Exploration of plasma exosomal miR-122-5p and its related targets KNG1 and C3 in the diagnosis of drug-resistant tuberculosis

Abstract

Drug-resistant tuberculosis (DR-TB) poses significant challenges not only to public health but also imposes substantial psychological and economic burdens on individuals and their families. As a severe infectious disease that jeopardizes both physical and mental well-being, DR-TB frequently spreads in underdeveloped regions due to inadequate diagnostic technologies.In this study, we validated the binding interaction between miR-122-5p and the proteins kininogen-1 (KNG1)/complement C3 using a dual-luciferase reporter assay. Furthermore, we employed liquid biopsy techniques to quantify miR-122-5p expression in plasma exosomes from DR-TB patients, alongside measuring plasma levels of KNG1, complement C3, and other coagulation and immune function parameters. This approach aims to identify efficient, non-invasive laboratory biomarkers for the early diagnosis of DR-TB. 50 patients with drug-susceptible tuberculosis (DS-TB) and 50 patients with DR-TB who were diagnosed in the nearby hospital between April 2024 and January 2025 were chosen. 51 healthy people who had physical exams over the same time frame were also selected as the control group. In the early morning, 5 ml of fasting venous blood was drawn from each of all subjects and centrifuged for standby. Informed consent was obtained from all Participants, who then signed the informed consent forms. We used Western blotting (WB), transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA) to find the biomarkers in the exosomes that were taken from each of the three groups' plasma. The dual-luciferase experiment was used to verify the targeting relationship between miR-122-5p and protein KNG1 and complement C3. The RNA level of the miR-122-5p gene in plasma exosomes was detected by real-time fluorescence quantitative PCR (qRT-PCR). The KNG1 level in the plasma of the subjects was measured by ELISA, and the clinical indicators of the patients were also collected. To assess the diagnostic effectiveness of the genes found in the plasma exosomes, we used the receiver operating characteristic (ROC) curve. We found that there is a targeting relationship between miR-122-5p and protein KNG1 as well as complement C3. Meanwhile, the level of miR-122-5p in the DR-TB group was significantly higher than that in the DS-TB group and the HCs group, indicating a relatively high diagnostic efficacy. A useful biomarker to enhance the diagnosis of DR-TB is the level of miR-122-5p in plasma exosomes.

Keywords: Biomarker; Diagnosis; Exosome; Tuberculosis; miR-122-5p.

Fig. 1

Exosome characterization. Plasma exosome characteristics. a Transmission electron microscopy (TEM) revealed the round shape of exosomes. The bar represents 100 nm. b. b(A): DR-TB, b(B): DS-TB, b(C): HCs The marker proteins of exosome (CD9、CD63 and TSG101) were detected by western blotting (WB). c. The Nanoparticle tracking analysis (NTA) presents the size and concentration distribution.

Fig. 2

Intergroup comparison of peripheral blood parameters. Comparison of peripheral blood index results among the three groups. ^*P<0.05;^**P < 0.01; ^***P<0.001.

Fig. 3

Expression levels of exosomal miR-122-5p in three groups. Relative expression levels of plasma exosomal miR-122-5p in DR-TB, DS-TB and HCs groups. ^***P<0.001.

Fig. 4

Expression levels of KNG1 in three groups. The expression levels of KNG1 in DR-TB, DS-TB and HCs groups. ^***P<0.001.

Fig. 5

Results of the Dual-Luciferase reporter assay. Results of dual-luciferase reporter gene assay. ^**P<0.01.

Fig. 6

ROC curves of biomarkers for diagnosing drug-resistant tuberculosis. Receiver operator characteristics (ROC) curves for prediction of DR-TB by the levels of plasma exosomal miR-122-5p alone or combined with biomarkers, KNG1 and C3 proteins.