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

畜牧兽医学报 ›› 2016, Vol. 47 ›› Issue (1): 183-189.doi: 10.11843/j.issn.0366-6964.2016.01.025

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

郝俊芳^1，2，黄凯²，王月影^1，2，杜丽丽^1，2，钟凯^1，2*

(1.河南农业大学农业部动物生化与营养重点开放实验室，郑州 450002；2.河南农业大学牧医工程学院，郑州 450002)

收稿日期:2015-05-23 出版日期:2016-01-23 发布日期:2016-01-23
通讯作者: 钟凯（1974-），女，河南郑州人，博士，副教授，主要从事泌乳生物化学研究，E-mail：zhongkai@henau.edu.cn
作者简介:郝俊芳（1989-），女，河南林州人，硕士生，主要从事泌乳生物化学研究，E-mail：haojunfang163@163.com
基金资助:
国家自然科学基金（31470120）

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

HAO Jun-fang^1，2，HUANG Kai²，WANG Yue-ying^1，2，DU Li-li^1，2，ZHONG Kai^1，2*

(1.Key Lab of Regulation on Animal Growth and Development of Agricultural Ministry of China，Henan Agricultural University，Zhengzhou 450002，China；2.College of Veterinary Medicine，Henan Agricultural University，Zhengzhou 450002，China)

Received:2015-05-23 Online:2016-01-23 Published:2016-01-23

摘要/Abstract

摘要：

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）48 h，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）；PB-miR-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。

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.

中图分类号:

S857.26

郝俊芳，黄凯，王月影，杜丽丽，钟凯. 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[J]. 畜牧兽医学报, 2016, 47(1): 183-189.

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.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/ 10.11843/j.issn.0366-6964.2016.01.025

https://www.xmsyxb.com/CN/Y2016/V47/I1/183

参考文献

［1］HUANG Y，SHEN X J，ZOU Q，et al.Biological functions of microRNAs：a review［J］.J Physiol Biochem，2011，67(1)：129-139.
［2］HE L，HANNON G J.MicroRNAs：small RNAs with a big role in gene regulation［J］.Nat Rev Genet，2004，5(7)：522-531.
［3］BARTEL D P.MicroRNAs：Target recognition and regulatory functions［J］.Cell， 2009，136(2)：215-233.
［4］HOGEVEEN H，HUIJPS K，LAM T J.Economic aspects of mastitis：new developments［J］.N Z Vet J，2011，59(1)：16-23.
［5］LAWLESS N，REINHARDT A T，BRYAN K，et al.MicroRNA regulation of bovine monocyte inflammatory and metabolic networks in an in vivo infection model［J］.G3(Bethesda)， 2014，4(6)：957-971.
［6］CHEN L，LIU X L，LI Z X，et al.Expression differences of miRNAs and genes on NF-κB pathway between the healthy and the mastitis Chinese Holstein cows［J］.Gene，2014，545(1)：117-125.
［7］JIN W，IBEAGHA-AWEMU E M，LIANG G，et al.Transcriptome microRNA profiling of bovine mammary epithelial cells challenged with Escherichia coli or Staphylococcus aureus bacteria reveals pathogen directed microRNA expression profiles［J］.BMC Genomics，2014，15：181.
［8］DILDA F，GIOIA G，PISANI L，et al.Escherichia coli lipopolysaccharides and Staphylococcus aureus enterotoxin B differentially modulate inflammatory microRNAs in bovine monocytes［J］.Vet J，2012，192(3)：514-516.
［9］NAEEM A，ZHONG K，MOISÁ S J，et al.Bioinformatics analysis of microRNA and putative target genes in bovine mammary tissue infected with Streptococcus uberis［J］. J Dairy Sci，2012，95(11)：6397-6408.
［10］IZUMl H，KOSAKA N，SHIMIZU T，et al.Bovine milk contains microRNA and messenger RNA that are stable under degradative conditions［J］.J Dairy Sci，2012，95(9)：4831-4841.
［11］ZADOKS R N，MIDDLETON J R，McDOUGALL S，et al.Molecular epidemiology of mastitis pathogens of dairy cattle and comparative relevance to humans［J］.J Mammary Gland Biol Neoplasia，2011，16(4)：357-372.
［12］陆承平.兽医微生物学［M］.4版.北京：中国农业出版社，2007.
LU C P.Veterinary microbiology［M］.4ed.Beijing：China Agriculture Press，2007.(in Chinese)
［13］WELLNITZ O，KERR D E.Cryopreserved bovine mammary cells to model epithelial response to infection［J］. Vet Immunol Immunopathol， 2004，101(3-4)：191-202．
［14］PAAPE M，MEHRZAD J，ZHAO X，et al.Defense of the bovine mammary gland by polymorphonuclear neutrophil leukocytes［J］.J Mammary Gland Biol Neoplasia，2002，7(2)：109-121.
［15］GOLDAMMER T，ZERBE H，MOLENAAR A，et al.Mastitis increases mammary mRNA abundance of beta-defensin 5，toll-like-receptor 2 (TLR2)，and TLR4 but not TLR9 in cattle［J］.Clin Diagn Lab Immunol，2004，11(1)：174-185．
［16］李金明.实时荧光PCR技术［M］.北京：人民军医出版社，2007：92-93.
LI J M.Real time fluorescent PCR technique［M］.Beijing：People’s Military Medical Press，2007：92-93.(in Chinese)
［17］LI L M，HUANG J，ZHANG X，et al.One SNP in the 3′-UTR of HMGB1 gene affects the binding of target bta-miR-223 and is involved in mastitis in dairy cattle［J］.Immunogenetics，2012，64(11)：817-824.
［18］JOHNNIDIS J B，HARRIS M H，WHEELER R T，et al.Regulation of progenitor cell proliferation and granulocyte function by microRNA-223［J］.Nature，2008，451(7182)：1125-1129.
［19］LENA A M，SHALOM-FEUERSTEIN R，RIVETTI DI VAL CERVO P，et al.miR-203 represses ‘stemness’ by repressing DeltaNp63［J］.Cell Death Differ， 2008，15(7)：1187-1195.
［20］MAILLOT G，LACROIX-TRIKI M，PIERREDON S，et al.Widespread estrogen-dependent repression of micrornas involved in breast tumor cell growth［J］.Cancer Res，2009，69(21)：8332-8340.
［21］YAO Y，SUO A L，LI Z F，et al.MicroRNA profiling of human gastric cancer［J］.Mol Med Rep，2009，2(6)：963-970.
［22］TAGANOV K D，BOLDIN M P，CHANG K J，et al.NF-kappaB-dependent induction of microRNA miR-146，an inhibitor targeted to signaling proteins of innate immune responses［J］.Proc Natl Acad Sci U S A，2006，103(33)：12481-12486.
［23］BOLDIN M P，TAGANOV K D，RAO D S，et al.miR-146a is a significant brake on autoimmunity，myeloproliferation，and cancer in mice［J］.J Exp Med，2011，208(6)：1189-1201.
［24］HE M，XU Z，DING T，et al.MicroRNA-155 Regulates inflammatory cytokine production in tumor-associated macrophages via targeting C/EBPbeta［J］.Cell Mol Immunol，2009，6(5)：343-352.
［25］BIGGER B W，TOLMACHOV O，COLLOMBET J M，et al.An araC-controlled bacterial cre expression system to produce DNA minicircle vectors for nuclear and mitochondrial gene therapy［J］.J Biol Chem，2001，276(25)：23018-23027.
［26］CHEN Z Y，HE C Y，EHRHARDT A，et al.Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo［J］.Mol Ther，2003，8(3)：495-500.
［27］JECHLINGER W，AZIMPOUR TABRIZI C，LUBITZ W，et al.Minicircle DNA immobilized in bacterial ghosts：in vivo production of safe non-viral DNA delivery vehicles［J］.J Mol Microbiol Biotechnol，2004，8(4)：222-231.
［28］ROBERTS P E，MATHER J P.Introduction to cell and tissue culture［M］.New York：Plenum Press，1998：77-78.

[1]	任晓丽, 范玉营, 皇甫和平, 刘云, 石冬梅. GSK126对犬乳腺肿瘤细胞上皮间质转化的影响[J]. 畜牧兽医学报, 2023, 54(4): 1721-1729.
[2]	胥辉豪, 刘江渝, 李启卷, 郑小波, 林珈好, 金艺鹏, 林德贵. 己糖激酶2在犬乳腺肿瘤中的表达及预后研究[J]. 畜牧兽医学报, 2023, 54(3): 1310-1324.
[3]	孟梅娟, 汪艳, 霍然, 李雪睿, 常广军, 沈向真. 抑制PERK对LPS诱导的奶牛乳腺上皮细胞自噬的影响[J]. 畜牧兽医学报, 2023, 54(1): 351-360.
[4]	罗仍卓么, 王晋鹏, 焦鹏, 李彦霞, 董益闻, 魏大为, 王兴平. 奶牛乳腺炎模型的建立及炎症相关因子基因mRNA转录水平的分析[J]. 畜牧兽医学报, 2022, 53(8): 2763-2772.
[5]	焦鹏, 王兴平, 汪书哲, 王晋鹏, 罗仍卓么, 贾立, 魏大为, 马云. 奶牛乳腺炎差异表达lncRNA BCL2对炎症及凋亡相关mRNA表达的影响[J]. 畜牧兽医学报, 2022, 53(7): 2160-2171.
[6]	李妍, 蓝婷英, 庞博, 杨晓农, 黄坚. NLRP3炎症小体及其下游炎症因子在犬乳腺肿瘤组织中的表达[J]. 畜牧兽医学报, 2022, 53(4): 1252-1258.
[7]	伍迎欢, 杨丹茹, 赵燕英. 异体移植炎症因子1通过核因子κB信号促进牛乳腺上皮细胞炎症因子的释放[J]. 畜牧兽医学报, 2021, 52(3): 782-788.
[8]	吴富鑫, 熊本海, 童津津, 蒋林树. 奶牛乳腺炎无乳链球菌毒力相关因子[J]. 畜牧兽医学报, 2020, 51(12): 2954-2963.
[9]	欧爱群, 王凯, 吴黎明, 李江红, 彭文君. 蜂胶对细菌脂多糖刺激下奶牛乳腺上皮细胞炎症相关基因mRNA转录水平和紧密连接蛋白的影响[J]. 畜牧兽医学报, 2020, 51(5): 1149-1157.
[10]	韩硕, 王宇轩, 刘娟, 邹紫雯, 李鑫丽, 罗林, 沈冰蕾. Bta-miR-223靶基因及调控网络的生物信息学分析[J]. 畜牧兽医学报, 2019, 50(10): 2171-2174.
[11]	刘镇, 饶家榕, 吕俊瑾, 芶思, 李惠珍, 林辉, 远立国. 犬乳腺肿瘤裸鼠模型建立及其生长特性[J]. 畜牧兽医学报, 2019, 50(4): 861-869.
[12]	刘畅, 唐鑫, 张文劲, 赵春阳, 郭爱珍, 胡长敏. 6-溴-靛玉红-3'-肟抑制脂多糖诱导的小鼠乳腺上皮细胞凋亡[J]. 畜牧兽医学报, 2018, 49(12): 2753-2761.
[13]	刁洪秀, 李艺, 林德贵, 张迪. 犬乳腺肿瘤中MUC1的表达及临床意义[J]. 畜牧兽医学报, 2018, 49(7): 1511-1516.
[14]	王晓，俞英. 中国北方奶牛金葡菌乳房炎感染现状及耐药性和流行类型研究进展[J]. 畜牧兽医学报, 2015, 46(9): 1477-1488.
[15]	王林会，李晓宇，金礼吉，李淑英，徐永平. 金黄色葡萄球菌外毒素及荚膜特异性卵黄抗体联合治疗奶牛乳房炎效果研究[J]. 畜牧兽医学报, 2013, 44(12): 2016-2021.

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

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价