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

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

小分子RNA(miRNA)是近年来发现的一类内源性、非编码单链小RNA,其在乳腺组织免疫防御过程中发挥的调控作用逐渐引起了人们的关注。作者拟用qRT-PCR检测炎症相关miRNAs在LPS诱导的小鼠乳腺上皮细胞炎症反应中的表达变化;构建3种miR-223真核表达载体并检验重组载体的转染效率。用不同浓度LPS处理小鼠乳腺上皮细胞(EpH4-Ev)48h,qRT-PCR检测miR-223、miR-146a、miR-181a和miR-155的表达变化。以EpH4-Ev细胞基因组DNA为模板,PCR扩增pre-miR-223序列,以质粒Minicircle、pcDNA3.1(+)和转座子piggyBac为母本构建3种miR-223真核表达载体,将其转染EpH4-Ev细胞,通过观察荧光、qRT-PCR检测miR-223的表达,评价重组质粒转染效率。结果:qRT-PCR检测结果表明,LPS诱导EpH4-Ev细胞炎症反应时,4种炎症相关miRNAs的表达均显著(P<0.05或P<0.01)增加,并呈现不同程度的剂量依赖性;重组质粒转染EpH4-Ev细胞后,qRT-PCR检测结果显示,Mini-miR-223质粒转染组的miR-223表达水平无统计学差异(P>0.05);PBmiR-223和pcDNA-miR-223转染EpH4-Ev细胞后,miR-223的表达均极显著(P<0.001)增加。本研究表明炎症相关miRNAs参与LPS诱导小鼠EpH4-Ev细胞的炎性反应过程。成功构建3种miR-223真核表达载体,为后续miRNAs在乳腺炎症反应中的功能研究奠定基础。

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 3kinds 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-146 a,miR-181 aand 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 withPCR.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 4kinds 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 PBmiR-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.