人支气管上皮hsa-miR-148a-3p 低表达细胞株的建立

doi:10.3969/j.issn.1004-616x.2014.03.010

Abstract

Abstract:

OBJECTIVE: To construct a 16HBE cell line with low expression of hsa-miR-148a-3p. METHODS：A pair of TuD RNA sequences of hsa-miR-148a-3p was designed according to information provided in miRBase， which was ligated to lentiviral vector pLKO.1-puro afterwards. The recombinant vector was transfected into 293FT cells for lentivirus packaging. The viruses were collected and used to infect normal 16HBE cells. The target cells were selected with Puromycin and was identified by quantitative PCR. The mRNA expression and the protein expression level of DNMT1 were then tested. RESULTS：Sequencing results indicated the successful construction of recombinant lentiviral vector with hsa-miR-148a-3p TuD RNA， quantitative PCR showed that the expression level of hsa-miR-148a-3p in the target cells was 44% lower than that of normal 16HBE cells(P<0.01)， whereas a 3.4-fold increase of the mRNA expression level and a two-fold increase of protein expression level of DNMT1 were observed in the cell line(P<0.01). CONCLUSION：Low expression of hsa-miR-148a-3p of 16HBE cell line was successfully constructed， and inhibition of hsa-miR-148a-3p could lead to higher expression and higher protein levels of DNMT1.

Key words: hsa-miR-148a-3p, DNMT1, 16HBE cells, lentivirus

XIE Xing， KE Yue-bin， LIU Qing-cheng， LIU Jian-jun， MAO Kan-lang，XU Xin-yun， XIA Bo， YANG Lin-qing. Construction of a human bronchial epithelial hsa-miR-148a-3p knockdown cell line[J]. Carcinogenesis, Teratogenesis & Mutagenesis, doi: 10.3969/j.issn.1004-616x.2014.03.010.

References

[1] Huang Y，Shen X，Zou Q. Biological functions of microRNAs：a review[J]. J Physiol Biochem，2011，67(1)： 129-139.

[2] Danish S，Maha A. MicroRNAs in development and disease[J]. Physiol Rev，2011，91(3)：827-887.

[3] Susan K，Oliver G，Cheryl S，et al. Cutting edge：the Foxp3 target miR-155 contributes to the development of regulatory T cells[J]. Immunol，2009(182)：2578-2582.

[4] Peter J. Functions of DNA methylation：islands，start sites， gene bodies and beyond[J]. Nat Rev Genet，2012 13(7)：484-492.

[5] 王志刚，吴建新. DNA甲基转移酶分类、功能及其研究进展[J]. 遗传，2009，31(9)：903-912.

[6] Pan Z，Gao Q，Yu M. MicroRNAs regulate CYP3A4 expression via direct and indirect targeting[J]. Drug Metabol Dis，2009，37(10)：2112-2117.

[7] Zhang H，Li Y，Huang Q，et al. MiR-148a promotes apoptosis by targeting Bcl-2 in colorectal cancer[J]. Cell Death Differ，2011，18(11)：1702-1710.

[8] Na?ma H，Yannick D，Arief S，et al. The silencing of microRNA 148a production by DNA hypermethylation is an early event in pancreatic carcinogenesis[J]. Clin Chem，2010， 56(7)：1107-1118.

[9] Lawrie C，Saunders J，Soneji S，et al. MicroRNA expression in lymphocyte development and malignancy[J]. Leukemia， 2008，22(7)：1440-1446.

[10] Pan W，Zhu S，Yuan M，et al. MicroRNA-21 and MicroRNA-148a contribute to DNA hypomethylation in lupus CD4+ T cells by directly and indirectly targeting DNA methyltransferase 1[J]. J Immunol，2010，184(12)：6773-6781.

[11] Anja D，Martijn K，Mariette S，et al. miR-148 targets human DNMT3b protein coding region[J]. RNA，2008，14(5)：872-877.

[12] Braconi C，Huang N，Patel T. MicroRNA-dependent regulation of DNA methyltransferase-1 and tumor suppressor gene expression by interleukin-6 in human malignant cholangiocytes[J]. Hepatology，2010，51(3)：881-890.

[13] Takeshi H，Yuka O，Hideo I. Vectors expressing efficient RNA decoys achieve the long-term suppression of specific microRNA activity in mammalian cells[J]. Nucleic Acids Res，2009，37(6)：43.

[14] 蔡剑锋，黄海燕，刘建军，等. 人支气管上皮细胞聚-ADP-核糖水解酶缺陷细胞株的构建及鉴定[J]. 毒理学杂志，2012，26(6)：415-419.

[15] Daniel T，Cameron B，Gregory G. Experimental strategies for microRNA target identification[J]. Nucleic Acids Res，2011， 39(16)：6845-6853.

[16] Francesca R，Christian B，Carlo C. Targeting microRNAs in neurons：Tools and perspectives[J]. Experiment Neurol， 2012，235(2)：419-426.

[17] Adam C，Tal K. Gene delivery by lentivirus vectors[J]. Mol Biotech，2007，36(3)：184-204.

[18] Daniel FI，Cherubini K，Yurgel LS，et al. The role of epigenetic transcription repression and DNA methyltransferases in cancer[J]. Cancer，2011，117(4)：677-687.

[19] 王海琴. DNA甲基转移酶1在结直肠癌DNA甲基化中作用机制的研究[D]. 湖南：中南大学湘雅二医院，2012：1-3.

[20] Muthusamy V，Bosenberg M，Wajapeyee N. Redefining regulation of DNA methylation by RNA interference[J]. Genomics，2010，96(4)：191-198.

[21] Xu Q，Jiang Y，Yin Y，et al. A regulatory circuit of miR-148a/152 and DNMT1 in modulating cell transformation and tumor angiogenesis through IGF-IR and IRS1[J]. J Mol Cell Biol，2013，5(1)：3-13.

[22] Kuhn E，Nuovo J，Terry V，et al. Chromosome 21-derived microRNAs provide an etiological basis for aberrant protein expression in human Down syndrome brains[J]. Biol Chem， 2010，285(2)：1529-1543.

[23] Ameres L，Horwich D，Hung H，et al. Target RNA-directed trimming and tailing of small silencing RNAs[J]. Science， 2010，328(5985)：1534-1539.

[24] Xie J，Ameres SL，Friedline R，et al. Long-term，efficient inhibition of microRNA function in mice using rAAV vectors[J]. Nat Methods，2012，9(4)：403-409.

Construction of a human bronchial epithelial hsa-miR-148a-3p knockdown cell line

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