HPPRRSmiRNA調控蛋白互作動態網絡系統生物學機制及miR-10b/125a/let-7let-7 c-Bcl-G-JAB1/MNSFβ凋亡新通路分析
Systems biology mechanism of dynamically miRNA-regulated PPI network of piglet infected with HPPRRSV and analysis of novel apoptosis pathway of miR-10b/125a/331/let-7c-Bcl-G-JAB1/MNSFβ involved in pathogenesis and immunity of infected PAM
選定共性導向、交叉融通科學問題屬性：
申請本項目，無論科學數據驅動動態網絡預測，還是科學假設驅動凋亡通路作用驗證，都在前期基礎上延續，並且相互促進完善系統生物學分子機制模型系統。
高致病性豬藍耳病（HPPRRS）感染是養豬業的首患，其持續性感染致病、急性免疫應答異常（初期過強）但隨後進入免疫抑制狀態的極其複雜機制的闡明是開闢防控研究新途徑的關鍵所在，而解決複雜性疾病機制正是系統生物學手段的優勢與強項。
在前期我們HPPRRS、甲型H1N1流感miRNA調控蛋白互作動態網絡的系統生物學分子機制預測研究基礎上，提出miR-10b/125a/331/let-7c-Bcl-G-JAB1/MNSFβ凋亡新通路，在HPPRRSV感染仔豬肺泡巨噬細胞（PAM）中的驗證，有助於認識該病毒侵染致病機制與複製增殖關係。
病毒感染、複製增殖與細胞凋亡是密切聯繫而又關係複雜的幾個事件。我們前期研究結果表明，細胞凋亡與HPPRRSV侵染致病機制關係密切，選用為研究對象順理成章。在HuN4株HPPRRSV感染仔豬肺泡巨噬細胞（PAM）病變中， miR-10b/125a/331/let-7c-Bcl-G-JAB1/MNSFβ凋亡新通路的作用，初期這些miR負調控相互作用促凋亡蛋白質複合體Bcl-G-JAB1/MNSFβ中的Bcl-G，從而抑制凋亡利於病毒增殖，隨著感染進程，miR負調作用減弱直至消失，甚至直接促進凋亡，蛋白複合體促凋亡作用恢復，理論上抑制病毒複製，實際上更利於病毒增殖，細胞通過凋亡而死亡，細胞通訊和病毒複製更高效，病毒感染增殖加劇，免疫抑制和免疫逃逸後持續性感染髮生，細胞組織急性死亡病毒失去有效複製場所，病毒複製增殖減弱，動物瀕死導致複製減弱直至停息，或耐過則免疫低下持續性感染。
基於此科學假設，此凋亡通路作用研究立論依據明確，得到證實對HPPRRS防控具有理論指導作用，或可確定靶點實際干預起到防治作用，價值意義明顯。
HPPRRSmiRNA調控蛋白互作動態網絡系統生物學機制及miR-10b/125a/let-7let-7 c-Bcl-G-JAB1/MNSFβ凋亡新通路分析
Systems biology mechanism of dynamically miRNA-regulated PPI network of piglet infected with HPPRRSV and analysis of novel apoptosis pathway of miR-10b/125a/331/let-7c-Bcl-G-JAB1/MNSFβ involved in pathogenesis and immunity of infected PAM
高致病性豬藍耳病(HPPRRS)的病原(HPPRRSV)專嗜豬肺泡巨噬細胞(PAM)，可感染肺泡II型上皮細胞，分子病理機制不明。以感染HuN4株HPPRRSV發病的仔豬作為研究模型，應用miR組系統生物學的策略和技術，分析仔豬從正常到感染髮病再到出現典型症狀的過程中，基於轉錄因
子與其它蛋白作用靶標的miR調控蛋白互作動態網絡的遺傳基礎並繪製圖譜，篩選與感染相關的目標miR；然後在細胞水平干預目標miR，研究其在感染過程中的作用，闡明miR調控病毒感染致病與免疫的系統生物學機制。在前期類似研究基礎上提出miR-10b/125a/331/let-7c-Bcl-
G-JAB1/MNSFβ凋亡新通路，本研究從HuN4株HPPRRSV體外感染原代細胞、穩轉細胞系與體內感染仔豬分離原代細胞3個層面研究感染早、中、晚期細胞凋亡通路在PAM損傷中的作用及分子機制。本項目的完成對該病的防治提供理論依據。
The highly pathogenic porcine reproductive and respiratory syndrome is an acute and high fatal disease, belongs to the strong natural immune response respiratory RNA virus disease. The pathogen (HPPRRSV) infects pig alveolar macrophage(PAM) and lymphocytes and also can infect pig alveolar type II epitheliu cells, causes serious lung injury, while its molecular pathology mechanism is unknown. Piglets
co-infected with HPPRRSV of HuN4 are used as research models, strategies and techniques of systems biology of miRNAome are applied to analyze the genetic basis of dynamic PPI network of miRNA molecules targeted on transcription factors and other important proteins of piglets from normal to infection and then to appearance of typical symptoms, and its regulation map will thus be drew. Target miRNAs related to HPPRRSV infection will be screened; and then target miRNAs will be intervened at cellular level to investigate their roles on the viral infection
clarify the systems biology mechanism of miRNAs controlling infection and immunity of HPPRRSV.
According to our past basis of similar investigaion, novel apoptosis pathway of miR-10b/125a/331/let-7c-Bcl-G-JAB1/MNSFβ is proposed for its regulated molecular mechenism involved in pathogenesis and immunity of infected PAM.
Over-expression of porcine BCL-G and JAB1 can alone or together promote apoptosis. The present study will explore the function and molecular mechanism of miR-10b/125a/331/let-7c-Bcl-G-JAB1/MNSFβ apoptosis pathway at early, middle and late period of HPPRRS through infecting PAM of primary cell in vitro, and of cell lines stably over-expressing Bcl-G,Bcl-2, JAB1, MNSFβ, MELK, miR-10a, miR-10b, miR-125a, miR-125b, miR-331and miR-let-7c with HPPRSV of HuN4 strain, and primary PAM cells isolated from piglets infected with HPPRSV of HuN4 strain, while miR-10a, miR-125b and Bcl-2 that owning opposite effect to miR-10b, miR-125a and Bcl-G will be used as controls, respectively. If miR-10b, miR-125a, miR-331 or miR-let-7c is proved to improve virus proliferation, chemical synthetic analogues RNAi will be enoculated to evaluate the blocking effect. Otherwise, if miR-10b, miR-125a, miR-331 or miR-let-7c is proved to inhibit virus proliferation, artificial miRNA will be inoculated to evaluate the inhibiting effect. HPPRRSV infecting piglets will injure PAM and lead to apoptosis or death, immunosuppression after persistent infection. Therefore, further investigation of the molecular mechanism of PAM injury is urgently needed. The completion of the project will provide the theoretical basis on prevention and treatment of HPPRRS.
