炎症相关miRNAs在LPS诱导的小鼠乳腺上皮细胞炎性反应中的表达及miR-223真核表达载体的构建

doi:10.11843/j.issn.0366-6964.2016.01.025

Abstract

Abstract:

Small RNA (miRNA) is a class of endogenous，single strand，small non-coding RNAs，which is recently discovered.Its regulation in the process of immune defense in mammary tissue gradually attracted people’s attention.Expression changes of inflammation-related miRNAs have been detected in mouse mammary epithelial cells when inflammation reaction induced with LPS occurred with qRT-PCR；we have also constructed 3 kinds of miR-223 eukaryotic expression vectors and tested recombinant vectors transfection efficiency.Mouse mammary epithelial cells (EpH4-Ev cells) have been incubated with different dose of LPS for 48 h，then，expressions of miR-223，miR-146a，miR-181a and miR-155 have been detected with qRT-PCR.Genomic DNA of EpH4-Ev cells were used as a template and pre-miR-223 cDNA sequence was amplificated with PCR.PCR product was combined with Minicircle，pcDNA3.1(+) and piggyBac transposon，separately，to construct three kinds of miR-223 eukaryotic expression vectors.Three kinds of recombined vectors were transfected into EpH4-Ev cells，respectively.Expression of miR-223 was measured and green fluorescence was observed in order to evaluate transfection efficiency of recombinant plasmid.qRT-PCR results showed that expression of 4 kinds of inflammation-related in EpH4-Ev cells significantly increased when inflammation induced by LPS (P<0.05 or P<0.01)，and the expression change was in a dose-dependent manner.qRT-PCR results showed that the expression level of miR-223 had no significant change (P>0.05) when Mini-miR-223 plasmid transfected into the EpH4-Ev cells，while the expression of miR-223 significantly increased when PB-miR-223 and pcDNA-miR-223 transfected EpH4-Ev cells (P<0.001).Inflammation-related miRNAs have been showed involved in the process of inflammation induced by LPS in mouse EpH4-Ev cells.Three kinds of miR-223 eukaryotic expression vectors have been constructed successfully，and may contribute to further research of the modulation significance of miRNAs in the mammary gland immune reaction.

CLC Number:

S857.26

HAO Jun-fang，HUANG Kai，WANG Yue-ying，DU Li-li，ZHONG Kai. Expression of Inflammation-related miRNAs in the LPS-induced Inflammation in Murine Mammary Epithelial Cells and Construction of miR-223 Eukaryotic Expression Vector[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(1): 183-189.

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Expression of Inflammation-related miRNAs in the LPS-induced Inflammation in Murine Mammary Epithelial Cells and Construction of miR-223 Eukaryotic Expression Vector

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