Academic Report on the Scientific Paper “circPTP4A2 Promotes Microglial Polarization in Ischemic Stroke via the miR-20b-5p/YTHDF1/TIMP2 Axis”

Background Introduction

Ischemic stroke (IS) is brain tissue necrosis caused by cerebral blood flow obstruction and is the second leading cause of disability and death globally. Current clinical treatment strategies mainly include intravenous thrombolysis and mechanical thrombectomy, but these methods have limited efficacy. Therefore, developing new effective treatment strategies is of great significance. Microglia, as resident macrophages in the brain, play an important role in acute and chronic neuroinflammatory responses. In the pathological stage of ischemic stroke, microglia can rapidly activate and differentiate into M1 or M2 phenotypes, which play different roles in tissue damage and repair, respectively.

Specifically, M1 microglia promote brain damage by secreting pro-inflammatory factors, while M2 microglia secrete anti-inflammatory factors and have neuroprotective effects. Therefore, promoting microglial polarization towards the M2 phenotype is considered one of the potential strategies for treating ischemic stroke, and understanding the mechanism of microglial M2 phenotype polarization is a key step in achieving this goal.

Paper Source

This paper was written by Xianxin Kang, Yanhui Cao, Guodong Sun, Dongsheng Fei, Kai Kang, Xianglin Meng, and Mingyan Zhao from the Department of Emergency Medicine, First Affiliated Hospital of Harbin Medical University, and was published online in the journal “Neuromolecular Medicine” on September 14, 2023.

Research Process

In the study, the authors explored the role of circPTP4A2 in regulating microglial polarization in ischemic stroke by constructing MCAO/R (middle cerebral artery occlusion/reperfusion) and OGD/R (oxygen-glucose deprivation/reoxygenation) models.

Experimental Steps

1. Clinical Sample Collection

   • Blood samples were collected from 30 ischemic stroke patients and 30 healthy volunteers.
   • Basic characteristic information, including whether it was the patient’s first onset, was included.

2. MCAO/R Model Establishment

   • 8-week-old C57BL/6 mice (32 in total) were divided into 4 groups: sham, MCAO/R, MCAO/R+sh-NC, and MCAO/R+sh-circPTP4A2.
   • MCAO/R model was constructed in mice, and shRNA interference was performed to knockdown circPTP4A2.

3. 2,3,5-Triphenyltetrazolium Chloride (TTC) Staining

   • Detected infarct volume in mouse brain tissue.
   • Assessed neurological injury through neurological function scoring.

4. Immunofluorescence Staining

   • Analyzed the expression levels of CD16 and CD206 in brain tissue.

5. Cell Culture and Treatment

   • Used human microglial cells (HMC3 cells) purchased from ATCC to construct an in vitro model under OGD/R conditions.
   • Conducted various transfection experiments, including shRNA interference and miR-20b-5p microRNA mimics and inhibitors.

6. Cell Counting Kit-8 (CCK-8) Assay

   • Measured cell viability.

7. Flow Cytometry

   • Detected the expression of cell surface CD16 and CD206.

8. RNA Immunoprecipitation (RIP) Experiment

   • Analyzed the interaction of circPTP4A2 with miR-20b-5p and YTHDF1 in cells.

9. RNA Pull-down Experiment

   • Further verified the binding between miR-20b-5p and YTHDF1.

10. Dual-Luciferase Reporter Gene Assay

    • Verified the binding sites of miR-20b-5p and YTHDF1.

11. Quantitative Real-time Polymerase Chain Reaction (qRT-PCR)

    • Detected the expression levels of target RNAs.

12. Western Blot

    • Detected the expression of related proteins in mice and cells, including TIMP2 and NF-κB pathway-related proteins.

Research Results

1. Expression of circPTP4A2 and miR-20b-5p

   • circPTP4A2 expression was significantly upregulated in the plasma of ischemic stroke patients, while miR-20b-5p was significantly downregulated.

2. Effect of circPTP4A2 Knockdown on MCAO/R-induced Brain Damage in Mice

   • TTC staining showed that knockdown of circPTP4A2 reduced MCAO/R-induced cerebral infarct volume in mice and alleviated neurological injury.
   • Immunofluorescence staining and Western blot analysis indicated that knockdown of circPTP4A2 promoted M2 polarization of microglia, reduced the expression of M1 markers (iNOS, CD16), and increased the expression of M2 markers (Arg1, CD206).

3. circPTP4A2 Regulates YTHDF1 Expression through miR-20b-5p

   • Dual-luciferase reporter gene, RNA pull-down, and RIP experiments verified that circPTP4A2 acts as a sponge for miR-20b-5p, regulating YTHDF1 expression.
   • Experimental results showed that knockdown of either circPTP4A2 or YTHDF1 could reduce TIMP2 expression and inhibit the activation of the NF-κB pathway.

4. miR-20b-5p Regulates OGD/R-induced Microglial Polarization by Modulating TIMP2

   • Overexpression of miR-20b-5p promoted M2 polarization of microglia and reduced TIMP2 expression, an effect that could be reversed by TIMP2 overexpression.

Conclusions and Significance

This study first discovered that circPTP4A2 promotes microglial polarization towards the M2 phenotype, reduces neuroinflammation, and alleviates neurological injury in the process of ischemic stroke by inhibiting miR-20b-5p and thereby regulating YTHDF1 expression. This finding provides a theoretical basis for new therapeutic strategies for ischemic stroke, where circPTP4A2, miR-20b-5p, and YTHDF1 can serve as potential therapeutic targets.

Research Highlights

1. Newly Discovered Regulatory Axis

   • The discovery of the circPTP4A2/miR-20b-5p/YTHDF1/TIMP2 axis reveals a new mechanism of microglial polarization.

2. Potential Clinical Applications

   • This research provides a foundation for developing new ischemic stroke treatment strategies, with circPTP4A2 potentially becoming a future therapeutic target.

3. Rich Experimental Design

   • Combining in vivo and in vitro models, the consistency of different experimental methods was verified, increasing the reliability of the conclusions.