Inhibiting effect of miR-18a on expression of extracellular matrix-related genes of NIH-3T3 by targeting <i>Notch2</i>

Jiaqi Hao; Jing Wen; Chuncheng Liu; Lu Cai

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Inhibiting effect of miR-18a on expression of extracellular matrix-related genes of NIH-3T3 by targeting Notch2

VernacularTitle:微小RNA-18a靶向Notch2抑制NIH-3T3细胞外基质相关基因的表达
Author: Jiaqi HAO ^{1
,

2} ; Jing WEN ³ ; Chuncheng LIU ^{1
,

2} ; Lu CAI ^{1
,

2}
Author Information

1. Inner Mongolia Key Laboratory of Functional Genome Bioinformatics, Inner Mongolia University of Science and Technology, Baotou, Inner Monoglia 014010, China
2. .
3. Inner Mongolia Key Laboratory of Functional Genome Bioinformatics, Inner Mongolia University of Science and Technology, Baotou, Inner Monoglia 014010, China.
Publication Type:Experiment
Keywords: silicosis; microRNA-18a; neurogenic locus notch homolog protein 2
From: Journal of Environmental and Occupational Medicine 2022;39(7):786-791
CountryChina
Language:Chinese
Abstract: Background The main pathological feature of silicosis is pulmonary fibrosis. Multiple miRNAs regulate the development of silicosis. Objective Using a fibroblast cell line, to explore the effect of miR-18a on the expression of extracellular matrix-related genes, and verify the mechanism. Methods The fibroblast cell line NIH-3T3 cells were transfected with miR-18a mimics or neurogenic locus notch homolog protein 2 (Notch2) small interfering RNA (siRNA). The mRNA expression changes of Acta2, Col1a1, and Notch2 were detected by real-time quantitative reverse transcription PCR (qRT-PCR), α-smooth muscle actin (α-SMA) and Notch2 were also detected at the protein level by Western blotting. To verify whether miR-18a could directly act on the complementary sequences of the Notch2 gene, human embryonic kidney HEK293T cells and the psiCHECK^TM-2 vector were used. Results The results of qRT-PCR showed that in NIH-3T3 cells, the over-expression of miR-18a mimics for 36 h inhibited the mRNA expression of Col1a1 and Acta2 (P<0.05). The results of Western blotting showed that the protein expression abundance of α-SMA was decreased at 48 h of miR-18a mimics over-expression. The qRT-PCR results showed that the over-expression of miR-18a for 36 h had no significant effect on Notch2 gene expression, but the Western blotting results showed that the over-expression of miR-18a mimics inhibited the expression of Notch2 at the protein level. The results of the dual luciferase reporter vector assay showed that in HEK293T cells, both over-expressed miR-18a mimics and inhibitors for 24 h demonstrated that Notch2 is a direct target gene of miR-18a. When Notch2 was inhibited for 36 h, the qRT-PCR results showed that Acta2 and Col1a1 were down-regulated (P < 0.05), and the results of Western blotting showed that α-SMA protein was also inhibited. Conclusion The findings indicate that miR-18a could inhibit the expression of extracellular matrix-related genes of NIH-3T3 cells by directly acting on the 3’UTR of target gene Notch2.