骨髓来源细胞MMP-10促进了细菌的定植和炎症。在IL-22-/-,MMP-10-/-和IL-22-/-MMP-10-/-小鼠中,Hp的定殖和炎症减弱。MMP-10诱导炎症的特征在于CD8+T细胞的大量涌入，其迁移是通过MMP-10-CXCL16轴由胃上皮细胞所诱导。同时,MMP-10导致Reg3a,E-cadherin和occludens-1蛋白的减少，导致宿主防御能力受损和Hp定植增加。本课题的研究成果不仅为胃癌诊断提供了新的生物标记物，也为治疗胃癌提供了可能的靶标。Neutrophils with an activated/immunosuppressive phenotype are highly enriched within GC and are associated with disease progression and are negatively correlated with patient survival.GCs prolonged neutrophil lifespan,and tumour-derived GM-CSF efficiently activated neutrophils and induced programmed death-ligand 1(PD-L1)expression on neutrophils by activating Janus kinase(JAK)-signal transducer and activator of transcription 3(STAT3)signalling pathways.Tumour-associated neutrophils suppress T-cell’s function in a PD-L1-dependent fashion,and,in doing so,contribute to the GC progression in vitro and in vivo.In this study,we showed that the percentages of NK cells in tumors were significantly decreased,and low percentages of tumor-infiltrating NK cells were positively correlated with poor survival and disease progression.Although the expression of activating and inhibitory receptors on NK cells was shown to be not different between tumor and nontumor tissues,NK cells in tumors had impaired effector functions,characterized by decreased IFNg,TNFa,and Ki-67 expression.We found that tumor-infiltrating monocytes/macrophages were physically close to NK cells,and their percentages negatively correlated with IFNgt and TNFat NK-cell percentages.Ex vivo study showed that isolated tumor-associated monocytes/macrophages could impair NK-cell expression of IFNg,TNFa,and Ki-67.Blockade of TGFb1 attenuated such monocytes/macrophages-mediated impairment of NK-cell function.We demonstrated a procolonization and proinflammation role of MMP-10 in H.pylori infection.MMP-10 is elevated in gastric mucosa and is produced by gastric epithelial cells synergistically induced by H.pylori and IL-22 via the ERK pathway.Human gastric MMP-10 was correlated with H.pylori colonization and the severity of gastritis.H.pylori colonization and inflammation were attenuated in IL-22?/?,MMP-10?/?,and IL-22?/?MMP-10?/?mice.MMP-10-associated inflammation is characterized by the influx of CD8+T cells,whose migration is induced via MMP-10-CXCL16 axis by gastric epithelial cells.Under the influence of MMP-10,Reg3a,E-cadherin,and zonula occludens-1 proteins decrease,resulting in impaired host defense and increased H.pylori colonization.Our results suggest that MMP-10 facilitates H.pylori persistence and promotes gastritis.

本研究通过筛选慢性萎缩性胃炎、胃癌、进展期胃癌及正常查体健康人的血清，发现ITGAM和ITGB3两个外泌体亚群与胃癌发生和转移相关，并通过LNC0208甲基化筛选出关键分子LINC在胃癌发生发展中起到重要作用。胃黏膜壁细胞是最主要的多巴胺能细胞，胃酸分泌促进多巴胺释放。D1R和D2R都参与多巴胺促进胃蛋白酶原分泌的调节。在格兰阴性菌脂多糖引起的NF-κB信号通路活化，多巴胺含量增加以及胃蛋白酶原分泌改变。幽门螺杆菌感染不同时期胃黏膜多巴胺系统及促胃液素水平发生明显改变，影响胃黏膜酸分泌及胃蛋白酶原分泌功能，进而影响胃炎进展及胃癌的发生。通过检测胃黏膜多巴胺含量及血清促胃液素水平可以预判胃黏膜的分泌功能，并对胃癌的预后具有指导意义，通过给予外源性多巴胺及其受体激动剂可能会改善胃黏膜分泌功能并延缓胃炎—胃癌的进展过程。In the present study,ITGAM and ITGB3 were found to be associated with the occurrence and metastasis of gastric cancer by screening serum from patients with chronic Atrophic gastritis,gastric cancer,advanced gastric cancer and normal controls.The LNC0208 methylation was used to screen the the key molecule LINC,which plays an important role in the occurrence and development of gastric cancer.Dopamine(DA)in the gastric juice is derived from parietal cells and is secreted along with gastric acid.Both D1R and D2R are involved in the regulation of DA promoting pepsin secretion.Activating NF-κB signaling pathway increases DA content and pepsin secretion induced by lipopolysaccharide in gram negative bacteria.The changes of DA and gastrin level in gastric mucosa at different stages of Helicobacter pylori infection affect gastric acid and pepsin secretion,and then affect gastritis development and gastric cancer.The content of DA in gastric mucosa and the level of serum gastrin can be used to predict the secretion function of gastric mucosa and to guide the prognosis of gastric cancer,administration of exogenous DA and its receptor agonists may improve gastric mucosal secretion and delay the progression of gastritis-gastric cancer.

Hp感染及其所诱导的慢性炎症微环境会影响胃黏膜上皮细胞膜上重要蛋白分子(例如离子通道)的表达水平、结构重塑及功能效应，通过调控胞内相关的信号网络、黏膜分泌及黏膜屏障而参与胃癌的发生；但众多离子通道及调控信号网络在Hp感染所致胃癌发生中的作用有待进一步阐明。因此，已我们前期运用多种组学技术筛选出的与胃癌相关的易感及驱动分子为基础；不仅进一步鉴定了新的离子通道/膜蛋白，并且阐明了它们在胃癌发生、发展中的重要作用及分子机制，为胃癌早期防治提供了新的理论和靶点:1)发现并解析了TRPV4/Ca2+作为细胞膜蛋白CaSR和VPAC1的下游信号分子促进胃癌进展的作用机制;2)揭示了辣椒素通过阻断胃肠道黏膜的TRPV4离子通道抑制Cl-分泌和促进Na+吸收，从而有可能成为一种防治消化道炎症及肿瘤安全有效的药物;3)揭示了巨噬细胞上的TRPV4离子通道对炎症小体及其M1极化的作用其机制，为进一步阐明TRPV4/Ca2+信号通过免疫调节，在胃肠道黏膜炎症及炎癌转化中的作用机制奠定了良好基础。4)发现并阐明了TRPV1通过Ca2+/CaMKKβ/AMPK信号通路抑制胃癌进展的作用机制;5)发现并阐明TRPV1/Ca2+/EDH改善胃肠道的血流灌注及其炎症微环境，促进黏膜损伤修复；表明TRPV1激动剂辣椒素可能作为防治胃肠道炎症的重要分子;6)发现并阐明了在Hp感染条件下NCX1促进胃癌发生发展的分子机制;7)阐明了SOCE/Ca2+信号在胃肠道炎症及肿瘤中的作用及分子机制。Hp infection-induced chronic inflammatory microenvironment may affect the expression and function of important membrane proteins(such as ion channels)on the gastric mucosal epithelial cells to remodel intracellular signal networks,mucosal ion secretion and mucosal barrier function,all of which are involved in the pathogenesis of GC.However,the role of ion channels and their downstream signal networks need to be elucidated.Our whole project has applied omics techniques to screen and identify several new key molecules related to the occurrence and development of GC.Based on these important molecules,we have not only clarified their roles but also the underlying molecular mechanisms in the pathogenesis of GC.We therefore have established novel theory basis and provided new targets for the early prevention and treatment of GC:1)Discovered and eluciated the mechanism of TRPV4/Ca2+as the downstream signaling molecule of cell membrane proteins CaSR and VPAC1 in promoting the progression of gastric cancer;2)Revealed that capsaicin inhibits Cl-secretion and promotes Na+by blocking the TRPV4 ion channel of the gastrointestinal mucosa,which may become a safe and effective drug for the prevention and treatment of digestive tract inflammation and tumors;3)Clarified the mechanism of the TRPV4 ion channel on macrophages on the inflammasome and M1 polarization,which laid a good foundation for further elucidating the mechanism of TRPV4/Ca2+signaling in gastrointestinal mucosal inflammation and inflammatory cancer transformation through immune regulation;4)Discovered and clarified the mechanism of TRPV1 inhibiting the progression of gastric cancer through the Ca2+/CaMKKβ/AMPK signaling pathway;5)Discovered and clarified that TRPV1/Ca2+/EDH improves the blood perfusion of the gastrointestinal tract and its inflammatory microenvironment,and promotes mucosal injury repair,which shows that the TRPV1 agonist capsaicin may act as an important molecule in the prevention and treatment of gastrointestinal inflammation;6)Discovered and clarified the molecular mechanism that NCX1 promotes the occurrence and development of gastric cancer under the condition of Hp infection;7)Clarified the SOCE/Ca2+signal in the gastrointestinal inflammation and tumors.

本课题将基于前期所鉴定出的胃炎—胃癌转化过程中关键的易感分子或驱动分子，通过染色质免疫共沉淀结合短序列测序技术和RNA 纯化的染色质分离技术以及核磁共振与质谱结合技术等手段，进行表观遗传组学及代谢组学的大数据筛选及生物信息分析，获得多个关键表观遗传调控因子。采用胃癌原代细胞及细胞系，通过功能缺失和恢复等技术手段，在 DNA 修饰、组蛋白修饰及染色质重塑、非编码RNA调控、细胞生物大分子代谢等方向进行表观遗传及代谢方面的详细探索，明确其所参与的信号通路，阐述其调控机理。进而通过建立小鼠原位及移植瘤模型，在体内水平研究这些因子对胃癌发生发展的促进作用。通过流式及组织芯片等技术在临床样本中验证此类表观调控因子参与的信号网络。本项目按照计划成功鉴定了多个表观调控因子(G9a、KDM2B、KDM6B等)与胃癌发生发展密切相关，通过研究发现这些表观调控因子可以通过组蛋白修饰、DNA修饰、非编码RNA调控等修饰方式影响胃癌细胞的增殖、迁移侵袭、自噬以及凋亡等功能，为胃癌临床诊断和治疗靶点的研发提供了新方向和新途径。此外，本项目研究发现多个小分子化合物可以靶向表观遗传调控因子影响胃癌的发生发展，对于胃癌临床化疗药物开发奠定了理论基础。This project will be based on the key susceptible molecules or driver molecules identified in the previous stage of the gastritis-gastric cancer transformation process,through chromatin immunoprecipitation combined with short-sequence sequencing technology and RNA purification chromatin separation technology.By means of epigenetics and metabolomics,big data screening and bioinformatics analysis have been carried out to obtain multiple key epigenetic regulatory factors.Using primary gastric cancer cells and cell lines,through technical means such as loss of function and restoration,epigenetic and metabolic aspects of DNA modification,histone modification and chromatin remodeling,non-coding RNA regulation,cell biological macromolecule metabolism,etc.The detailed exploration,clarify the signal pathways it participates in,and explain its regulation mechanism.Furthermore,through the establishment of mouse orthotopic and transplanted tumor models,the promotion of these factors on the occurrence and development of gastric cancer was studied in vivo.The signal network involved in such apparent regulatory factors is verified in clinical samples through technologies such as flow cytometry and tissue microarray.According to the plan,this project successfully identified a number of apparent regulatory factors(G9a,KDM2B,KDM6B,etc.)that are closely related to the occurrence and development of gastric cancer.Through research,it has been found that these apparent regulatory factors can be modified through histone modification,DNA modification,and non-coding RNA Modification methods such as regulation affect the functions of gastric cancer cell proliferation,migration,invasion,autophagy and apoptosis,and provide new directions and new ways for the development of clinical diagnosis and treatment targets for gastric cancer.In addition,the research of this project found that multiple small molecule compounds can target epigenetic regulatory factors to affect the occurrence and development of gastric cancer,laying a theoretical foundation for the development of clinical chemotherapeutic drugs for gastric cancer.

在2019年度的研究工作中,VPAC1/TRPV4和MMP-10分子与胃癌关系的发现和阐释是其中两项最为突出的成果。VIP可能参了消化道肿瘤的发生、发展过程，但其原理和机制并不清楚。本年度研究中，我们首次揭示了VIP受体VPAC1通过TRPV4/Ca2+/AKT/β-Catenin信号通路促进胃癌发生、发展的新原理和机制，而且提示临床上VIP/VPAC1过度活化可能经此机制加重胃癌的进一步恶化。另一方面，尽管近年来通过口服抗生素根除幽门螺杆菌的治疗取得了长足的进展，但由于细菌对抗生素的耐药性增强，使得幽门螺杆菌在胃内的定植和慢性胃炎通常会持续进展，但分子机制并不清楚。本年度研究中，我们首次发现MMP-10分子在幽门螺杆菌感染后，促进其在胃内定植和慢性胃炎的作用及机制。因此MMP-10可能成为在幽门螺杆菌感染所致慢性胃炎中新的治疗靶标。 In 2019,the discovery and interpretation of the relationship between VPAC1/TRPV4 and MMP-10 and gastric cancer were two outstanding findings.Although vasoactive intestinal peptide(VIP)is important in gastrointestinal physiology,the involvements of VIP and its receptor VPAC1 in progression of gastrointestinal tumor have not been explored.Here,we demonstrate that VPAC1/TRPV4/Ca2+signaling axis could enforce a positive feedback regulation in gastric cancer progression.Our study suggests that VPAC1/TRPV4/Ca2+may serve as potential prognostic markers and therapeutic targets for gastric cancer.Although eradication therapy for H.pylori by oral antibiotics has progressed in recent years,it is noteworthy that chronic gastritis and H.pylori colonization commonly persist because of increased antimicrobial resistance.Our results indicate that MMP-10 facilitates H.pylori persistence and promotes chronic gastritis and suggest that MMP-10 is a possible therapeutic target for chronic gastritis.