circRNA_SLC8A1 promotes the survival of mycobacterium tuberculosis in macrophages by upregulating expression of autophagy-related protein SQSTM1/p62 to activate the NF-κB pathway

Abstract

Macrophages act as the first immune defense line of the host against Mycobacterium tuberculosis (Mtb). A previous study showed that circRNA_SLC8A1 was significantly upregulated in Mtb-infected macrophages, but its regulatory mechanism in anti-tuberculosis infection is unclear. Therefore, this study aimed to investigate the role of circRNA_SLC8A1 in the anti-tuberculosis activity of macrophages. We showed that circRNA_SLC8A1 was upregulated in tuberculosis patients. Moreover, the binding sites of miR-20b-5p on circRNA_SLC8A1 and Sequestosome 1 (SQSTM1/p62) mRNA were predicted by StarBase and verified by the double luciferase reporter gene assay. Next, we found that miR-20b-5p expression was decreased, while SQSTM1 protein expression was increased in a time- and dose-dependent manner in the human macrophage U937 in response to Mtb infection. Furthermore, circRNA_SLC8A1 overexpression vector (circRNA_SLC8A1) or shRNA (sh-circRNA_SLC8A1) and/or miR-20b-5p mimic or inhibitor and/or SQSTM1 overexpression vector (SQSTM1) or small interfering RNA (si-SQSTM1) or its corresponding control were transfected into Mtb-infected macrophages. Results showed that overexpression of circRNA_SLC8A1 or miR-20b-5p inhibitor promoted the secretion of pro-inflammatory factors IL-1β, IL-6, and TNF-α, increased Nitric Oxide (NO) content and inducible nitric oxide synthase (iNOS) expression, inhibited Reactive oxygen species (ROS) production. Cleaved-caspase-3 protein expression, and cell apoptosis, and promoted Mtb survival. Silencing SQSTM1 inhibited secretion of pro-inflammatory factors and activation of the NF-κB pathway. Overexpression of miR-20b-5p blocked the promoting of circ-SLC8A1 on SQSTM1 protein expression. In summary, circRNA_SLC8A1 sponged miR-20b-5p to upregulate SQSTM1/p62 expression and promoted Mtb survival in macrophages through the NF-κB signaling pathway.

Keywords: Macrophages; Mycobacterium tuberculosis; SQSTM1/p62; The NF-κB signaling pathway; circRNA_SLC8A1; miR‐20b‐5p.

Figure 1

circRNA_SLC8A1 as a sponge for miR-20b-5p is upregulated in tuberculosis patients (A). The expression of circRNA_SLC8A1 in the peripheral blood of active pulmonary tuberculosis (PTB), latent pulmonary tuberculosis infection (LPTB), extra-pulmonary tuberculosis infection (EPTB) patients, and healthy volunteers (HC) (B). The expression of circRNA_SLC8A1 in the peripheral blood of PTB patients with different disease progression (C). The binding sequence diagram of circRNA_SLC8A1 and miR-20b-5p (D). HEK-293 T cells were co-transfected with circRNA_SLC8A1-wild type or circRNA_SLC8A1-mutant and NC mimic or miR-20b-5p mimic. Firefly and Renilla luciferase activities were determined (E). The macrophages were transfected with circRNA_SLC8A1 or sh-circRNA_SLC8A1 or its corresponding control, and then infected with 5 MOI of Mtb for 12 h. The expression of circRNA_SLC8A1 was detected with RT-qPCR (F). The macrophages were transfected with circRNA_SLC8A1 or/and miR-20b-5p mimic, and then infected with 5 MOI of Mtb for 12 h. The expression of miR-20b-5p was detected with RT-qPCR (G). The expression of miR-20b-5p in Mtb-infected macrophages with different MOI was detected with RT-qPCR (H). The macrophages were infected with Mtb for 0, 12, 24 and 48 h, respectively, and the expression of miR-20b-5p in macrophages was detected with RT-qPCR. Mtb: Mycobacterium tuberculosis. Data were shown as mean ± SEM of one representative experiment, similar results were obtained from three independent experiments. N = 5, *P < 0.05, **P < 0.01.

Figure 2

Overexpression of circRNA_SLC8A1 promotes the expression of inflammatory mediators and inhibits ROS production in Mtb-infected macrophages. The macrophages were transfected with circRNA_SLC8A1 or sh-circRNA_SLC8A1 or its corresponding control, and then infected with 5 MOI of Mtb or PBS for 12 h (A-C). The concentrations of IL-1β (A), IL-6 (B) and TNF-α (C) in the supernatant were measured with ELISA. (D) The content of NO was detected with Griess assay (E). The mRNA level of iNOS was measured with RT-qPCR (F). The production of ROS was measured with the flow cytometry. Mtb: Mycobacterium tuberculosis. Data were shown as mean ± SEM of one representative experiment, similar results were obtained from three independent experiments. N = 5, *P < 0.05, **P < 0.01.

Figure 3

Knock-down of circRNA_SLC8A1 induces the apoptosis of Mtb-infected macrophages and inhibits the survival of Mtb in macrophages. The macrophages were transfected with circRNA_SLC8A1 or sh-circRNA_SLC8A1 or its corresponding control, and then infected with 5 MOI of Mtb or PBS for 12 h (A). The apoptosis of macrophages was measured with the flow cytometry (B). The protein levels of Cleaved-caspase-3 were detected with Western blotting (C). The viability of macrophages was measured with CCK-8 (D). The survival of Mtb in macrophages was measured with CFU assay. Mtb: Mycobacterium tuberculosis. Data were shown as mean ± SEM of one representative experiment, similar results were obtained from three independent experiments. N = 5, *P < 0.05, **P < 0.01.

Figure 4

Overexpression of miR-20b-5p inhibits the expression of inflammatory mediators and promotes ROS production in Mtb-infected macrophages. The macrophages were transfected with miR-20b-5p mimic or inhibitor, and then infected with 5 MOI of Mtb or PBS for 12 h (A). The expression of miR-20b-5p was detected with RT-qPCR (B). The concentrations of IL-1β, IL-6 and TNF-α in the supernatant were measured with ELISA. C. The content of NO was detected with Griess assay. D. The mRNA level of iNOS was measured with RT-qPCR. E. The production of ROS in macrophages was measured with the flow cytometry. Mtb: Mycobacterium tuberculosis. Data were shown as mean ± SEM of one representative experiment, similar results were obtained from three independent experiments. N = 5, *P < 0.05, **P < 0.01.

Figure 5

circRNA_SLC8A1 inhibits the apoptosis of Mtb-infected macrophages by targeting miR-20b-5p. The macrophages were transfected with circRNA_SLC8A1 overexpression vector (circRNA_SLC8A1) and/or miR-20b-5p mimic, and then infected with 5 MOI of Mtb for 12 h (A). The apoptosis of macrophages was measured with flow cytometry (B). The protein levels of Cleaved-caspase-3 were detected with Western blotting (C). The viability of macrophages was measured with CCK-8 (D). The survival of Mtb in macrophages was measured with CFU assay. Mtb: Mycobacterium tuberculosis. Data were shown as mean ± SEM of one representative experiment, similar results were obtained from three independent experiments. N = 5, *P < 0.05, **P < 0.01.

Figure 6

miR-20b-5p targets the 3’-UTR of SQSTM1/p62 and inhibits its expression post transcriptionally.(A) The binding sequence diagram of miR-20b-5p and SQSTM1/p62 (B) HEK-293 T cells were co-transfected with SQSTM1/p62-wild type or SQSTM1/p62-mutant and NC mimic or miR-20b-5p mimic. Firefly and Renilla luciferase activities were determined (C-D). The macrophages were infected with Mtb at 5 MOI for 0, 12, 24 and 48 h, respectively, and the protein levels of SQSTM1/p62 were detected with immunofluorescence analysis (E). The macrophages were transfected with miR-20b-5p mimic or miR-20b-5p inhibitor or its corresponding control, and then infected with 5 MOI of Mtb for 12 h. The mRNA (E) and protein (F) levels of SQSTM1/p62 were detected with RT-qPCR and Western blotting, respectively (G). The macrophages were transfected with circRNA_SLC8A1 overexpression vector (circRNA_SLC8A1) and/or miR-20b-5p mimic, and then infected with 5 MOI of Mtb for 12 h. The protein levels of SQSTM1/p62 were detected with Western blotting. Mtb: Mycobacterium tuberculosis. Data were shown as mean ± SEM of one representative experiment, similar results were obtained from three independent experiments. N = 5, compared with 0 h *P < 0.05, **P < 0.01.

Figure 7

Overexpression of SQSTM1/p62 promotes the expression of inflammatory mediators and activates the NF-κB pathway in Mtb-infected macrophages.The macrophages were transfected with SQSTM1/p62 overexpression vector (SQSTM1/p62) or SQSTM1/p62 siRNA (si-SQSTM1/p62), and then infected with 5 MOI of Mtb for 12 h (A-B). The mRNA (A) and protein (B) expression of SQSTM1/p62 was detected with RT-qPCR and Western blotting, respectively (C-E) The concentrations of IL-1β (C), IL-6 (D) and TNF-α (E) in the supernatant were measured with ELISA (F). The content of NO was detected with Griess assay (G). The mRNA level of iNOS was measured with RT-qPCR (H-I). The protein levels of p-NF-κB(p-p65) in nuclear of macrophages were measured with Immunofluorescence analysis. Mtb: Mycobacterium tuberculosis. Data were shown as mean ± SEM of one representative experiment, similar results were obtained from three independent experiments. N = 5, *P < 0.05, **P < 0.01.

Figure 8

miR-20b-5p targets SQSTM1/p62 to promote macrophages to resist Mtb infection through the NF-kB signaling pathway. The macrophages were transfected with miR-20b-5p mimic and/or SQSTM1/p62 overexpression vector (SQSTM1/p62) and treated with or without TNF-α, and then infected with Mtb for 12 h (A). The expression of miR-20b-5p was detected with RT-qPCR (B-C). The mRNA (B) and protein (C) expression levels of SQSTM1/p62 were detected with RT-qPCR and Western blotting, respectively (D-E). The protein levels of p-NF-κB (p-p65) in nuclear of macrophages were measured with Immunofluorescence analysis. (F) The viability of macrophages was measured with CCK-8 (G). The survival of Mtb in macrophages was measured with CFU assay (H). The protein levels of Cleaved-caspase-3 were detected with Western blotting. (I-J) The apoptosis of macrophages was measured with the flow cytometry. Mtb: Mycobacterium tuberculosis. Data were shown as mean ± SEM of one representative experiment, similar results were obtained from three independent experiments. N = 5, *P < 0.05, **P < 0.01.

Figure 9

The mechanistic schematic model of action of circRNA_SLC8A1 in macrophages against Mtb infection. Briefly, circRNA_SLC8A1 sponged miR‐20b‐5p to upregulate SQSTM1/p62 expression, further activating the NF-κB signaling pathway to inhibit macrophage apoptosis and promote Mtb survival in macrophages.