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. 2024;27(16):2323-2334.
doi: 10.2174/0113862073243966231030093213.

Differential miRNA Profiling Reveals miR-4433a-5p as a Key Regulator of Chronic Obstructive Pulmonary Disease Progression via PIK3R2- mediated Phenotypic Modulation

Siming Tao  1 , Chunyan Liao  1 , Yide Wang  1 , Dan Xu  2 , Zheng Li  2 , Fengsen Li  1
Affiliations

Differential miRNA Profiling Reveals miR-4433a-5p as a Key Regulator of Chronic Obstructive Pulmonary Disease Progression via PIK3R2- mediated Phenotypic Modulation

Siming Tao et al. Comb Chem High Throughput Screen. 2024.

Abstract

Objective: In this study, a high-throughput sequencing technology was used to screen the differentially expressed miRNA in the patients with "fast" and "slow" progression of chronic obstructive pulmonary disease (COPD). Moreover, the possible mechanism affecting the progression of COPD was preliminarily analyzed based on the target genes of candidate miRNAs.

Methods: The "fast" progressive COPD group included 6 cases, "slow" and Normal progressive COPD groups included 5 cases each, and COPD group included 3 cases. The peripheral blood samples were taken from the participants, followed by total RNA extraction and high throughput miRNA sequencing. The differentially expressed miRNAs among the progressive COPD groups were identified using bioinformatics analysis. Then, the candidate miRNAs were externally verified. In addition, the target gene of this miRNA was identified, and its effects on cell activity, cell cycle, apoptosis, and other biological phenotypes of COPD were analyzed.

Results: Compared to the Normal group, a total of 35, 16, and 7 differentially expressed miRNAs were identified in the "fast" progressive COPD, "slow" progressive COPD group, and COPD group, respectively. The results were further confirmed using dual-luciferase reporter assay and transfection tests with phosphoinositide- 3-kinase, regulatory subunit 2 (PIK3R2) as a target gene of miR-4433a-5p; the result showed a negative regulatory correlation between the miRNA and its target gene. The phenotype detection showed that the activation of the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) signaling pathway might participate in the progression of COPD by promoting the proliferation of inflammatory A549 cells and inhibiting cellular apoptosis.

Conclusions: MiR-4433a-5p can be used as a marker and potential therapeutic target for the progression of COPD. As a target gene of miR-4433a-5p, PIK3R2 can affect the progression of COPD by regulating phenotypes, such as cellular proliferation and apoptosis.

Keywords: COPD; biological phenotypes.; cell transfection; dual-luciferase reporter assay; miRNA; progressive development.

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Conflict of interest statement

The authors declare no conflict of interest, financial or otherwise.

Figures

Fig. (1)
Fig. (1)
A flowchart of screening and mechanism of miRNA as a biomarker for patients with different progressive COPD. Notes: This research mainly includes three parts, which are as follows: High throughput miRNA sequencing、Target gene verification, and preliminary functional analysis of miR-4433a-5p、High throughput miRNA sequencing.
Fig. (2)
Fig. (2)
Differential analysis between the Normal group and “fast” and “slow” progression of COPD groups. Notes: (A) Volcanic maps of miRNA differences between the COPD group and control group; (B) Volcanic maps of miRNA between the slow COPD group and the control group; (C) Volcanic maps of miRNA between the fast COPD group and the control group; (D) Heat maps of different miRNAs between the three groups; (E) Wayne diagram of differential miRNAs between the three groups.
Fig. (3)
Fig. (3)
Network prediction and enrichment analysis of the genes targeted by differentially expressed miRNAs. Notes: (A) Heat map of differential expression between COPD and control groups; (B) Network prediction of the genes targeted by differentially expressed miRNAs. The green square represents miRNA; The purple circles represent miRNA target genes.
Fig. (4)
Fig. (4)
GO and KEGG pathways enrichment analyses. Notes: (A) GO enrichment analyses of differential genes. (B) KEGG pathways enrichment analyses of differential genes.
Fig. (5)
Fig. (5)
qRT-PCR results of the selected miRNAs. Notes: (A) The differential expression of miR4433a-5p between groups; (B) The differential expression of miR-1246 between groups; (C) The differential expression of miR-1290 between groups; (D) The differential expression of miR-375 between groups; (E) The differential expression of miR-129-5p between groups.
Fig. (6)
Fig. (6)
Correlation analysis of CSE concentration and proliferation inhibition of A549 cells over time. Notes: This picture includes both a visual display and a quantitative analysis of the bar chart display.
Fig. (7)
Fig. (7)
Target gene analysis and biological phenotype detection using dual-luciferase reporter assay and transfection test. Notes: (A) Dual-luciferase reporter assay results of miR-4433a-5p and PIK3R2. (B) Expression levels of miR-4433a-5p in the different transfection groups were detected using qRT-PCR. (C) Expression levels of PIK3R2 in the different transfection groups were detected using qRT-PCR. (D) Effects of miR-4433a-5p transfection on the proliferation of inflammatory cells. (E) Expression levels of PIK3R2 in the different transfection groups were detected using Western blot. (F) Expression levels of PIK3R2 in patients with “slow” and “fast” progression of COPD. (G) siRNA of optimized target sequence PIK3R2. (H) Expression levels in different transfection groups after knockdown of PIK3R2; (I) and (J) Apoptosis of A549 cells in the different transfection groups.
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