NNT-AS1, A Long Non-coding RNA with Therapeutic Promise in Mycoplasma Pneumoniae Pneumonia via the Mir-410-3p/TMEM14A/Wnt/ΒCatenin Signalling Pathway.

Background: Long non-coding RNA (lncRNA) NNT-AS1 is involved in the progression of various lung and inflammatory diseases, and has been reported to be upregulated in patients with Mycoplasma pneumoniae pneumonia (MPP). However, its role in MPP progression remains largely unknown.

Objectives: We aimed to investigate the function and underlying mechanism of action for lncRNA NNT-AS1 in MPP.

Materials and methods: Mycoplasma pneumonia (MP)-stimulated mouse models and MP-induced A549 cells were established to explore the effects of NNT-AS1 knockdown on MPP progression. The levels of inflammatory cytokines including TNF-α, IL-1β, and IL-6 were detected using ELISA. Histological changes in mouse lung tissues were examined using H&E Staining. The expression of NNT-AS1 in mouse lung tissues or A549 cells was determined by RT-qPCR. CCK-8 and colony formation assays were used to detect the viability and proliferative capacity of A549 cells, and flow cytometry analysis was applied to determine A549 cell apoptosis. The interaction between RNA and downstream targets was explored using RNA Pull-down, RIP, and dual-Luciferase Reporter Assay assays. Western blot was used to measure the levels of downstream target TMEM14A as well as key proteins on the Wnt/βcatenin pathway.

Results: NNT-AS1 was upregulated in MPP mice and MP-stimulated A549 cells, accompanied by increased levels of inflammatory mediators, which were suppressed by NNT-AS1 silencing. Besides, NNT-AS1 deficiency elevated the proliferation while inhibiting apoptosis in MP-induced A549 cells. Additionally, through online databases and mechanical assays, we verified that NNT-AS1 served as a miRNA sponge for miR-410-3p, and transmembrane protein 14A (TMEM14A) was targeted by miR-410-3p. NNT-AS1 elevated TMEM14A expression by interacting with miR-410-3p. Moreover, NNT-AS1 promoted the activation of the Wnt/β-catenin pathway via elevating TMEM14A expression. Further rescue experiments validated that miR-410-3p inhibition or TMEM14A elevation rescued the impacts of NNT-AS1 silencing on proliferation, apoptosis, as well as inflammation in MP-stimulated A549 cells.

Conclusions: Our study unveiled that NNT-AS1 facilitated inflammatory injury in MP-stimulated A549 cells via the miR-410-3p/TMEM14A/Wnt/βcatenin signalling pathway, which might provide a novel therapeutic target for MPP.