Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (8): 1606-1619.doi: 10.3864/j.issn.0578-1752.2024.08.014

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles     Next Articles

lincRNA Cox2 Regulates BCG-infected Macrophages Glycolysis by miR-129-5p/AMPK

XU Lei(), YU JiaLin, LIU Li, DENG GuangCun, WU XiaoLing()   

  1. School of Life Sciences, Ningxia University/Key Lab of Ministry of Education for Protection and Utilization of Special Biological Resources in Western China, Yinchuan 750021
  • Received:2023-10-27 Accepted:2024-01-31 Online:2024-04-16 Published:2024-04-24
  • Contact: XU Lei, WU XiaoLing

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Abstract:

【Objective】 The aim of this study was to investigate the regulatory role of lincRNA Cox2 in the glycolysis of RAW264.7 macrophages infected by Bacillus Calmette-Guerin (BCG), and to elucidate the interaction between Mtb and macrophages, so as to provide a new target for the diagnosis and treatment of tuberculosis. 【Method】 RNA interference technique was used to knock down the expression of lincRNA Cox2, and miR-129-5p mimics were used to overexpress miR-129-5p. QPCR was performed to measure the lincRNA Cox2, miR-129-5p and proinflammatory cytokine ( IL-1β, TNF-α, and IL-6 ) expression after BCG infection. The expression of Lactic Acid was detected by Lactic Acid assay kit. The bacterial load was measured bacterial load in BCG-infected macrophages. Dual luciferase reporter gene system validation experiments were carried out on lincRNA Cox2 and miR-129-5p, or miR-129-5p and AMPK relationships. The expression of AMPK (AMP activated protein kinase), HK1 (Hexokinase 1), PKM2 (pyruvate kinase M2), and LDHA (Lactate dehydrogenase A) were detected by Western blotting. 【Result】 The expression of lincRNA Cox2 was significantly upregulated (P=0.000013) after BCG infection in RAW264.7 macrophages for 12 h. Compared with the BCG group, the siRNA+BCG group had significantly upregulated the expression of AMPK (P=0.000771), HK1 (P=0.00323), PKM2 (P=0.000135), LDHA (P=0.002532), and the secretion of LD (P=0.020802), but the expression of IL-1β (P=0.000451), TNF-α (P=0.000147), IL-6 (P=0.0001) was significantly reduced. The lincRNA Cox2 knockdown caused a significant reduce of bacterial load in BCG-infected macrophages (P=0.000127). Dual luciferase reporter gene system were performed to the co-localized of lincRNA Cox2 and miR-129-5p, and targeting AMPK. The expression of miR-129-5p was significantly reduced (P=0.000156) after BCG infection in RAW264.7 macrophages for 12 h. Compared with the BCG group, the miR-129-5p mimics+BCG group had significantly reduced the expression of AMPK (P=0.000262), HK1 (P=0.019524), PKM2 (P=0.001658), LDHA (P=0.000887), and the secretion of LD (P=0.044952). 【Conclusion】 lincRNA Cox2 promoted BCG-infected RAW264.7 macrophages glycolysis process by sponging miR-129-5p and targeting AMPK.

Key words: lincRNA Cox2, miR-129-5p, AMPK, BCG, macrophages, glycolysis

Table 1

The sequences for the specific primers used in real time PCR"

引物名称 Primer 引物序列Sequence（5'→3'）
lincRNA Cox2 F: AAGGAAGCTTGGCGTTGTGA
R: GAGAGGTGAGGAGTCTTATG
miR-129-5p F: ACACTCCAGCTGGCCTTTTTCGCCAGACC CGAA
R: TGGTGTCGTGGAGTCG
IL-1β F: CAGGCAGGCAGTATCACTCATTG
R: CGTCACACACCAGCAGGTTATC
TNF-α F: TAGCCCATGTTGTAGCAAACC
R: ATGAGGTACAGGCCCTCTGAT
IL-6 F: GACAGCCACTCACCTCTTCAG
R: CATCCATCTTTTTCAGCCATC
β-actin F: GTGCTATGTTGCTCTAGACTTCG
R: ATGCCACAGGATTCCATACC
U6 F: CTCGCTTCGGCAGCACA
R: AACGCTTCACGAATTTGCGT

Fig. 1

The expression of lincRNA Cox2 in RAW264.7 macrophages after BCG infection for different lengths of time"

Fig. 2

The expression of the vital enzymes of glycolysis in RAW264.7 macrophages after BCG infection for different lengths of time"

Fig. 3

The expression of AMPK in RAW264.7 macrophages with BCG infection A: The expression of AMPK in RAW264.7 macrophages after BCG infection for different lengths of time with Western blot; B: The silencing efficiency of lincRNA Cox2 was evaluated with qPCR detection; C: RAW264.7 macrophages were transfected with NC or si-lincRNA Cox2 for 24 h and then infected with BCG for 12 h. The expression of AMPK was detected by Western blotting; D: Immunofluorescence assay for detecting the expression level of AMPK. Green dots represent AMPK protein and blue dots represent the nucleus (630×)"

Fig. 4

Effects of lincRNA Cox2 knockdown on the process of glycolytic and the expression of proinflammatory cytokine A: RAW264.7 macrophages were transfected with si-lincRNA Cox2 for 24 h and then infected with BCG for 12 h. The expression of the key enzymes of glycolytic were detected by Western blot; B: Quantification of HK1 expression in RAW264.7 macrophages; C: Quantification of PKM2 expression in RAW264.7 macrophages; D: Quantification of LDHA expression in RAW264.7 macrophages; E: The effect of overexpression of miR-129-5p on LD secretion; F: The expression of IL-1β with qPCR detection; G: The expression of TNF-α with qPCR detection; H: The expression of IL-6 with qPCR detection"

Fig. 5

Effect of lincRNA Cox2 knockdown on RAW264.7 macrophage bacterial load *** P<0.001"

Fig. 6

lincRNA Cox2 sponges miR-129-5p in RAW264.7 macrophages A: The binding site of lincRNA Cox2 and miR-129-5p; B: Verification of the binding site of lincRNA Cox2 to miR-129-5p by dual luciferase reporter system; C: The expression of miR-129-5p in RAW264.7 macrophages after BCG infection for different lengths of time with qPCR detection; D: qPCR detection the influence of interference lincRNA Cox2 on miR-129-5p"

Fig. 7

Overexpression of miR-129-5p inhibits AMPK and t glycolytic in RAW264.7 macrophages A: The binding site of miR-129-5p and AMPK; B: Verification of the binding site of miR-129-5p to AMPK by dual luciferase reporter system; C: RAW264.7 macrophages were transfected with miR-129-5p mimics for 24 h and then infected with BCG for 12 h. The expression of AMPK and the key enzymes of glycolytic were detected by Western blot; D: Quantification of AMPK expression in RAW264.7 macrophages; E: Quantification of HK1 expression in RAW264.7 macrophages; F: Quantification of PKM2 expression in RAW264.7 macrophages; G: Quantification of LDHA expression in RAW264.7 macrophages; H: The effect of overexpression of miR-129-5p on LD secretion"

Fig. 8

The picture of lincRNA Cox2 regulates BCG-infected macrophages glycolysis by miR-129-5p/AMPK"

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