Introduction and objective:
Mycoplasma pneumoniae infection (MPI) can lead to extrapulmonary diseases, in which diarrhea occurs frequently. The role and impact of microRNA-95 (miR-95) in MPI and MPI with diarrhea (MPI+DIA) remain elusive. The aim of the study is to investigate miR-95 expression and its functional impact in paediatric MPI with diarrhea.

Material and methods:
The study enrolled 80 MPI patients (53 without diarrhea, 27 with diarrhea) and 80 matched healthy controls. Expression of miR-95 was quantified using reverse transcription quantitative polymerase chain reaction (RT -qPCR). Predictors of disease status were analyzed through binary logistic regression. An in vitro pneumonia model (MP-BEAS-2B) was generated by infecting BEAS-2B cells with Mycoplasma pneumoniae (MP). In parallel, an intestinal epithelial injury model was generated by treating Caco-2 cells with 4 μg/mL lipid-associated membrane proteins (LAMPs). Cell proliferation was measured with cell counting kit-8 (CCK-8), apoptosis by flow cytometry, and interleukin -8 (IL-8) and tumour necrosis factor-alpha (TNF-α) levels were determined via RT-qPCR.

Results:
The study revealed that miR-95 was significantly elevated in the MPI group, with a further increase observed in MPI patients complicated with diarrhea. Additionally, miR-95 served as a discriminator among healthy controls, MPI patients, and MPI+DIA patients. In MP-BEAS-2B and LAMPs-Caco-2 cells, miR-95 was highly expressed. However, downregulating miR-95 significantly enhanced cell proliferation, inhibited apoptosis, and reduced the expression of IL-8 and TNF-α.

Conclusions:
MiR-95 expression was significantly elevated in MPI patients, and further increased in those with MPI complicated by diarrhea. Downregulating miR-95 significantly protected BEAS-2B and Caco-2 cells from MP damage and alleviated cellular inflammation.