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Correlative Effects of PARP12 mRNA Knockdown on Pro-inflammatory Gene Expression and Alternative Splicing in BV2
Bang Hu1,2,3,4, WenLi Zhou1,2,3,4, Hua Chen5,6,7
1Hubei University of Chinese Medicine, Wuhan, 430065, People's Republic of China.
Abstract:
Neuroinflammation is a key mechanism underlying neuropathic pain, in which microglial cells play a central role. Poly(ADP-ribose) polymerase 12 (PARP12) is an interferon-stimulated gene (ISG) containing a predicted RNA-binding domain and may be involved in inflammatory regulation and RNA metabolism; however, its posttranscriptional regulatory functions in microglia remain to be fully elucidated. PARP12 was stably knocked down in BV2 microglial cells using lentiviral shRNA. Quantitative PCR confirmed a significant reduction in mRNA levels (knockdown efficiency 78.3%), but Western blot analysis showed no successful downregulation of PARP12 protein levels. Transcriptomic and alternative splicing changes were systematically analyzed using RNA sequencing (RNA-seq), and selected key genes were validated by RT-qPCR. Functional enrichment analysis was performed on differentially expressed genes (DEGs) and regulated alternative splicing events (RASEs). Expression changes of homeostatic microglial markers and classical polarization markers were analyzed from the existing RNA-seq data. Comparisons were made with public proinflammatory microglial signature gene sets. PARP12 mRNA knockdown led to substantial transcriptomic remodeling: 276 DEGs (107 upregulated and 169 downregulated) and 287 significant RASEs were identified. Downregulated DEGs were significantly enriched in inflammatory pathways such as NF-κB, TNF, and chemokine signaling; most core immune genes (including Ccl2, Ptgs2, and C3) showed coordinated downregulation, whereas a smaller subset (Ccl5, Cd81, and S1pr1) was upregulated. Homeostatic microglial markers (Tmem119, P2ry12, Cx3cr1, Sall1, etc.) did not differ significantly between shPARP12 and shNC cells. Splicing analysis revealed altered splicing of immune/metabolism-related genes such as Fyb, Tnfaip8, Lrch4, and Pkm, but changes in protein isoform ratios were not validated. The overall proinflammatory signature gene set was downregulated after PARP12 knockdown. PARP12 mRNA knockdown is closely associated with attenuation of the proinflammatory transcriptional program and widespread alternative splicing changes in BV2 microglial cells. However, given the unsuccessful knockdown at the protein level, the observed changes cannot be directly attributed to loss of PARP12 protein function. Further mechanistic studies combining CRISPR/Cas9 gene knockout and in vivo pathological models are warranted.
Insights
Knocking down Poly(ADP-ribose) polymerase 12 (PARP12) mRNA in microglial cells altered gene expression and splicing, reducing inflammatory responses. However, protein levels were unaffected, limiting direct conclusions about PARP12
Area of Science:
- Neuroscience
- Immunology
- Molecular Biology
Background:
- Neuroinflammation, driven by microglial cells, is central to neuropathic pain.
- Poly(ADP-ribose) polymerase 12 (PARP12), an interferon-stimulated gene, has potential roles in inflammation and RNA metabolism within microglia.
- Its precise posttranscriptional regulatory functions in microglia require further elucidation.
Purpose of the Study:
- To investigate the posttranscriptional regulatory functions of PARP12 in microglia.
- To analyze transcriptomic and alternative splicing changes following PARP12 knockdown in BV2 microglial cells.
- To assess the impact of PARP12 knockdown on microglial inflammatory gene expression.
Main Methods:
- Stable knockdown of PARP12 in BV2 microglial cells using lentiviral shRNA.
- Quantitative PCR and Western blot to confirm knockdown efficiency at mRNA and protein levels.
- RNA sequencing (RNA-seq) for transcriptomic and alternative splicing analysis, with RT-qPCR validation.
Main Results:
- PARP12 mRNA knockdown resulted in 276 differentially expressed genes and 287 regulated alternative splicing events.
- Downregulated genes were enriched in inflammatory pathways (NF-κB, TNF, chemokine signaling), with core immune genes largely downregulated.
- The overall proinflammatory microglial signature gene set was attenuated, despite unsuccessful PARP12 protein knockdown.
Conclusions:
- PARP12 mRNA knockdown is associated with transcriptomic remodeling and reduced proinflammatory gene expression in microglial cells.
- Observed changes cannot be directly attributed to loss of PARP12 protein function due to failed protein knockdown.
- Further studies using gene knockout and in vivo models are necessary to clarify PARP12's role.