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Knockdown of circ-UQCRC2 ameliorated lipopolysaccharide-induced injury in MRC-5 cells by the miR-326/PDCD4/NF-κB
Gang Zhou1, Yunyan Duan1, Chi Lu2
1Department of Pediatrics, Hubei Provincial Hospital of Traditional Chinese Medicine, Wuhan, Hubei, China.
Background:
Circular RNAs (circRNAs) have been shown as important modulators in the pathogenesis of pediatric pneumonia. In this paper, we focused on the molecular basis of circRNA ubiquinol-cytochrome c reductase core protein 2 (circ-UQCRC2, circ_0038467) in lipopolysaccharide (LPS)-induced cell injury.
Methods:
Quantitative real-time polymerase chain reaction (qRT-PCR) was used to gauge the levels of circ-UQCRC2, microRNA (miR)-326 and programmed cell death 4 (PDCD4) mRNA. PDCD4 protein expression and the activation of the NF-κB signaling pathway were evaluated by western blot. Ribonuclease R (RNase R) assay was performed to assess the stability of circ-UQCRC2. Cell viability and apoptosis were detected by the Cell Counting Kit-8 (CCK-8) and flow cytometry assays, respectively. The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 were measured by the enzyme-linked immunosorbent assay (ELISA). Targeted relationship between miR-326 and circ-UQCRC2 or PDCD4 was confirmed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays.
Results:
Our data showed the up-regulation of circ-UQCRC2 level in pneumonia serum and LPS-treated MRC-5 cells. The silencing of circ-UQCRC2 attenuated LPS-induced MRC-5 cell injury. Mechanistically, circ-UQCRC2 directly targeted miR-326, and circ-UQCRC2 regulated PDCD4 expression through miR-326. MiR-326 was a downstream effector of circ-UQCRC2 function, and PDCD4 was a functional target of miR-326 in regulating LPS-induced MRC-5 cell injury. Additionally, circ-UQCRC2 knockdown inactivated the NF-κB signaling pathway by regulating the miR-326/PDCD4 axis.
Conclusion:
Our findings demonstrated a novel regulatory network, the miR-326/PDCD4/NF-κB pathway, for the function of circ-UQCRC2 in LPS-induced cell injury in MRC-5 cells.
Insights
Circular RNA ubiquinol-cytochrome c reductase core protein 2 (circ-UQCRC2) is upregulated in pneumonia and exacerbates lipopolysaccharide-induced cell injury. Silencing circ-UQCRC2 inhibits this injury by regulating the miR-326/PDCD4/NF-κB pathway.
Area of Science:
- Molecular Biology
- Cell Biology
- Immunology
Background:
- Circular RNAs (circRNAs) play a role in pediatric pneumonia pathogenesis.
- This study investigates circRNA ubiquinol-cytochrome c reductase core protein 2 (circ-UQCRC2) in lipopolysaccharide (LPS)-induced cell injury.
Purpose of the Study:
- To elucidate the molecular mechanism of circ-UQCRC2 in LPS-induced cell injury.
- To identify the regulatory network involving circ-UQCRC2, microRNA-326 (miR-326), and programmed cell death 4 (PDCD4).
Main Methods:
- Quantitative real-time polymerase chain reaction (qRT-PCR) for gene expression analysis.
- Western blot for protein expression and signaling pathway activation.
- Cell Counting Kit-8 (CCK-8) and flow cytometry for cell viability and apoptosis.
- Enzyme-linked immunosorbent assay (ELISA) for cytokine levels.
- Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays for molecular interactions.
Main Results:
- Circ-UQCRC2 was upregulated in pneumonia serum and LPS-treated cells.
- Silencing circ-UQCRC2 reduced LPS-induced cell injury.
- Circ-UQCRC2 directly targeted miR-326, which in turn regulated PDCD4.
- Circ-UQCRC2 knockdown inactivated the NF-κB pathway via the miR-326/PDCD4 axis.
Conclusions:
- Circ-UQCRC2 promotes LPS-induced cell injury in MRC-5 cells.
- A novel regulatory network involving circ-UQCRC2, miR-326, PDCD4, and the NF-κB pathway was identified.
