| Background:Tuberculosis(TB)is a zoonotic respiratory infection contagious infectious disease mainly caused by Mycobacterium tuberculosis(Mtb).The host cell of Mtb is Alveolar macrophages and the interaction between Mtb and macrophage is quiet complicated.After Mtb infection,macrophages eliminate the free pathogens in the cytoplasm through apoptosis process and inhibit the proliferation of Mtb in host In the meantime,Mtb could evade immune killing of host cells in variety ways such as inhibiting apoptosis process and inducing necrosis.The process of apoptosis and necrosis is regulated by many factors.Among those factors,long non-coding RNA(LncRNA)has received extensive attention.Due to the function in regulating apoptosis and necrosis,LncRNA-Cox2 is one of the most studied LncRNA.However,the role of LncRNA-Cox2 in regulating macrophage apoptosis and necrosis during Mtb infection remains unclear.Therefore,It is of significance to explore the function of LncRNA in regulating apoptosis and necrosis during macrophage defense process.Purpose and methods:To probe the molecular mechanism of LncRNA-Cox2 in regulating apoptosis and necrosis process during Mycobacterium tuberculosis infection,murine alveolar macrophage RAW264.7 cell line was transfected with siRNA-LncRNACox2 and then followed Bacillus Calmette Guerin(BCG)vaccine infection.Flow cytometry,transmission electron microscopy,qRT-PCR,Western Blotting,ELISA,fluorescent in situ hybridization were used to investigate the regulation of LncRNA-Cox2 on apoptosis and necrosis.The results are shown as below:Results:(1)Compared with the control group,the expression of LncRNA-Cox2 was significantly higher after treating with 12 hours of infection(p<0.001)and small interfering RNA can significantly down-regulate the expression of LncRNA-Cox2 in BCG-infected macrophages(p<0.001).Fluorescence in situ hybridization assay showed that the expression of LncRNA-Cox2 both in nucleus and cytoplasm.Further studies showed that LncRNA-Cox2 could be activate by TLR2 agonist Pam3CSK4,The results indicated that the high expression of LncRNA-Cox2 is associated with activation of the TLR2 pathway after BCG infection.(2)Compared with the control group,the expression of apoptosis rate was significantly higher 12h after BCG infection(p<0.001)and siRNA-LncRNA-Cox2 could depress the expression of LncRNA-Cox2 notably(p<0.001).The depression of LncRNA-Cox2 upregulated cell apoptotic rate(p<0.001),and promoted the expression of pro-apoptosis factors Caspase3,Cytochrome c in mRNA and protein level,down-regulated the expression levels of anti-apoptosis protein MCL-1.Further research found that siRNA-LncRNA-Cox2 can significantly increase intracellular Ca2+concentration(p<0.001)and upregulate the expression of ER stress factors PERK p-eIF2a and Chop(p<0.001).The results were further confirmed by TUDCA which is an ER stress inhibitor.After TUDCA treatment,LncRNA-Cox2 knockdown could not promote BCG-induced apoptosis.In a word,LncRNA-Cox2 knockdown could promote BCG-induced apoptosis through enhancing ER stress.(3)Compared with the control group,the expression of necrosis rate was significantly higher 12h after BCG infection(p<0.001);siRNA-LncRNA-Cox2 significantly reduced the rate of cell necrosis(p<0.001)and inhibited the expression of necrosis-related factors RIP1,RIP3 and MLKL in mRNA and protein level(p<0.001).It is suggested that the knock down of LncRNA-Cox2 influenced BCG-infected macrophage necrosis by up-regulating the expression of RIP 1,RIP3 and MLKL.Conclusion:BCG infection upregulated the expression of LncRNA-Cox2 and promotes macrophages apoptosis and necrosis;Further research confirmed that the konck down of LncRNA-Cox2 could promote apoptosis via ER stress pathway during BCG infection.Meanwhile,interference with LncRNA-Cox2 can inhibit BCG induced necrosis by restraining the activation of RIP1/RIP3/MLKL axis. |
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