Detection Of Differential Expression Of MicroRNA In Spinal Tuberculosis And Screening Of Molecular Markers For Early Diagnosis Of Spinal Tuberculosis

Objectives:Spinal tuberculosis is a common disease in clinic, with high rate of disability. The early diagnosis is very important for the development and outcome. The diagnosis is mainly based on clinical manifestations, images and laboratory tests. It is not difficult to diagnose typical spinal tuberculosis, but in the early stage of the disease, while the clinical manifestation is not typical, lacking specificity of imaging and laboratory examination. So there is still a major problem in early diagnosis.To establish molecular markers for early diagnosis of spinal tuberculosis, by screening microRNA in peripheral blood mononuclear cells of peripheral blood mononuclear cells of patients with spinal tuberculosis.Materials and methods:According to the clinical manifestations, imaging data and related laboratory examination and biopsy or surgical specimens histopathological examination (if necessary) to confirm the spinal tuberculosis cases. The mononuclear cell extraction in peripheral blood were obtained before anti-tuberculosis treatment. In healthy volunteers as control group, the peripheral blood mononuclear cells were obtained as well. Total RNA was extracted by Trizol method and the quality control was completed by NanoDrop-1000 system. The TB case group (n= 4) and control group (n=3) samples were detected. The fold change (fold change, FC more than or equal to 2 times) and significant P values (P<0.05) were detected by exiqon miRCURY LNATM microRNA (human) chip to screen differentially expressed microRNAs, meanwhile previous results of pulmonary tuberculosis related research were compared. The expression of miR-18b-5p, miR-520g-3p and miR-19a-3p and miR-1470 were detected by PCR real-time in 32 independent samples (case group n=12, control group n=20). The reliability of the chip data was verified. At the same time, combined with Miranda, MirBase and TargetScan database, the target genes were predicted and analyzed by GO, Network and Pathway in the differential expression of microRNA. By analyzing the specificity and sensitivity of miR-19a-3p to the detection of spinal tuberculosis, the receiver operating characteristic curve (ROC curve) was established, and the diagnostic efficiency of the spinal tuberculosis was evaluated by the area index (AUC) of the curve. RT-PCR was applied to detect tumor necrosis factor (TNF-a) mRNA expression in all the 32 independent samples, combined with the data of miR-19a-3p, analyzing relationship between these two genes, and scatter diagram was applied showing the relationship.Result:All the 7 samples test results were qualified. In 3100 microRNA probes, a total of 58 microRNA showed differential expression (in which 36 up-regulated genes; 22 down-regulated genes), and its multiple changes in 2.07-13.8. Compared with the previous studies of pulmonary tuberculosis, only 4 microRNA (has-miR-296, has-miR-377, has-miR-145 and has-miR-744) were differentially expressed. RT-PCR detection results showed that the expression of miR-18b-5p and miR-19a-3p in case group was decreased, and the expression of miR-520g-3p and miR-1470 increased in case group. And this results were basically consistent with the results of the chip test. Bioinformatics analysis showed that the differentially expressed microRNAs involved multiple target genes. miR-19a-3p in three databases were target gene number 208 (which contains TNF) and in the center position of network. Target gene function GO analysis mainly involved MAKP kinase activity, cell metabolism regulation and macrophage chemical chemotaxis. Pathway analysis target gene function mainly involved MAKP, TNF and Rapl signal pathway. The sensitivity of miR-19a-3p for patients with spinal tuberculosis was 80%, the specificity was 66.7%, and the AUC was 0.733. There is a negative correlation between miR-19a-3p and TNF-a (uperman ’s’ Rho=0.602, p= 0.014).Conclusions:There is a clear difference microRNA profile between Spinal tuberculosis and pulmonary tuberculosis; Target gene function, primary concern of MAKP, Rapl and TNF signaling pathway, with the results of previous research on tuberculosis are similar. MiR-19a-3p may become one of biomaker of the early stage of the spinal tuberculosis diagnosis; it can regulate the expression of TNF-a involved in the pathological process of spinal tuberculosis. But this conclusion has yet to be in a larger sample of validation and experimental verification such as luciferase.