国际口腔医学杂志 ›› 2018, Vol. 45 ›› Issue (6): 628-634.doi: 10.7518/gjkq.2018.06.002

• RNA专栏 • 上一篇    下一篇

长链非编码RNA lnc-p26090对口腔鳞状细胞癌细胞糖酵解及增殖的影响

李媛媛,程斌,王韵()   

  1. 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 广州 510055
  • 收稿日期:2018-04-02 修回日期:2018-08-10 出版日期:2018-11-01 发布日期:2018-11-15
  • 通讯作者: 王韵 E-mail:wangyun23@mail.sysu.edu.cn
  • 作者简介:李媛媛,学士,Email: liyuany6@mail2.sysu.edu.cn
  • 基金资助:
    国家自然科学基金(81502356);广州市科技计划项目(201804010040)

Effects of long non-coding RNA lnc-p26090 on the glycolysis and proliferation in oral squamous cell carcinoma

Yuanyuan Li,Bin Cheng,Yun Wang()   

  1. Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China
  • Received:2018-04-02 Revised:2018-08-10 Online:2018-11-01 Published:2018-11-15
  • Contact: Yun Wang E-mail:wangyun23@mail.sysu.edu.cn
  • Supported by:
    This study was supported by National Natural Science Foundation of China(81502356);Science and Technology Program of Guangzhou(201804010040)

摘要:

目的 检测长链非编码RNA lnc-p26090在口腔鳞状细胞癌(OSCC)组织及细胞系中的表达情况,探讨lnc-p26090对OSCC细胞糖酵解和生长增殖的影响。方法 利用荧光实时定量聚合酶链式反应(RT-qPCR)技术检测OSCC组织及细胞系中lnc-p26090的表达水平;利用UCSC和LNCipedia数据库网站获取lnc-p26090的染色体定位信息及蛋白质编码潜能,并在HEK 293T细胞中转染重组质粒验证其蛋白质编码能力。在OSCC细胞系HSC-3和SCC-25中转染lnc-p26090后,采用葡萄糖/乳酸测定试剂盒及RT-qPCR技术检测细胞糖酵解能力改变,同时采用细胞计数试剂盒、流式细胞术及蛋白质印迹法技术检测细胞生长增殖能力改变。结果 与正常口腔角化上皮细胞及配对的癌旁组织相比,OSCC细胞系及癌组织中lnc-p26090的表达水平显著上调(P<0.05)。通过数据库查询及重组质粒转染验证,lnc-p26090是位于人类染色体2p11.2上的长链非编码RNA。在HSC-3及SCC-25细胞中转染lnc-p26090,可显著抑制细胞葡萄糖消耗量和乳酸产生量,同时细胞中糖酵解相关基因M2型丙酮酸激酶、人磷酸果糖激酶、己糖激酶2、人葡萄糖转运蛋白1和乳酸脱氢酶A基因的表达显著降低。另外,转染lnc-p26090后细胞增殖水平也受到显著抑制,同时细胞被阻滞在G1期,细胞中周期相关蛋白G1/S-特异性周期蛋白D1和人周期素依赖性激酶4表达显著降低,p21表达显著升高。结论 lnc-p26090在OSCC组织及细胞系中表达上调,是位于人类染色体2p11.2上的长链非编码RNA,并参与调控OSCC细胞糖酵解和生长增殖过程。

关键词: 口腔鳞状细胞癌, 长链非编码RNA, 糖酵解, 细胞增殖

Abstract:

Objective To investigate the long non-coding RNA lnc-p26090 expression level in oral squamous cell carcinoma (OSCC) tissues and cell lines. To explore the effect of lnc-p26090 on OSCC cells glycolysis and proliferation.Methods Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the relative expression level of lnc-p26090 in OSCC tissues and cell lines. Then we searched the UCSC and LNCipedia websites to obtain the chromosome location and protein coding capacity of lnc-p26090. Moreover, we transfected the plasmid into HEK 293T cell to confirm the protein coding capacity of lnc-p26090. After silencing lnc-p26090 in OSCC cell lines, we detected the glucose consumption, lactic acid production and expression level of glycolysis related genes. Furthermore, cell counting kit-8 (CCK-8) assay, flow cytometry and western blot were performed to examine the effect of lnc-p26090 on proli-feration and cell cycle distribution.Results lnc-p26090 was highly expressed both in OSCC tissues and cell lines. lnc-p26090 located in chro-mosome 2p11.2, without protein coding capacity. Silencing lnc-p26090 significantly repressed the glucose consumption, lactic acid production of cells. Moreover, the expression level of glycolysis related genes: pyruvate kinase M2, phosphofructokinase M, hexokinase 2, glucose transporter 1 and lactate dehydrogenase A were decreased after lnc- p26090 knockdown in cells. Furthermore, when downregulating lnc-p26090 in cells, proliferation was inhibited, and cell-cycle was blocked in G1 phase. At the meanwhile, cyclin D1 and cyclin-dependent kinase 4 expression level was decreased and p21 was increased significantly.Conclusion lnc-p26090, which located in chromosome 2p11.2, was upregulated in OSCC tissues and cell lines. It could be involved in the glycolysis and proliferation of OSCC.

Key words: oral squamous cell carcinoma, long non-coding RNA, glycolysis, proliferation

中图分类号: 

  • R739.8

表 1

RT-qPCR引物及扩增产物信息"

基因 上游引物(5’- 3’) 下游引物(5’- 3’) 扩增产物大小/bp
lnc-p26090 TGCCCTGTGATTATCCGCAAAC CAGATGGCGGGAAGATGAAGAC 124
PKM2 CCACTTGCAATTATTTGAGGAA GTGAGCAGACCTGCCAGACT 153
PFKM GCCAGTCTAATTGCCGTTCC TACCAACTCGAACCACAGCC 174
HK2 TCAGATTGAGAGTGACTGCC TTTCTCGTATCCTGTCCACC 179
GLUT1 CCATCCTTCCTGCTATCCTAC GACATCCTTGCACTCTCATC 178
LDHA GTGGCTTGGAAGATAAGTGG CATACAGGCACACTGGAATC 167
GAPDH ACCACAGTCCATGCCATCAC TCCACCACCCTGTTGCTGTA 165

图 1

lnc-p26090的表达情况 A:在NOK细胞及OSCC细胞中的表达,*P<0.05,**P<0.01,***P<0.001;B:在14例OSCC组织及其癌旁组织中的表达。"

图 2

lnc-p26090在染色体中的定位及编码蛋白质能力A:UCSC网站显示lnc-p26090在人类染色体上的具体定位;B:LNCipedia网站分析lnc-p26090编码蛋白质潜能;C:HEK293T细胞转染3种重组质粒后GFP的表达情况。"

图 3

lnc-p26090对HSC-3、SCC-25细胞糖酵解的影响 A:转染效率;B:转染对细胞糖酵解相关基因表达的影响;C:转染后细胞的葡萄糖消耗量和乳酸产量;*P<0.05,**P<0.01,***P<0.001。"

图 4

lnc-p26090对HSC-3、SCC-25细胞生长增殖的影响 A:CCK-8法检测结果;B:流式细胞术检测结果;C:蛋白质印迹法检测结果;*P<0.05,**P<0.01,***P<0.001。"

[1] Choi S, Myers JN . Molecular pathogenesis of oral squamous cell carcinoma: implications for therapy[J]. J Dent Res, 2008,87(1):14-32.
doi: 10.1177/154405910808700104 pmid: 18096889
[2] Ayala FR, Rocha RM, Carvalho KC , et al. GLUT1 and GLUT3 as potential prognostic markers for oral squamous cell carcinoma[J]. Molecules, 2010,15(4):2374-2387.
doi: 10.3390/molecules15042374 pmid: 20428049
[3] Wang Y, Zhang X, Zhang Y , et al. Overexpression of pyruvate kinase M2 associates with aggressive clini-copathological features and unfavorable pro-gnosis in oral squamous cell carcinoma[J]. Cancer Biol Ther, 2015,16(6):839-845.
doi: 10.1080/15384047.2015.1030551 pmid: 25970228
[4] Bergmann JH, Spector DL . Long non-coding RNAs: modulators of nuclear structure and function[J]. Curr Opin Cell Biol, 2014,26:10-18.
doi: 10.1016/j.ceb.2013.08.005
[5] Cech TR, Steitz JA . The noncoding RNA revolution-trashing old rules to forge new ones[J]. Cell, 2014,157(1):77-94.
doi: 10.1016/j.cell.2014.03.008 pmid: 24679528
[6] Martens-Uzunova ES, Böttcher R, Croce CM , et al. Long noncoding RNA in prostate, bladder, and kid-ney cancer[J]. Eur Urol, 2014,65(6):1140-1151.
doi: 10.1016/j.eururo.2013.12.003 pmid: 24373479
[7] Zhang X, Li Y, Li X , et al. Long non-coding RNA P4713 contributes to the malignant phenotypes of oral squamous cell carcinoma by activating the JAK/STAT3 pathway[J]. Int J Clin Exp Pathol, 2017,10(11):10947-10958.
[8] Hanahan D, Weinberg RA . Hallmarks of cancer: the next generation[J]. Cell, 2011,144(5):646-674.
doi: 10.1016/j.cell.2011.02.013 pmid: 21376230
[9] El Mjiyad N, Caro-Maldonado A, Ramírez-Peinado S , et al. Sugar-free approaches to cancer cell killing[J]. Oncogene, 2011,30(3):253-264.
doi: 10.1038/onc.2010.466 pmid: 20972457
[10] Levine AJ, Puzio-Kuter AM . The control of the meta- bolic switch in cancers by oncogenes and tumor sup-pressor genes[J]. Science, 2010,330(6009):1340-1344.
doi: 10.1126/science.1193494
[11] Gammon L, Biddle A, Heywood HK , et al. Sub-sets of cancer stem cells differ intrinsically in their pat-terns of oxygen metabolism[J]. PLoS One, 2013,8(4):e62493.
doi: 10.1371/journal.pone.0062493 pmid: 23638097
[12] Bochenek G, Häsler R, El Mokhtari NE , et al. The large non-coding RNA ANRIL, which is associated with atherosclerosis, periodontitis and several forms of cancer, regulates ADIPOR1, VAMP3 and C11ORF10[J]. Hum Mol Genet, 2013,22(22):4516-4527.
doi: 10.1093/hmg/ddt299
[13] Goncalves MD, Cantley LC . A glycolysis outsider steps into the cancer spotlight[J]. Cell Metab, 2018,28(1):3-4.
doi: 10.1016/j.cmet.2018.06.017
[14] Coelho RG, Fortunato RS, Carvalho DP . Metabolic reprogramming in thyroid carcinoma[J]. Front Oncol, 2018,8:82.
doi: 10.3389/fonc.2018.00082
[15] Rupaimoole R, Lee J, Haemmerle M , et al. Long noncoding RNA ceruloplasmin promotes cancer growth by altering glycolysis[J]. Cell Rep, 2015,13(11):2395-2402.
doi: 10.1016/j.celrep.2015.11.047 pmid: 4691557
[16] Bian Z, Zhang J, Li M , et al. LncRNA-FEZF1-AS1 promotes tumor proliferation and metastasis in co-lorectal cancer by regulating PKM2 signaling[J]. Clin Cancer Res, 2018. doi: 10.1158/1078-0432.CCR-17- 2967.
doi: 10.1158/1078-0432.CCR-17-2967 pmid: 29914894
[17] Johnson DG, Walker CL . Cyclins and cell cycle checkpoints[J]. Annu Rev Pharmacol Toxicol, 1999,39:295-312.
doi: 10.1146/annurev.pharmtox.39.1.295 pmid: 10331086
[18] Bloom J, Cross FR . Multiple levels of cyclin speci-ficity in cell-cycle control[J]. Nat Rev Mol Cell Biol, 2007,8(2):149-160.
doi: 10.1038/nrm2105 pmid: 17245415
[19] Yao F, Zhao T, Zhong C , et al. LDHA is necessary for the tumorigenicity of esophageal squamous cell carcinoma[J]. Tumour Biol, 2013,34(1):25-31.
doi: 10.1007/s13277-012-0506-0 pmid: 22961700
[20] Xiao H, Wang J, Yan W , et al. GLUT1 regulates cell glycolysis and proliferation in prostate cancer[J]. Prostate, 2018,78(2):86-94.
doi: 10.1002/pros.23448 pmid: 29105798
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