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中华口腔医学研究杂志(电子版) ›› 2015, Vol. 09 ›› Issue (02) : 106 -112. doi: 10.3877/cma.j.issn.1674-1366.2015.02.004

所属专题： 文献；

基础研究

微小RNA-221/222海绵体载体的构建及验证
周李杰1, 赵玮1, 江方方1, 刘子锋1, 欧阳颖1, 余东升1,†()   
  1. 1. 510055　广州，中山大学光华口腔医学院·附属口腔医院，广东省口腔医学重点实验室
  • 收稿日期:2014-09-09 出版日期:2015-04-01
  • 通信作者: 余东升
  • 基金资助:
    国家自然科学基金(81272554、81472526); 广东省自然科学基金(9151008901000187、S20110200 03247); 广东省科技计划国际合作项目(2011B050400030、2012B031800387)

Construction and validation of microRNA-221/222 sponge vector

Lijie Zhou1, Wei Zhao1, Fangfang Jiang1, Zifeng Liu1, Ying Ou-Yang1, Dongsheng Yu1,†()   

  1. 1. Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China
  • Received:2014-09-09 Published:2015-04-01
  • Corresponding author: Dongsheng Yu
  • About author:
    Corresponding author: Yu Dongsheng, Email: , Tel: 020-83862543
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引用本文：

周李杰, 赵玮, 江方方, 刘子锋, 欧阳颖, 余东升. 微小RNA-221/222海绵体载体的构建及验证[J]. 中华口腔医学研究杂志(电子版), 2015, 09(02): 106-112.

Lijie Zhou, Wei Zhao, Fangfang Jiang, Zifeng Liu, Ying Ou-Yang, Dongsheng Yu. Construction and validation of microRNA-221/222 sponge vector[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2015, 09(02): 106-112.

目的

构建微小RNA（miR）-221/222海绵体载体并初步探讨其在口腔鳞状细胞癌（OSCC）细胞中的作用。

方法

利用miRNA海绵体技术设计合成含3个has-miR-221和3个has-miR-222反义序列的DNA片段，经酶切连入pDC316-mCMV-EGFP质粒载体，合成miR-221/222海绵体载体。将miR-221/222海绵体载体转染入OSCC细胞系UM1，检测转染效率，实时荧光定量聚合酶链反应（PCR）检测miR-221和miR-222表达水平，Western blot检测转染后PTEN蛋白表达水平，Transwell实验检测细胞的侵袭能力。

结果

成功构建miR-221/222海绵体载体，测序验证与设计序列完全一致；UM1各组细胞转染效率均在70%以上；实时荧光定量PCR显示，miR-221/222海绵体载体组miR-221和miR-222表达水平较pDC316质粒组和UM1细胞组明显降低（t1 = 111.69，P1 = 0.001；t2 = 6.98，P2 = 0.002；t3 = 236.55，P3 = 0.001；t4 = 22.06，P4 = 0.001）；Western blot显示，海绵体转染组较对照组PTEN蛋白水平表达明显升高；Transwell实验显示，海绵体转染组细胞侵袭能力较对照组减弱。

结论

实验成功构建了miR-221/222海绵体载体，并初步验证其对OSCC细胞侵袭性的抑制作用，为后续miR-221/222的功能研究奠定基础。

关键词: 微小RNA, 海绵体载体, 第10号染色体缺失的磷酸酶及张力蛋白同源物基因, 癌，鳞状细胞
Objective

To construct microRNA (miR) -221/222 sponge vector and investigate the effect of sponge transfection in oral squamous cell carcinoma cells.

Methods

Using the technology of miRNA sponge, a DNA fragment contained three has-miR-221 and three has-miR-222 antisense sequences was designed and synthesized. The DNA fragment was cloned into the pDC316-mCMV-EGFP plasmid vector by enzyme digestion to construct the miR-221/222 sponge vector. Then the miR-221/222 sponge vector was transfected into UM1 cells, and the transfection efficiency was detected. Real time quantitative PCR was used to test the expression level of the miR-221 and miR-222. The protein expression of phosphatase and tension homolog deleted on chromosome 10 (PTEN) was analyzed by Western blot and Transwell assay to measure the invasion ability of cells.

Results

The miR-221/222 sponge vector was constructed successfully, and it was validated to be entirely consistent with the designed sequence. The transfection efficiency of UM1 cells in each group was more than 70%. The results of real time quantitative PCR showed that the expression levels of the miR-221 and miR-222 in sponge transfected group were decreased compared to the positive control group and the negative control group (t1 = 111.69, P1 = 0.001; t2 = 6.98, P2 = 0.002; t3 = 236.55, P3 = 0.001; t4 = 22.06, P4 = 0.001) . Western blot showed that the expression of PTEN in the transfected sponge group was significantly increased compared with mock group and non-transfected group. Transwell assay indicated that cell invasion was inhibited by miR-221/222 sponge transfection.

Conclusions

The miR-221/222 sponge vector was successfully constructed and its inhibitory effect of the invasion of OSCC cells has been validated. This lays the foundation for further study on the function of miR-221/222.

Key words: MicroRNA, Sponge vector, Phosphatase and tension homolog deleted on chromosome 10, Carcinoma, squamous cell
图1 miR-221/222海绵体序列模式图
图2 miR-221/222海绵体载体测序图
图3 UM1细胞转染pDC316-mCMV-EGFP质粒和海绵体载体转染效率检测（倒置显微镜）
图4 各组细胞中miR-221和miR-222的相对表达量
图5 各组细胞转染后PTEN、pAkt、Akt和GAPDH蛋白表达水平
图6 各组细胞转染后细胞侵袭能力比较（结晶紫× 100）
[1]
Garofalo M,Quintavalle C,Romano G, et al. miR221/222 in cancer: their role in tumor progression and response to therapy[J]. Curr Mol Med, 2012, 12(1): 27-33.
[2]
Chen JC,Su YH,Chiu CF, et al. Suppression of Dicer increases sensitivity to gefitinib in human lung cancer cells[J]. Ann Surg Oncol, 2014, 21(Suppl 4): S555-S563.
[3]
Wang M,Zhao C,Shi H, et al. Deregulated microRNAs in gastric cancer tissue-derived mesenchymal stem cells: novel biomarkers and a mechanism for gastric cancer[J]. Br J Cancer, 2014, 110(5): 1199-1210.
[4]
Hwang MS,Yu N,Stinson SY, et al. miR-221/222 targets adiponectin receptor 1 to promote the epithelial-to-mesenchymal transition in breast cancer[J]. PLoS One, 2013, 8(6): e66502.
[5]
Chen Y,Zaman MS,Deng G, et al. MicroRNAs 221/222 and genistein-mediated regulation of ARHI tumor suppressor gene in prostate cancer[J]. Cancer Prev Res(Phila), 2011, 4(1): 76-86.
[6]
Quintavalle C,Mangani D,Roscigno G, et al. MiR-221/222 target the DNA methyltransferase MGMT in glioma cells[J]. PLoS One, 2013, 8(9): e74466.
[7]
Dettmer MS,Perren A,Moch H, et al. MicroRNA profile of poorly differentiated thyroid carcinomas: new diagnostic and prognostic insights[J]. J Mol Endocrinol, 2014, 52(2): 181-189.
[8]
Rommer A,Steinleitner K,Hackl H, et al. Overexpression of primary microRNA 221/222 in acute myeloid leukemia[J]. BMC Cancer, 2013(13): 364.
[9]
江方方,赵玮,胡逢春, 等.反义miR-222靶向上调PUMA基因表达促进口腔鳞状细胞癌凋亡[J/CD].中华口腔医学研究杂志:电子版, 2013, 7(3): 201-206.
[10]
Jiang F,Zhao W,Zhou L, et al. miR-222 regulates the cell biological behavior of oral squamous cell carcinoma by targeting PUMA[J]. Oncol Rep, 2014, 31(3): 1255-1262.
[11]
Zhang CZ,Han L,Zhang AL, et al. MicroRNA-221 and microRNA-222 regulate gastric carcinoma cell proliferation and radioresistance by targeting PTEN[J]. BMC Cancer, 2010(10): 367.
[12]
Ebert MS,Neilson JR,Sharp PA. MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells[J]. Nat Methods, 2007, 4(9): 721-726.
[13]
Calin GA,Sevignani C,Dumitru CD, et al. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers[J]. Proc Natl Acad Sci U S A, 2004, 101(9): 2999-3004.
[14]
Kozomara A,Griffiths-Jones S. miRBase: annotating high confidence microRNAs using deep sequencing data[J]. Nucleic Acids Res, 2014, 42(Database issue): D68-D73.
[15]
Friedman RC,Farh KK,Burge CB, et al. Most mammalian mRNAs are conserved targets of microRNAs[J]. Genome Res, 2009, 19(1): 92-105.
[16]
Kim SY,Nam SY,Choi S, et al. Prognostic value of lymph node density in node-positive patients with oral squamous cell carcinoma[J]. Ann Surg Oncol, 2011, 18(8): 2310-2317.
[17]
DE Lima PO,Jorge CC,Oliveira DT, et al. Hypoxic condition and prognosis in oral squamous cell carcinoma[J]. Anticancer Res, 2014, 34(2): 605-612.
[18]
李劲松,黄洪章,潘朝斌, 等.微小RNA-21反义寡核苷酸对舌鳞癌细胞增殖和凋亡的调控作用[J/CD].中华口腔医学研究杂志:电子版, 2009, 3(2): 140-150.
[19]
Gombos K,Horváth R,Szele E, et al. miRNA expression profiles of oral squamous cell carcinomas[J]. Anticancer Res, 2013, 33(4): 1511-1517.
[20]
Yang CJ,Shen WG,Liu CJ, et al. miR-221 and miR-222 expression increased the growth and tumorigenesis of oral carcinoma cells[J]. J Oral Pathol Med, 2011, 40(7): 560-566.
[21]
Krützfeldt J,Rajewsky N,Braich R, et al. Silencing of microRNAs in vivo with 'antagomirs’[J]. Nature, 2005, 438(7068): 685-689.
[22]
Ebert MS,Sharp PA. MicroRNA sponges: progress and possibilities[J]. RNA, 2010, 16(11): 2043-2050.
[23]
Kluiver J,Slezak-Prochazka I,Smigielska-Czepiel K, et al. Generation of miRNA sponge constructs[J]. Methods, 2012, 58(2): 113-117.
[24]
Liu Y,Cui H,Wang W, et al. Construction of circular miRNA sponges targeting miR-21 or miR-221 and demonstration of their excellent anticancer effects on malignant melanoma cells[J]. Int J Biochem Cell Biol, 2013, 45(11): 2643-2650.
[25]
Xie Q,Yan Y,Huang Z, et al. MicroRNA-221 targeting PI3-K/Akt signaling axis induces cell proliferation and BCNU resistance in human glioblastoma[J]. Neuropathology, 2014, 34(5): 455-464.
[26]
Wang H,Xu C,Kong X, et al. Trail resistance induces epithelial-mesenchymal transition and enhances invasiveness by suppressing PTEN via miR-221 in breast cancer[J]. PLoS One, 2014, 9(6): e99067.
[27]
Li J,Yen C,Liaw D, et al. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer[J]. Science, 1997, 275(5308): 1943-1947.
[28]
Calderaro J,Rebouissou S,de Koning L, et al. PI3K/AKT pathway activation in bladder carcinogenesis[J]. Int J Cancer, 2014, 134(8): 1776-1784.
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