miR-133a-3p targeted regulating CD2AP gene reduces RSV-infected damage of human bronchial epithelial 16-HBE cell line

Abstract

Abstract: Objective To explore the effect of miR-133a-3p on the apoptosis and inflammatory response of bronchial epithelial cells infected by respiratory syncytial virus (RSV) and its regulatory effect on CD2-associated protein (CD2AP). Methods The bronchial mucosal tissue specimens from 59 children with bronchial asthma were collected as the AsP group and 59 bronchial mucosal tissue specimens from non-asthmatic children with bronchiec- tasis were selected as the NC group. RT-qPCR method was used to detect the expression of miR-133a-3p and CD2AP in the tissues. RSV infected bronchial epithelial cells (16-HBE) were used to establish a cell injury model (RSV group). miR-NC, miR-133a-3p mimics, si-NC, si-CD2AP, pcDNA-NC, pcDNA-CD2AP, miR-133a-3p mimics and pcDNA-NC, miR-133a-3p mimics and pcDNA-CD2AP were respectively transferred into 16-HBE cells infected with RSV. RT-qPCR and Western blot were used to detect the expression of miR-133a-3p and CD2AP in the cells. ELISA was used to detect the level of TNF-α, IL-6, and IL-1α. Flow cytometry was used to detect apoptosis . The dual luciferase reporter experiment was used to detect the targeting relationship between miR-133a-3p and CD2AP. Western blot method was used to detect the protein expression of cleaved-caspase-3. Results Compared with NC group, the expression of miR-133a-3p in the bronchial mucosa tissue of the AsP group was decreased (P＜0.05), and the expression of CD2AP mRNA was increased (P＜0.05). The expression of miR-133a-3p in RSV-infected 16-HBE cells was decreased (P＜0.05), and the expression level of CD2AP, TNF-α, IL-6 and IL-1α was increased (P＜0.05), the apoptosis rate and the protein level of cleaved-caspase-3 were increased(P＜0.05). Transfection of miR-133a-3p mimics or transfection of si-CD2AP into RSV-infected 16-HBE cells significantly reduced the level of TNF-α, IL-6, IL-1α, the apoptosis rate and the protein level of cleaved-caspase-3 (P＜0.05). The dual luciferase report experiment confirmed that CD2AP was the target gene of miR-133a-3p. Co-transfection of miR-133a-3p mimics and pcDNA-CD2AP significantly reversed the effects of miR-133a-3p mimics transfection on 16-HBE cell apoptosis and inflammation. Conclusions The over-expression of miR-133a-3p inhibits the inflammatory response and apoptosis of RSV-infected bronchial epithelial cells by negatively regulating the expression of CD2AP and thereby reducing cell damage.

Key words: miR-133a-3p, CD2AP, respiratory syncytial virus, bronchial epithelial cells, apoptosis, inflammatory reaction

CLC Number:

LIANG Min, LI Wan, XIONG Shi-si, BIAN Jun-mei. miR-133a-3p targeted regulating CD2AP gene reduces RSV-infected damage of human bronchial epithelial 16-HBE cell line[J]. Basic & Clinical Medicine, 2021, 41(10): 1463-1469.

References

[1]张博达, 李俊玲, 金典, 等. 孟鲁司特抑制哮喘模型大鼠气道黏液高分泌[J]. 基础医学与临床, 2019, 39: 1699-1703.
[2]Othumpangat S, Walton C, Piedimonte G. microRNA-221 modulates RSV replication in human bronchial epithelium by targeting NGF expression[J]. PLoS One, 2012, 7: e30030-e30040.
[3]Kim J, Kim DY, Heo HR, et al. Role of miRNA-181a-2-3p in cadmium-induced inflammatory responses of human bronchial epithelial cells[J]. J Thorac Dis, 2019, 11: 3055-3069.
[4]Huang H, Lu H, Liang L, et al. microRNA-744 inhibits proliferation of bronchial epithelial cells by regulating smad3 pathway via targeting transforming growth factor-β1 (TGF-β1) in severe asthma[J]. Med Sci Monit, 2019, 25: 2159-2168.
[5]Huang Y, Wang Y, Lin L, et al. Overexpression of miR-133a-3p inhibits fibrosis and proliferation of keloid fibroblasts by regulating IRF5 to inhibit the TGF-β/Smad2 pathway[J]. Mol Cell Probes, 2020, 52: 101563-101573.
[6]Ming L, Ning J, Ge Y, et al. Excessive apoptosis of podocytes caused by dysregulation of microRNA-182-5p and CD2AP confers to an increased risk of diabetic nephropathy[J]. J Cell Biochem, 2019, 120: 16516-16523.
[7]秦岭, 冯俊涛, 胡成平, 等. 呼吸道合胞病毒感染支气管上皮细胞介导IL-8致Th17/Treg失衡[J]. 中南大学学报(医学版), 2016, 41: 337-344.
[8]Yao L, Zhou B, You L, et al. LncRNA MIAT/miR-133a-3p axis regulates atrial fibrillation and atrial fibrillation-induced myocardial fibrosis[J]. Mol Biol Rep, 2020, 47: 2605-2617.
[9]Shi L, Yu C, Tian X, et al. Effect of microRNA-133a-3p/matrix metalloproteinase-9 axis on the growth of atherosclerotic vascular smooth muscle cells[J]. Exp Ther Med, 2019, 18: 4356-4362.
[10]Xu H, Li H, Zhu P, et al. Tanshinone IIA Ameliorates progression of CAD Through regulating cardiac H9c2 cells proliferation and apoptosis by miR-133a-3p/EGFR Axis[J]. Drug Des Devel Ther, 2020, 14: 2853-2863.
[11]江敏时, 檀卫平, 郭奕文, 等. 孟鲁司特抑制RSV感染人支气管上皮细胞所致炎性作用研究[J]. 中国现代药物应用, 2017, 11: 148-149.
[12]周洁, 吉玲, 王新华, 等. 细胞间黏附分子-1在支气管哮喘儿童支气管黏膜中的表达及其对支气管上皮细胞凋亡的影响[J]. 新乡医学院学报, 2019, 36: 31-35.
[13]Saurus P, Tolvanen TA, Lindfors S, et al. Inhibition of SHIP2 in CD2AP-deficient podocytes ameliorates reactive oxygen species generation but aggravates apoptosis[J]. Sci Rep, 2017, 7: 10731-10741.
[14]Wang JY, Zhang DQ, Cao Q, et al. miR-939-5p decre-ases the enrichment of RNA polymerase II in the promoter region of CD2AP involved in nephrotic syndrome[J]. J Cell Biochem, 2019, 1: 1-9.
[15]Park HY, Seong SB, Min SY, et al. CD2-associated protein/phosphoinositide 3-kinase signaling has a preventive role in angiotensin II-induced podocyte apoptosis[J]. Int J Biochem Cell Biol, 2016, 79: 370-381.