Inhibiting miR-155-5p promotes proliferation of human submandibular gland epithelial cells in primary Sjogren's syndrome by negatively regulating the PI3K/AKT signaling pathway via PIK3R1

doi:10.12122/j.issn.1673-4254.2025.01.09

Abstract

Abstract:

Objective To investigate the mechanism mediating the regulatory effect of miR-155-5p on proliferation of human submandibular gland epithelial cells (HSGECs) in primary Sjogren's syndrome (pSS). Methods Dual luciferase reporter assay was used to verify the targeting relationship between miR-155-5p and the PI3K/AKT pathway. In a HSGEC model of pSS induced by simulation with TRAIL and INF-γ, the effects of miR-155-inhibitor-NC or miR-155 inhibitor on cell viability, cell cycle, apoptosis and proliferation were evaluated using CKK8 assay, flow cytometry and colony formation assay. ELISA and RT-PCR were used to detect the expressions of inflammatory cytokines and miR-155-5p mRNA in the cells; Western blotting was performed to detect the expressions of proteins in the PI3K/AKT signaling pathway. Results Dual luciferase assay showed that miR-155-5p targets the PI3K/AKT pathway via PIK3R1 mRNA. The HSGEC model of pSS showed significantly decreased cell viability, cell clone formation ability and expressions IL-10 and IL-4 and increased cell apoptosis, cell percentage in G2 phase, expressions of TNF‑α, IL-6, miR-155-5p and PIK3R1 mRNA, p-PI3K/PI3K ratio, p-Akt/AKT ratio, and PIK3R1 protein expression. Treatment of the cell models with miR-155 inhibitor significantly increased the cell viability, G1 phase cell percentage, colony formation ability, and expressions of IL-10 and IL-4 levels, and obviously reduced cell apoptosis rate, G2 phase cell percentage, expressions of TNF-α, IL-6, miR-155-5p and PIK3R1 mRNA, p-PI3K/PI3K ratio, p-AKT/AKT ratio, and PIK3R1 protein expression. Conclusion In HSGEC model of pSS, inhibition of miR-155-5p can promote cell proliferation and reduced cell apoptosis by targeting PI3K1 mRNA to negatively regulate the overexpression of PI3K/AKT signaling pathway.

Key words: primary Sjogren's syndrome, miR-155-5p, PIK3R1, PI3K/AKT signaling pathway, inflammatory factors

Yuru ZHANG, Lei WAN, Haoxiang FANG, Fangze LI, Liwen WANG, Kefei LI, Peiwen YAN, Hui JIANG. Inhibiting miR-155-5p promotes proliferation of human submandibular gland epithelial cells in primary Sjogren's syndrome by negatively regulating the PI3K/AKT signaling pathway via PIK3R1[J]. Journal of Southern Medical University, 2025, 45(1): 65-71.

Figures/Tables 11

Tab.1 Ingredients of Liquid A and B

246 μL serum-free

medium+4 μg plasmid

240 μLserum-free

medium+10 μL

DNA

Lipofectamine 2000

Fig.1 Double luciferase detection results and binding sites. **P<0.01.

Fig.2 Effect of miR-155 inhibitor on viability of HSGEC model of pSS (pSS-HSGECs). **P<0.01 vs Blank group; ##P<0.01 vs Model group; ΔΔP<0.01 vs Idling group.

Fig.3 Effect of miR-155 inhibitor on apoptosis of pSS-HSGECs. **P<0.01 vs Blank group; ##P<0.01 vs Model group; ΔΔP<0.01 vs Idling group.

Fig.4 Effect of miR-155p inhibitor on cell cycle distribution in pSS-HSGECs. **P<0.01 vs the blank group; ##P<0.01 vs the Model group; ΔΔP<0.01 vs the Idling group.

Fig.5 Flow cytometric analysis of cell cycle distribution in the cells.

Fig.6 Effect of miR-155p inhibitor on proliferation of pSS-HSGECs. **P<0.01 vs Blank group; ##P<0.01 vs Model group; ΔΔP<0.01 vs Idling group.

Fig.7 Cytokine levels in pSS-HSGECs in each group. **P<0.01 vs Blank group; ##P<0.01 vs Model group; ΔΔP<0.01 vs Idling group.

Fig.8 Expression of miR-155-5p and PIK3R1 mRNA in pSS-HSGECs in each group. **P<0.01 vs Blank group; ##P<0.01 vs Model group; ΔΔP<0.01 vs Idling group.

Fig.9 Protein expressions of the PI3K/AKT pathway in pSS-HSGECs in each group. A: Blank group. B: Model group. C: Idling group. D: miR-155 inhibition group.

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