切换至 "中华医学电子期刊资源库"

中华口腔医学研究杂志(电子版) ›› 2017, Vol. 11 ›› Issue (05) : 266 -272. doi: 10.3877/cma.j.issn.1674-1366.2017.05.002

所属专题: 文献

基础研究

沉默c-myc基因对成釉细胞瘤hTERT+-AM永生化细胞株中基质金属蛋白酶表达及侵袭能力的影响
梁启祥1, 张彬2,(), 梁衍灿2, 谢宏亮3   
  1. 1. 510630 广州,中山大学附属第三医院口腔科
    2. 510120 广州,中山大学孙逸仙纪念医院口腔颌面外科
    3. 518000 深圳市人民医院口腔科
  • 收稿日期:2017-05-31 出版日期:2017-10-01
  • 通信作者: 张彬
  • 基金资助:
    国家自然科学基金(81072229); 广东省自然科学基金(2014A030313025)

Effect of c-myc silence on MMPs expression and invasion of immortalized ameloblastoma cell line hTERT+-AM

Qixiang Liang1, Bin Zhang2,(), Yancan Liang2, Hongliang Xie3   

  1. 1. Department of Stomatology, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China
    2. Department of Oral and Maxillofacial Surgery, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China
    3. Department of Stomatology, Shenzhen People′s Hospital, Shenzhen 518000, China
  • Received:2017-05-31 Published:2017-10-01
  • Corresponding author: Bin Zhang
  • About author:
    Corresponding author: Zhang Bin, Email:
引用本文:

梁启祥, 张彬, 梁衍灿, 谢宏亮. 沉默c-myc基因对成釉细胞瘤hTERT+-AM永生化细胞株中基质金属蛋白酶表达及侵袭能力的影响[J]. 中华口腔医学研究杂志(电子版), 2017, 11(05): 266-272.

Qixiang Liang, Bin Zhang, Yancan Liang, Hongliang Xie. Effect of c-myc silence on MMPs expression and invasion of immortalized ameloblastoma cell line hTERT+-AM[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2017, 11(05): 266-272.

目的

探讨沉默癌基因c-myc对人成釉细胞瘤细胞株hTERT+-AM的基质金属蛋白酶(MMP)表达及细胞侵袭力的影响。

方法

利用慢病毒载体Lenti-shc-myc-eGFP/puro稳定转染hTERT+-AM细胞,成功构建c-myc沉默稳转细胞株。利用定量反转录聚合酶链反应(RT-PCR)检测细胞中c-mycMMP-2MMP-9的mRNA表达情况。蛋白印迹法(Western blot)检测细胞中c-myc、MMP-2、MMP-9的蛋白表达情况,利用Transwell实验检测其迁移侵袭能力的变化。所有数据采用SPSS 13.0统计软件包进行统计学分析。

结果

沉默c-myc基因后hTERT+-AM细胞中c-myc的mRNA相对表达量为0.41 ± 0.02,下降约60%(LSD-t=-0.591,P<0.001),而其蛋白的表达也下调约50%(LSD-t=-0.461,P<0.001),MMP-2MMP-9的mRNA相对表达量分别为0.79 ± 0.05和0.76 ± 0.01,均下降约20%(LSD-tMMP-2=-0.206,LSD-tMMP-9=-0.242,P<0.001),而MMP-2、MMP-9的蛋白表达均约下调了50%(LSD-tMMP-2=-0.257,LSD-tMMP-9=-0.261,P<0.001),细胞迁移(±s=24.9 ± 4.5,LSD-t=-17.000,P= 0.021)、侵袭(±s=10.8 ± 4.1,LSD-t=-22.250,P= 0.011)能力均下降。

结论

c-myc基因参与人成釉细胞瘤AM局部侵袭的调节,沉默c-myc基因后,继而下调MMP-2MMP-9,抑制AM细胞的迁移和侵袭能力,c-myc可能成为人成釉细胞瘤AM侵袭防治的新靶点。

Objective

To investigate the effect of c-myc silence on MMPs expression and invasion of immortalized ameloblastoma cell line hTERT+-AM.

Methods

Functional characteristics of immortalized ameloblastoma cell line hTERT+-AM with stable c-myc silence included migration, invasion, and regulation of matrix metalloproteinases MMP-2, MMP-9 by qRT-PCR and Western blotting. All data were analyzed by SPSS 13.0 software package.

Results

After gene silencing of c-myc in hTERT+-AM cells, the mRNA relative expression of c-myc, MMP-2 and MMP-9 was (0.41 ± 0.02) , (0.79 ± 0.05) and (0.76 ± 0.01) respectively, declined by about 60% (LSD-t=-0.591, P<0.001) and 20% (LSD-tMMP-2=-0.206, LSD-tMMP-9=-0.242, P<0.001) , the protein expression of c-myc, MMP-2 and MMP-9 was reduced by about 50% (LSD-tc-myc=-0.461, LSD-tMMP-2=-0.257, LSD-tMMP-9=-0.261, P<0.001) , the capacity of migration (±s=24.9 ± 4.5, LSD-t=-17.000, P= 0.021) and invasion (±s=10.8 ± 4.1, LSD-t=-22.250, P= 0.011) was decreased.

Conclusions

Silence of c-myc gene significantly inhibits MMPs expression and cell invasive ability of hTERT+-AM cells, suggesting c-myc may be a new target for ameloblastoma treatment.

表1 定量RT-PCR引物序列
图1 稳定沉默前后c-myc的mRNA的表达变化
图2 稳定沉默前后c-myc的蛋白表达变化
图3 稳定沉默c-myc前后MMP-2、MMP-9的蛋白表达变化
图4 稳定沉默c-myc前后MMP-2的mRNA表达变化
图5 稳定沉默c-myc前后MMP-9的mRNA表达变化
图6 稳定沉默c-myc前后hTERT+-AM细胞迁移能力的变化
图7 稳定沉默c-myc前后hTERT+-AM细胞侵袭能力的变化
[1]
Luo HY, Li TJ. Odontogenic tumors:a study of 1309 cases in a Chinese population[J]. Oral Oncol,2009,45(8):706-711.
[2]
Morgan PR. Odontogenic tumors:a review[J]. Periodontol 2000,2011,57(1):160-176.
[3]
Sampson DE, Pogrel MA. Management of mandibular ameloblastoma:the clinical basis for a treatment algorithm[J]. J Oral Maxillofac Surg,1999,57(9):1074-1077,discussion 1078-1079.
[4]
Hemann MT, Bric A, Teruya-Feldstein J,et al. Evasion of the p53 tumour surveillance network by tumour-derived MYC mutants[J]. Nature,2005,436(7052):807-811.
[5]
Shachaf CM, Felsher DW. Tumor dormancy and MYC inactivation:pushing cancer to the brink of normalcy[J]. Cancer Res,2005,65(11):4471-4474.
[6]
李自娟,钟鸣,王洁,等. c-myc mRNA在成釉细胞瘤中的表达[J].上海口腔医学,2004,13(6):515-518.
[7]
Zhang B, Zhang J, Huang HZ,et al. Expression and role of metalloproteinase-2 and endogenous tissue regulator in ameloblastoma[J]. J Oral Pathol Med,2010,39(3):219-222.
[8]
Tekkesin MS, Mutlu S, Olgac V. The role of RANK/RANKL/OPG signalling pathways in osteoclastogenesis in odontogenic keratocysts,radicular cysts,and ameloblastomas[J]. Head Neck Pathol,2011,5(3):248-253.
[9]
Kumamoto H, Yamauchi K, Yoshida M,et al. Immunohistochemical detection of matrix metalloproteinases(MMPs)and tissue inhibitors of metalloproteinases(TIMPs)in ameloblastomas[J]. J Oral Pathol Med,2003,32(2):114-120.
[10]
Tao Q, Lv B, Qiao B,et al. Immortalization of ameloblastoma cells via reactivation of telomerase function:Phenotypic and molecular characteristics[J]. Oral Oncol,2009,45(12):e239-e244.
[11]
Barsyte-Lovejoy D, Mao DY, Penn LZ. c-Myc represses the proximal promoters of GADD45a and GADD153 by a post-RNA polymerase II recruitment mechanism[J]. Oncogene,2004,23(19):3481-3486.
[12]
Fernandez PC, Frank SR, Wang L,et al. Genomic targets of the human c-Myc protein[J]. Genes Dev,2003,17(9):1115-1129.
[13]
Dews M, Homayouni A, Yu D,et al. Augmentation of tumor angiogenesis by a Myc-activated microRNA cluster[J]. Nat Genet,2006,38(9):1060-1065.
[14]
Mizukami Y, Fujiki K, Duerr EM,et al. Hypoxic regulation of vascular endothelial growth factor through the induction of phosphatidylinositol 3-kinase/Rho/ROCK and c-Myc[J]. J Biol Chem,2006,281(20):13957-13963.
[15]
Gomez-Roman N, Grandori C, Eisenman RN,et al. Direct activation of RNA polymeraseⅢ transcription by c-Myc[J]. Nature,2003,421(6920):290-294.
[16]
Haggerty TJ, Zeller KI, Osthus RC,et al. A strategy for identifying transcription factor binding sites reveals two classes of genomic c-Myc target sites[J]. Proc Natl Acad Sci U S A,2003,100(9):5313-5318.
[17]
de Alborán IM, Baena E, Martinez-A C. c-Myc-deficient B lymphocytes are resistant to spontaneous and induced cell death[J]. Cell Death Differ,2004,11(1):61-68.
[18]
Soucie EL, Annis MG, Sedivy J,et al. Myc potentiates apoptosis by stimulating Bax activity at the mitochondria[J]. Mol Cell Biol,2001,21(14):4725-4736.
[19]
Bhandari DR, Seo KW, Jung JW,et al. The regulatory role of c-MYC on HDAC2 and PcG expression in human multipotent stem cells[J]. J Cell Mol Med,2011,15(7):1603-1614.
[20]
Kaur S, Soto-Pantoja DR, Stein EV,et al. Thrombospondin-1 signaling through CD47 inhibits self-renewal by regulating c-Myc and other stem cell transcription factors[J]. Sci Rep,2013(3)1673.
[21]
Dang CV. c-Myc target genes involved in cell growth,apoptosis,and metabolism[J]. Mol Cell Biol,1999,19(1):1-11.
[22]
He C, Jiang H, Geng S,et al. Expression of c-Myc and Fas correlates with perineural invasion of pancreatic cancer[J]. Int J Clin Exp Pathol,2012,5(4):339-346.
[23]
Han G, Wang Y, Bi W. C-Myc overexpression promotes osteosarcoma cell invasion via activation of MEK-ERK pathway[J]. Oncol Res,2012,20(4):149-156.
[24]
Li ZR, Wu YF, Ma CY,et al. Down-regulation of c-Myc expression inhibits the invasion of bile duct carcinoma cells[J]. Cell Biol Int,2011,35(8):799-802.
[25]
Cho HJ, Oh YJ, Kwon J,et al. c-Myc stimulates cell invasion by inhibiting FBX8 function[J]. Mol Cells,2010,30(4):355-362.
[26]
Cui J, Dong BW, Liang P,et al. Effect of c-myc,Ki-67,MMP-2 and VEGF expression on prognosis of hepatocellular carcinoma patients undergoing tumor resection[J]. World J Gastroenterol,2004,10(10):1533-1536.
[27]
Lu Q, Hong W. Bcl2 enhances c-Myc-mediated MMP-2 expression of vascular smooth muscle cells[J]. Cell Signal,2009,21(7):1054-1059.
[28]
Hatakeyama S, Watanabe M, Fujii Y,et al. Targeted destruction of c-Myc by an engineered ubiquitin ligase suppresses cell transformation and tumor formation[J]. Cancer Res,2005,65(17):7874-7879.
[29]
Zhang B, Zhang J, Huang HZ,et al. Inhibition of ameloblastoma invasion in vitro and in vivo by inhibitor of metalloproteinase-2 activity[J]. J Oral Pathol Med,2009,38(9):731-736.
[1] 祝雅, 赵晖, 董莲莲, 金燕. 亚低温治疗对心肺复苏后兔脑组织中基质金属蛋白酶9和神经元特异性烯醇化酶水平及脑复苏的影响[J]. 中华危重症医学杂志(电子版), 2021, 14(05): 374-379.
[2] 孙清华, 尹格平, 陈铭, 武爱芳, 袁峥, 梁静. 子宫内膜癌中血管生成拟态及基质金属蛋白酶-2表达情况研究[J]. 中华妇幼临床医学杂志(电子版), 2016, 12(01): 29-34.
[3] 马丽千, 唐富波, 王海滨, 李琰光, 张慧苹, 胡森. 丁酸钠对50%总体表面积烫伤大鼠肺血管内皮糖萼层保护作用和机制研究[J]. 中华损伤与修复杂志(电子版), 2020, 15(01): 37-44.
[4] 王越, 李贤, 王瑶, 高占红, 赵伟, 孙素娟, 陈紫叶, 张超. Ⅳ期压疮愈合过程中创面渗出液基质金属蛋白酶-9、基质金属蛋白酶抑制剂-1的表达水平变化的研究[J]. 中华损伤与修复杂志(电子版), 2019, 14(01): 34-38.
[5] 任国强, 李炳辉, 李恭驰, 张静, 祝友鹏, 邹新华. 糖尿病创面基质金属蛋白酶9对血管内皮生长因子表达的影响[J]. 中华损伤与修复杂志(电子版), 2017, 12(02): 123-127.
[6] 牛丽佳, 夏义欣, 宋琪, 张晓莉. 足月胎膜早破孕妇羊水中β人绒毛膜促性腺激素、基质金属蛋白酶9及核苷酸结合寡聚化结构域受体2水平变化及与羊膜感染的相关性[J]. 中华实验和临床感染病杂志(电子版), 2021, 15(05): 311-316.
[7] 欧发荣, 廖贵清, 纪焕中, 张深榕, 苏凯. 下颌骨转移性成釉细胞瘤伴右颌下区淋巴结转移一例[J]. 中华口腔医学研究杂志(电子版), 2022, 16(05): 309-312.
[8] 肖路, 薛同敏, 王敏, 刘伟, 张攀, 张培建. 缺氧调控人肝癌细胞核转录因子NF-κBp65/基质金属蛋白酶9表达相关机制的研究[J]. 中华普通外科学文献(电子版), 2015, 09(04): 276-279.
[9] 尚培中, 李晓武, 蔡大伟, 赵伟男, 温艳春, 谷化平. 基质金属蛋白酶9在胰腺癌组织中的表达及其临床意义[J]. 中华普通外科学文献(电子版), 2013, 07(06): 436-440.
[10] 刘立, 陈诚, 李新科, 刘凯, 屠昌明. 血清IL-6、hs-CRP、MMP-9联合检测在腹股沟疝无张力修补术预后评价中的价值分析[J]. 中华疝和腹壁外科杂志(电子版), 2023, 17(04): 405-409.
[11] 黄潘文, 张平, 方年新. 噻托溴铵对COPD患者痰液炎症细胞计数、炎症因子及MMP-9水平的影响[J]. 中华肺部疾病杂志(电子版), 2019, 12(03): 339-341.
[12] 梁美莲, 陈小燕, 张凡, 潘兴飞. IL-32γ活化Akt通路诱导肝星状细胞LX-2表达MMP2[J]. 中华肝脏外科手术学电子杂志, 2017, 06(04): 328-331.
[13] 吴震宇, 胡亚芬, 董晓芬, 马远方. 血清CTGF、TGF-β1、MMP2水平对糖尿病肾病肾间质纤维化的预测分析[J]. 中华肾病研究电子杂志, 2022, 11(06): 332-337.
[14] 杜非凡, 王雪, 吴志鸿. 基质金属蛋白酶2、基质金属蛋白酶抑制剂2和可溶性CD44在高度近视眼合并原发性开角型青光眼患者房水中的定量研究[J]. 中华眼科医学杂志(电子版), 2019, 09(06): 335-341.
[15] 杨雨鑫, 梁育飞, 史永红, 郑国启. MMP-9和EGFR在上皮型恶性腹膜间皮瘤的表达及意义[J]. 中华消化病与影像杂志(电子版), 2017, 07(03): 108-113.
阅读次数
全文


摘要