Function And Mechanisms Of XLOC₀04122,LincRNA-Cox2 And SIRPα In The Development Of Mycobacterium Tuberculosis Infection

Tuberculosis（TB） is a chronic infectious disease caused by MTB（Mycobacterium tuberculosis, MTB）. Of the 9.6 million new TB cases, 58% were in the South-East Asia and Western Pacific region and 28% were in Africa. India, Indonesia, and China had the largest number of TB cases: 23%, 10% and 10% of the global total, respectively. In recent years, TB prevalence was gradually lower than before, but it is a severe challenge for us to defeat TB due to emergence of multidrug-resistant and extensively drug-resistant TB. Recently, along with constantly updated diagnostic techniques, the treatment success rate and recurrence rates for tuberculosis have been greatly improved than before, but some problemssuch as difficulties in early diagnosis, low sensitivity, poor specificity and false positive, are still dissatisfied. For instance, a part of children with latently M. tuberculosis infection may miss the best treatment time because of the lack of clinical signs and imaging findings. So, accurate, rapid and practicable biomarkers of diagnosing M. tuberculosis are crucial for TB control.In recent years, the appearance of next-generation sequencing and microarray technology bring us a new direction for the discovery of new TB diagnostic marker. Thus, in previous experiments, we identified a series of anti-TB immune response closely related m RNA, micro RNA and lnc RNA in TB-infectious macrophage models by whole-genome sequencing. Based on bioinformatics analysis and preliminary experiment results, we selected XLOC₀04122, linc RNA-Cox2 and SIRPα as the main study object.For XLOC₀04122, firstly, we tested differential expression of XLOC₀04122 for TB-infectious group and the normal group by real-time PCR. Then, GO（Gene ontology, GO） analysis and KEGG（Kyoto Encyclopedia of Genes and Genomes, KEGG） pathway based approaches were used to investigate biological functions of XLOC₀04122. For linc RNA-Cox2, firstly, we detected the expression changes of linc RNA-Cox2 in RAW264.7 stimulated with BCG for different time by RT-PCR. Then, we detected the protein expression levels of LC3, P38, ERK, JNK in RAW264.7 treated with Si RNA for 48 h by western blot.For SIRPα, firstly we detected the expression changes of SIRPα stimulated with BCG for different time by flow cytometry, RT-PCR and western blot. Secondly, western blot and RT-PCR were used to investigate its impact on autophagy-related factor（LC3Ⅱ） and linc RNA-Cox2 expression fromboth cells and animals. Finally, PBMC（peripheral blood mononuclear cell, PBMC） was collected from peripheral blood of patients to detect the expression level of SIRPα by Flow Cytometry, and we evaluated the relationship of SIRPα and TB patients’ prognosis.The main research results are as follows:1 The relationship between XLOC₀04122 and MTB infectionThe expression level of XLOC₀04122 was significantly down-regulated in TB alveolar macrophages; Target gene of XLOC₀04122 was related with activity of many ion channels, particularly Ca2 + channel signal pathway.2 The relationship between Linc RNA-Cox2 and MTB infection（1） The expression level of linc RNA-Cox2 was gradually increased in RAW264.7 stimulated with BCG, which reached to the highest value in 24 h and recovered to the initial level in 48 h after stimulation.（2）Knockdown of linc RNA-Cox2 could up-regulate the phosphorylation of ERKand P38,whereas JNK phosphorylation level and the expression level of LC3Ⅱhad no change;3 The relationship between SIRPα and MTB infection（1） The expression level of SIRPα was gradually decreased in RAW264.7 stimulated with BCG, which reached to the lowest value in 12 h and increased gradually after 24 h.（2） The expression levels of LC3Ⅱwere increased after knockout of SIRPα treated with BCG both in cells and animals.（3） Linc RNA-Cox2 expression in the lungs had an increased trend after knockout of SIRPα, but it did not change significantly in the spleen, liver.（4） Expression level of SIRPα in TB patients was higher than in healthy people. We divided tuberculosis patients into high SIRPα expression group and low SIRPα expression group and there were significant differences on disseminated blood type tuberculosis, sputum, fever, lung cavity between SIRPα high group and SIRPα low group.In summary, MTB infection had a significant impact on expression of XLOC₀04122, linc RNA-Cox2 and SIRPα. It played a key role in the body’s immune response to TB that Ca2 + channel signal pathway participated by XLOC₀04122, the up-regulated phosphorylation levels of ERK, P38 after knockdown of linc RNA-Cox2 and autophagy activation by knocked out SIRPα, which provide a theoretical basis on gene diagnosis on TB,suggesting that they have the potential to become new biomarkers for diagnosis of tuberculosis infection.