探讨微小RNA(miR)-199a-3p调控CD4^＋T细胞表达，参与肺炎支原体肺炎(MPP)患儿免疫反应及其发病机制。

选择2018年1月至2020年12月重庆市第七人民医院收治的500例MPP患儿为研究对象，并将其纳入观察组，将同期在该院进行健康体检的200例儿童纳入对照组。将进行体外试验的THP-1细胞培养后，随机分为miR-199a-3p siRNA组(n＝3，转染miR-199a-3p siRNA)，miRNA mimic组(n＝3，转染miRNA mimic)及未处理组(n＝3，等量RPMI 1640完全培养基处理)，采用外购肺炎支原体(MP)中提取的脂质相关膜蛋白(LAMP)，刺激上述3组THP-1细胞后，进行相关试验。采用独立样本t检验，对观察组及对照组受试儿的外周血单核细胞中miR-199a-3p/U6、CD4^＋T细胞比例，以及血清白细胞介素(IL)-6、IL-17、IL-23及粒细胞-巨噬细胞集落刺激因子(GM-CSF)水平进行统计学比较。采用单因素方差分析及LSD-t检验，对3组THP-1细胞中IL-6、IL-17、IL-23及GM-CSF表达水平进行总体及两两比较。采用Pearson相关性分析法，对LAMP刺激后THP-1细胞中CD4^＋T细胞表达水平与miR-199a-3p表达水平的相关性进行分析。本研究经重庆市第七人民医院伦理委员会批准(审批文号：LLH201708-02)。所有受试儿监护人对本研究均知情同意，并签署知情同意书。

①观察组患儿外周血单核细胞中miR-199a-3p/U6为0.55±0.12，低于对照组的0.87±0.09；外周血单核细胞中CD4^＋T细胞比例，以及血清IL-6、IL-17、IL-23及GM-CSF水平分别为(14.4±3.8)%、(0.52±0.08) ng/L、(36.2±5.1) ng/L、(2.89±0.21) ng/mL、(273.8±30.2) mg/L，均分别高于对照组的(6.7±2.0)%、(0.41±0.09) ng/L、(23.9±5.9) ng/L、(1.56±0.14) ng/mL、(163.3±13.8) mg/L，并且上述差异均有统计学意义(t＝38.44、34.30、9.29、15.56、44.23、66.24，均为P<0.001)。②LAMP刺激后，miRNA mimic组THP-1细胞中IL-6、IL-17、IL-23及GM-CSF表达水平，均分别高于miR-199a-3p siRNA组及未处理组，并且差异均有统计学意义(P<0.05)。③LAMP刺激后，THP-1细胞中CD4^＋T细胞相对表达水平与miR-199a-3p相对表达水平呈负相关关系(r＝—0.643，P＝0.003)。

miR-199a-3p可能通过调控CD4^＋T细胞的表达，参与MPP患儿免疫反应。

To investigate the mechanism by which microRNA(miR)-199a-3p regulates the expression of CD4⁺ T cells and participates in the immune response of children with Mycoplasma hyopneumoniae pneumonia (MPP).

From January 2018 to December 2020, a total of 500 children with MPP admitted to the Seventh People′s Hospital of Chongqing were selected as the study subjects and included in observation group. And 200 children who underwent health examinations at the same hospital during the same period were included in control group. After the THP-1 cells for in vitro experimentation were cultured, they were randomly divided into miR-199a-3p siRNA group (n=3, transfected miR-199a-3p siRNA), miRNA mimic group (n=3, transfected miRNA mimic) and untreated group (n=3, treated with equal RPMI 1640 complete medium), and the lipid-associated membrane proteins (LAMP) extracted from purchased Mycoplasma pneumoniae(MP) were used to stimulate the above three groups of THP-1 cells for related experiments. The miR-199a-3p/U6 and proportion of CD4⁺ T cells in peripheral blood mononuclear cells, and serum interleukin (IL)-6, IL-17, IL-23 and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels between observation group and control group were statistically compared by independent-samples t test. One-way ANOVA and LSD-t test were used to overall and pairwise compare the expression levels of IL-6, IL-17, IL-23 and GM-CSF in three groups of THP-1 cells. Pearson correlation analysis was used to analyze the correlation between CD4⁺ T cell expression levels and miR-199a-3p expression levels in THP-1 cells after stimulated by LAMP. This study was approved by the Ethics Committee of the Seventh People′s Hospital of Chongqing (Approval No. LLH201708-02). All the guardians of the children gave informed consent to the study and signed the informed consent forms.

① miR-199a-3p/U6 in peripheral blood mononuclear cells of children in observation group was 0.55±0.12, which was lower than that of 0.87±0.09 in control group; The proportion of CD4⁺ T cells in peripheral blood mononuclear cells, and the serum levels of IL-6, IL-17, IL-23 and GM-CSF were (14.4±3.8)%, (0.52±0.08) ng/L, (36.2±5.1) ng/L, (2.89±0.21) ng/mL and (273.8±30.2) mg/L, respectively, which were higher than those of (6.7±2.0)%, (0.41±0.09) ng/L, (23.9±5.9) ng/L, (1.56±0.14) ng/mL and (163.3±13.8) mg/L, respectively in control group, and the above differences were statistically significant (t=38.44, 34.30, 9.29, 15.56, 44.23, 66.24; all with P<0.001). ② After LAMP stimulation, the expression levels of IL-6, IL-17, IL-23 and GM-CSF in THP-1 cells in miRNA mimic group were higher than those in miR-199a-3p siRNA group and untreated group, respectively, and the differences were statistically significant (P<0.05). ③The expression level of CD4⁺ T cells in THP-1 cells after LAMP stimulation was negatively correlated with the expression level of miR-199a-3p (r=—0.643, P=0.003).

miR-199a-3p may be involved in the immune response of children with MPP by regulating the expression of CD4⁺ T cells.