国际口腔医学杂志

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
  • 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
  • Supported by:
    This study was supported by National Natural Science Foundation of China(81502356);Science and Technology Program of Guangzhou(201804010040)

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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。"

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