MicroRNA-21 for regulation of TLR4 in Hela cells

doi:10.3969/j.issn.2095-4344.2015.02.011

Abstract

Abstract:

BACKGROUND: MicroRNAs (miRNA) through regulating specific target gene mRNA expression play important roles in different processes of diseases.
OBJECTIVE: To study the interaction of miRNA-21 with its target gene TLR4 in Hela cells.
METHODS: The candidate target gene of miRNA-21 was determined according to miRNA analysis databases. The constructed recombinant adenovirus vector carrying pri-miRNA-21 gene was used, which could package and amplify viruses to transfect Hela cells. Then, the expression of fluorescent proteins was detected. Forty-eight hours after transfection of miRNA-21 or control, extracted proteins were used for detection of TLR4 protein using western blot assay.
RESULTS AND CONCLUSION: Recombinant adenoviruses pAd/pri-miRNA-21 and pAd/neg at 100 MOI could successfully infect Hela cells. Bioinformatic analysis suggested several possible binding sites between miRNA-21 and TLR4. The experimental results showed that miRNA-21 down-regulated TLR4 at protein levels, indicating that miRNA-21 can interfere with the expression of TLR4 target gene.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: Genes, Toll-Like Receptor 4, Hela Cells, MicroRNAs

CLC Number:

R318

Zhao Jing, Yue Peng, Huang Jia-fang, Wang Yu-tao, Ma Ting, Chen Bao-rong. MicroRNA-21 for regulation of TLR4 in Hela cells[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(2): 220-224.

Figures/Tables 1

2.1 miRNA-21靶基因的预测结果通过网站预测的方法得到了miRNA-21可能的靶基因TLR4，然后通过blast数据库比对发现miRNA-21的5’端区域与TLR4的3’UTR区存在互补的碱基(图1)。 2.2 Triton X-114液相分离法去除质粒内毒素结果因重组的腺病毒载体质粒较大约42 kb，所以采用手工法提取质粒，Triton X-114液相分离法去除质粒内毒素。经后期实验证实该方法提取的质粒浓度明显高于试剂盒，并能有效降低质粒在提取过程中断裂的可能。且经Triton X-114液相分离法去除质粒内毒素对后期质粒的转染及病毒的扩增未见明显影响(图2)。 2.3 线性化重组腺病毒pAd/pri-miRNA-21、pAd/neg质粒结果经Pac Ⅰ酶切37 ℃ 3 h后取5 µL进行琼脂糖凝胶电泳，可见到重组质粒分别被切成35 kb和4.5 kb两条片断(图3)。 2.4 重组腺病毒的包装成功检测荧光表达将线性化带有绿色荧光蛋白的重组腺病毒pAd/pri-miRNA-21、pAd/neg质粒分别转染293A细胞后观察荧光表达，随着转染天数的增加，带有绿色荧光的细胞逐渐增多，转染第7天视野下可见成片细胞变绿(图4A，B)。转染第10天细胞大部分出现病变，表现为变圆、贴壁性差，呈葡萄串样脱落(图4C，D)，此时成功收获第一代病毒颗粒，并经过两轮扩增后，TCID50法测定重组腺病毒滴度为1× 1 010 pfu/mL，获得了可以高效转导pri-miRNA-21基因的腺病毒。 2.5 重组腺病毒感染靶细胞Hela结果感染Hela细胞 24 h后开始动态观察绿色荧光蛋白的表达，发现重组腺病毒pAd/pri-miRNA-21、pAd/neg可以成功感染Hela细胞(图5)，随着病毒颗粒的增多，感染效率逐渐增加；且在MOI值为1的培养孔里未见pAd/pri-miRNA-21、pAd/neg重组病毒增殖，证实所得病毒正确，无野生型病毒扩增。 2.6 Western blotting检测结果分别提取感染48 h pAd/pri-miRNA-21和pAd/neg重组腺病毒的Hela细胞总蛋白，检测TLR4基因在蛋白水平上表达的不同，发现感染pAd/pri-miRNA-21组与对照组pAd/neg及空白组相比，TLR4蛋白的表达量明显降低(图6)。"

References

[1] Shukla GC, Singh J, Barik S.MicroRNAs: Processing, Maturation, Target Recognition and Regulatory Functions. Mol Cell Pharmacol.2011;3(3): 83-92.
[2] Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell.2009;136(2):215-233.
[3] Jiang M, Zhang P, Hu G, et al. Relative expressions of miR-205-5p, miR-205-3p, and miR-21 in tissues and serum of non-small cell lung cancer patients. Mol Cell Biochem. 2013; 383 (1-2): 67-75.
[4] Kumar S, Keerthana R, Pazhanimuthu A,et al. Overexpression of circulating miRNA-21 and miRNA-146a in plasma samples of breast cancer patients. Indian J Biochem Biophys.2013;50(3):210-214.
[5] Pan X, Wang ZX, Wang R. MicroRNA-21: a novel therapeutic target in human cancer. Cancer Biol Ther.2010;10(12): 1224-1232.
[6] Carthew RW, Sontheimer EJ. Origins and Mechanisms of miRNAs and siRNAs. Cell. 2009;136 (4): 642-655.
[7] Sharma M,Juvvuna PK, Kukreti H, et al. Mega roles of microRNAs in regulation of skeletal muscle health and disease. Front Physiol.2014;5:239.
[8] Kim YJ, Park SJ, Choi EY, et al. PTEN modulates miR-21 processing via RNA-regulatory protein RNH1. PLoS One. 2011; 6 (12): e28308.
[9] Ludwig K,Fassan M, Mescoli C, et al. PDCD4/miR-21 dysregulation in inflammatory bowel disease-associated carcinogenesis. Virchows Arch.2013;462(1):57-63.
[10] Torres A, Torres K, Paszkowski T, et al. Highly increased maspin expression corresponds with up-regulation of miR-21 in endometrial cancer: a preliminary report. Int J Gynecol Cancer.2011;21(1):8-14.
[11] Ferraro A, Kontos CK, Boni T, et al.Epigenetic regulation of miR-21 in colorectal cancer: ITGB4 as a novel miR-21 target and a three-gene network (miR-21-ITGBeta4-PDCD4) as predictor of metastatic tumor potential. Epigenetics.2014;9(1): 129-141.
[12] Wang N, Zhang CQ, He JH, et al. MiR-21 down-regulation suppresses cell growth, invasion and induces cell apoptosis by targeting FASL, TIMP3, and RECK genes in esophageal carcinoma. Dig Dis Sci.2013;58(7):1863-1870.
[13] Vicinus B, Rubie C, Stegmaier N, et al. miR-21 and its target gene CCL20 are both highly overexpressed in the microenvironment of colorectal tumors: significance of their regulation. Oncol Rep.2013;30(3):1285-1292.
[14] 钟智伟.肺癌中miR-31miR-21对错配修复蛋白的调控以及对细胞增殖的影响[D].宁波大学, 2012.
[15] 包龙龙.miR-21调控肝癌细胞增殖转移分子机制的研究[D].苏州大学, 2013.
[16] Wang X, Gu J, Zhang MQ, Li Y. Identification of phylogenetically conserved microRNA cis-regulatory elements across 12 Drosophila species. Bioinformatics. 2008; 24(2):165-171.
[17] Gammell P. MicroRNAs: recently discovered key regulators of proliferation and apoptosis in animal cells : Identification of miRNAs regulating growth and survival. Cytotechnology. 2007; 53(1-3):55-63.
[18] Stark A, Brennecke J, Russell RB,et al. Identification of Drosophila MicroRNA targets. PLoS Biol.2003;1(3):E60.
[19] 张朵,李博,兰风华.以TritonX-114液相分离法去除质粒溶液中的内毒素[J].生物技术通讯, 2007,(6):0971-0902.
[20] Meng F, Henson R, Wehbe-Janek H, et al. MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology, 2007;133(2): 647-658.
[21] Asangani IA, Rasheed SA, Nikolova DA,et al.MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer. Oncogene.2008;27(15): 2128-2136.
[22] Ren W, Qiang C, Gao L, et al. Circulating microRNA-21 (MIR-21) and phosphatase and tensin homolog (PTEN) are promising novel biomarkers for detection of oral squamous cell carcinoma. Biomarkers.2014;19(7): 590-596.
[23] Zhu S, Wu H, Wu F, et al. MicroRNA-21 targets tumor suppressor genes in invasion and metastasis. Cell Res. 2008; 18(3):350-359.
[24] Chen Y, Liu W, Chao T, et al. MicroRNA-21 down-regulates the expression of tumor suppressor PDCD4 in human glioblastoma cell T98G. Cancer Lett.2008;272(2):197-205.
[25] Schramedei K, Morbt N, Pfeifer G, et al. MicroRNA-21 targets tumor suppressor genes ANP32A and SMARCA4. Oncogene. 2011;30(26):2975-2985.
[26] Yao Q, Xu H, Zhang QQ, Zhou H,et al. MicroRNA-21 promotes cell proliferation and down-regulates the expression of programmed cell death 4 (PDCD4) in HeLa cervical carcinoma cells. Biochem Biophys Res Commun.2009;388 (3): 539-542.