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2018, 35(6):479-484.doi:10.13213/j.cnki.jeom.2018.17712

Mechanism of LincRNA-p21 on inhibiting proliferation of esophageal cancer cells

Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing, Jiangsu 210009, China

Accepted: 2017-12-05;  Published: 2018-07-06

Corresponding Author: YIN Li-hong, Email: lhyin@seu.edu.cn  

[Objective] To investigate the underlying mechanisms of long intergenic non-coding RNA (LincRNA)-p21 on the inhibition of esophageal cancer cell proliferation.

[Methods] The expression of LincRNA-p21 in two esophageal cancer cell lines (EC109 and EC9706), a human immortalized esophageal epithelial cell line (Het-1A), and the tissues and paired adjacent non-tumor mucosa of 64 cases of esophageal cancer were detected by real-time quantitative reverse-transcription polymerase chain reaction (RT-qPCR). After being transfected successfully with lentivirus packaging full-length LincRNA-p21 gene, the cell cycle distribution of EC109 cells was detected by flow cytometry, and the proliferation of EC109 cells by EdU staining. Meanwhile, RT-qPCR and Western blot were used to detect the mRNA and protein expression levels of p21 and the protein expression level of cyclin D.

[Results] The LincRNA-p21 expression levels were 0.18 and 0.32 folds in EC109 and EC9706 cells as that in Het-1A cells, respectively (P < 0.05), and the p21 mRNA levels were 0.42 and 0.48 folds, respectively. After transfection with lentivirus, the relative expression level of LincRNA-p21 increased by about 14 860 folds (P < 0.05) and that of p21-mRNA increased by 1.83 folds (P < 0.05). The results of flow cytometry showed that in the LincRNA-p21 transfection group, cells in G1 phase increased, cells in S phase and G2 phase decreased (P < 0.05), and the proliferation index was lower than that in the negative control group (40.4% vs. 48.2%) (P < 0.05). The results of EdU staining showed that the cell proliferation rate decreased after up-regulating LincRNA-p21. The results of Western blot showed that overexpression of LincRNA-p21 down-regulated the protein expression level of cyclin D.

[Conclusion] LincRNA-p21 promotes the expression of p21 and inhibits the proliferation of human esophageal cancer EC109 cells by inhibiting the expression of cyclin D and contributing to G1/S arrest.

Key Words: esophageal cancer;  long non-coding RNA;  LincRNA-p21 p21 G1/S arrest 

表 1


Table 1 Primer sequences for RT-qPCR analysis

图 1


Figure 1 p21 protein levels in esophageal cancer cells and immortalized esophageal epithelial cells

图 2


Figure 2 Transfection of LincRNA-p21 under microscope

[注]A:转染组;B:阴性对照组。1:倒置相差显微镜;2:荧光显微镜。 [Note] A: Transfection group; B: Negative control group. 1: Inverted phase contrast microscope; 2: Fluorescence microscope.
图 3


Figure 3 Effect of LincRNA-p21 on p21 protein expression

[注]A:阴性对照组;B:LincRNA-p21过表达组。 [Note]A: Negative control group; B: LincRNA-p21 overexpression group.
图 4


Figure 4 EdU staining assay for cell proliferation (fluorescence microscope, ×200)

[注]A:阴性对照组;B:LincRNA-p21过表达组。1:EdU;2:DAPI;3:重叠。红色荧光表示增殖期细胞,蓝色荧光表示活细胞。 [Note] A: Negative control group; B: LincRNA-p21 overexpression group. 1: EdU; 2: DAPI; 3: Overlay. Red fluorescence indicates proliferating cells and blue fluorescence indicates live cells.
表 2


Table 2 Effect of LincRNA-p21 on the cell cycle distribution of esophageal cancer cells

图 5

LincRNA-p21cyclin D蛋白表达的影响

Figure 5 Effect of LincRNA-p21 on the protein expression of cyclin D

[注]A:阴性对照组;B:LincRNA-p21过表达组。 [Note]A: Negative control group; B: LincRNA-p21 overexpression group.



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食管癌是一种常见的恶性肿瘤,位居全球死因顺位第六位,我国恶性肿瘤发病第四位[1-4]。食管癌患者的总体五年生存率为15%~25% [1],其预后差,五年生存率低,对人类健康的危害极大。

基因间的长链非编码RNA(long intergenic noncoding RNA,LincRNA)-p21是p53基因下游的转录本,是近年研究较多的LincRNA之一,位于细胞周期调节基因p21/Cdkn1A上游约15 kb处,长度约3.0 kb,是细胞增殖、凋亡和DNA损伤应答的调节因子[5-6]。p21蛋白是p53下游的细胞周期蛋白依赖性激酶(cyclindependent protein kinases,CDK)抑制剂,p21蛋白可以抑制cyclin D1-CDK4和cyclin E-CDK2复合物的活性,进而阻止成视网膜细胞瘤蛋白的磷酸化,导致细胞周期阻滞在G1期[7],从而抑制肿瘤的发生。食管癌与p21表达关系密切[8]。多项研究表明LincRNA-p21可能与多种恶性肿瘤(结肠癌、肝癌、非小细胞肺癌、膀胱癌、皮肤癌等)的增殖、侵袭、转移等密切相关[9-13],但是LincRNA-p21与食管癌的研究尚无报道。本研究检测了食管癌细胞EC109、EC9706和永生化食管上皮细胞Het-1A、食管癌组织及癌旁组织中LincRNA-p21水平,并将携带LincRNA-p21基因全长的缺陷型慢病毒载体转染EC109细胞,研究稳转细胞株Ad-LincRNA-p21-EC109中LincRNA-p21p21的表达,观察细胞功能变化,探讨LincRNA-p21通过p21调控细胞周期抑制EC109细胞增殖的机制。

1   材料与方法

1.1   细胞


1.2   组织样本


1.3   主要仪器


1.4   主要试剂


1.5   慢病毒转染

将EC109置于含100 U/mL青霉素、100 μg/mL链霉素,10%(体积分数)胎牛血清的RPMI-1640培养液中,于37℃、5%(体积分数)的CO2细胞培养箱常规培养。EC109细胞培养至对数期,以细胞密度5×104个/孔接种于12孔板中,每孔1 mL完全培养基,继续培养24 h。按照转染复数(multiplicity of infection,MOI)=20计算需要的病毒量,携带LincRNA-p21基因和空载体的慢病毒液各10 μL,RPMI-1640稀释助染剂至6 μg/mL,分别转染EC109细胞[转染时使用1%(体积分数)的FBS培养基]。培养8~12 h,观察细胞状态并更换新鲜的完全培养基,转染36~96 h。实时定量逆转录-聚合酶链反应(RT-qPCR)验证转染效率。慢病毒载体上携带绿色荧光蛋白(GFP)基因,LincRNA-p21基因进入细胞内表达时会发出绿色荧光,采用流式仪计数带荧光的细胞。

1.6   总RNA提取

细胞培养至对数期,PBS清洗3遍,800×g离心5 min,收集细胞,200 μL PBS重悬,加入1mL Trizol,吹打裂解细胞,室温静置5min,加入氯仿200μL,颠倒混匀,涡旋振荡15s,于4℃,12000×g离心10min,取等体积上层水相与异丙醇(4℃预冷)至新EP管,轻柔颠倒混匀,室温10min,12000×g离心10min;弃上清,75%乙醇1 mL洗涤(2次),4℃,800×g离心5 min,弃上清,室温干燥5 min,无酶水溶解RNA,控制RNA含量在2 000 ng/μL。

取0.5cm3组织剪碎,放入1.5mL EP管中,加入1mL的Trizol,置于组织研磨机研磨(70 Hz,10 min);室温静置5 min,后续步骤同细胞RNA提取。

1.7   RNA逆转录

PCR小管中依次加入5×逆转录酶混合物4 μL,1 μg总RNA,补无酶水至总体积20 μL。反应条件:37℃、15 min,85℃、5 s,4℃保存。

1.8   RT-qPCR分析LincRNA-p21p21表达水平

LincRNA-p21p21设计特异性引物(见表 1β-actin为内参),将反转录成的cDNA进行荧光定量分析。反应体系包括:SYBR Premix Ex TaqII 10 μL,Rox Reference Dye 0.4 μL,10 μmol/L引物各0.8 μL,cDNA 2 μL,无酶水补足体积至20 μL。反应条件:95℃预变性30 s,95℃、5 s,60℃、30 s,40个循环。



Table1.Primer sequences for RT-qPCR analysis

1.9   细胞增殖测定


1.10   细胞周期检测

细胞培养至对数期,收集单细胞悬液,PBS洗涤2次后,加入70%(体积分数)的预冷乙醇,于4℃固定过夜,离心收集细胞,1 mL PBS洗细胞1次,100 μg/mL RNase A 100 μL、37 ℃水浴30 min,加入400 μL PI染色混匀,室温避光孵育30 min,1 h内用流式细胞仪分析细胞周期。计算增殖指数=(S+G2)/(G1+S+G2)×100%。

1.11   蛋白表达测定

采用Western blot法检测p21蛋白和cyclin D蛋白。稳转的EC109培养至对数期,消化计数1×107个细胞,离心收集(2 000×g,5 min),预冷的PBS洗2次,转至1.5 mL EP管,顺序加入放射免疫沉淀法裂解试剂1 mL、蛋白酶抑制剂10 μL、磷酸酶抑制剂1 μL。涡旋15 s,混匀,冰上裂解15 min后,于4℃、12 000×g离心10 min。将上清转移至新的预冷EP管。二喹啉甲酸试剂盒测定蛋白浓度,加入适量6×十二烷基硫酸钠上样缓冲液煮沸变性。将等量的蛋白质(20 μg)加入到10%(质量浓度)十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中,然后转移至聚偏二氟乙烯膜上,在5%(质量浓度)脱脂乳中封闭2 h,一抗(1:1 000)稀释,4℃孵育过夜,二抗(1:5 000)稀释,室温孵育1 h,免疫印迹化学发光试剂显影曝光。

1.12   统计学分析

使用SPSS 13.0和Excel 2010分析数据,使用GraphPad Prism 5.0作图。每项实验重复3次,结果用均数±标准差表示。两个独立样本均数比较采用t检验。检验水准α=0.05。

2   结果

2.1   细胞株和组织中LincRNA-p21p21的表达

RT-qPCR检测显示,食管癌细胞EC109、EC9706的LincRNA-p21相对表达水平分别为永生化食管上皮细胞Het-1A的18%和32%,p21的mRNA相对表达水平分别为后者的42%和48%,食管癌细胞株的LincRNA-p21p21表达均下调(P < 0.05)。Western blot检测结果(图 1)显示食管癌细胞株p21蛋白水平也较Het-1A降低。RT-qPCR检测还显示,食管癌组织中的LincRNA-p21表达水平是其配对的癌旁组织的31%(t=5.0,P < 0.001)。

图 1


2.2   LincRNA-p21转染效率

转染组及阴性对照组慢病毒转染效率大于80%(图 2),可进行后续实验。LincRNA-p21稳定表达后,RT-qPCR检测LincRNA-p21表达水平,结果显示:转染组LincRNA-p21水平是阴性对照组的14 860倍(P < 0.05,后文中将转染组称为LincRNA-p21过表达组),差异有统计学意义。

图 2


2.3   LincRNA-p21过表达对p21表达的影响

RT-qPCR检测结果显示:LincRNA-p21过表达组p21的mRNA相对表达水平比阴性对照组上调1.83倍(P < 0.05)。Western blot法检测结果显示(图 3):LincRNA-p21过表达组的p21蛋白表达高于阴性对照组。

图 3


2.4   LincRNA-p21对食管癌细胞增殖的影响

EdU染色法结果显示见图 4LincRNA-p21过表达组、阴性对照组细胞增殖率分别为(44.4±1.7)%、(64.7±3.8)%,过表达组细胞增殖率明显降低(P < 0.05)。

图 4


2.5   LincRNA-p21对食管癌细胞周期的影响

采用流式细胞仪分析EC109细胞的周期变化,结果见表 2LincRNA-p21过表达组EC109细胞G1期、S期、G2期细胞构成比分别为(59.6±0.2)%、(24.8± 0.3)%、(15.6±0.4)%,增殖指数为40.4%;阴性对照组对应指标分别为(51.8±1.3)%、(30.7±0.6)%、(17.6±0.8)%、48.2%。LincRNA-p21过表达组的G1期细胞增多,S和G2期细胞减少(P < 0.05),增殖指数明显低于阴性对照,EC109细胞周期被阻滞在G1/S期,分裂活动受阻,抑制肿瘤细胞增殖。



Table2.Effect of LincRNA-p21 on the cell cycle distribution of esophageal cancer cells

2.6   LincRNA-p21对cyclin D蛋白表达的影响

Western blot法检测cyclin D蛋白表达结果见图 5,软件比色校正后发现LincRNA-p21过表达组细胞周期蛋白cyclin D的表达明显低于阴性对照组。

图 5

LincRNA-p21cyclin D蛋白表达的影响

3   讨论


LincRNA-p21首次在小鼠成纤维细胞中被发现,HUARTE等[5]指出LincRNA-p21p53的直接靶标,p53诱导LincRNA-p21转录,突变p53基因抑制LincRNA-p21表达;进一步采用RNA干扰技术介导LincRNA-p21p53敲除实验,发现LincRNA-p21参与下游p53依赖性转录抑制。多项研究指出,LincRNA-p21参与p53转录网络中的多种调控机制,包括:抑制基因表达,调节mRNA翻译和稳定蛋白质。DIMITROVA等[18]和ZHENG等[19]研究发现,LincRNA-p21在调节p21水平方面具有重要作用。LincRNA-p21招募核糖核蛋白K(heterogeneous nuclear ribonucleoprotein K,hnRNPK)到p21的启动子区,促进p53p21启动子结合以初始化p21转录,因此LincRNA-p21可以激活p21的表达[19]。然而,HUARTE等[5]和BAO等[20]LincRNA-p21低表达的小鼠成纤维细胞模型中没有检测到p21的降低,LincRNA-p21p21调控与否仍无定论。本研究发现食管癌细胞株EC109和EC9706中LincRNA-p21p21表达降低,食管癌细胞株中p21蛋白表达也降低。进一步扩大到食管癌组织样本,发现食管癌组织中LincRNA-p21的水平也异常下调,提示LincRNA-p21可能是抑癌因子。使用携带LincRNA-p21基因全长的缺陷型慢病毒转染食管癌细胞EC109,过表达LincRNA-p21后,EC109细胞株中p21 mRNA和蛋白水平均明显增加,说明LincRNA-p21促进食管癌细胞中p21的表达,与多项报道结果一致[18-19]

食管癌起源于食管黏膜上皮,细胞周期失调是肿瘤发生发展的一个常见因素。p21作为细胞周期蛋白激酶抑制因子家族成员之一的细胞周期蛋白激酶的抑制剂,具有广泛激酶抑制活性。p21通过抑制CDK起作用,促进DNA的损伤修复,抑制肿瘤的发生[21]。据DIMITROVA等[18]报道,LincRNA-p21与hnRNPK协同作用以促进p53介导的p21表达,LincRNA-p21通过顺式激活p21以促进其表达并作用于G1/S检查点。ZHENG等[19]证实了p21参与LincRNA-p21对细胞周期的调控,诱导G1期阻滞,介导原代肝癌细胞增殖的抑制,由于p21控制细胞周期从G1到S期过渡,因此LincRNA-p21具有调节p21水平的关键作用。现认为p21发挥功能的可能机制是p21分别结合cyclin、CDK,广泛地抑制多种cyclin-CDK复合物,较常见的是位于G1/S转换期的cyclin D1-CDK4和cyclin E-CDK2的活性被抑制,进而导致细胞G1期阻滞[22-24]。Cyclin D是调节G1/S期的关键因子,可推动细胞周期由G1期进入到S期。本研究结果显示,LincRNA-p21过表达组细胞增殖率低于对照组,细胞周期分析发现G1期延长,S期和G2期缩短,增殖指数明显低于阴性对照。LincRNA-p21促进p21表达,p21蛋白负向调节cyclin D蛋白水平,低水平cyclin D无法推动细胞周期由G1期进入到S期,细胞进程受阻,抑制增殖。该结果与DIMITROVA等[18]和ZHENG等[19]发现的过表达LincRNA-p21抑制小鼠成纤维细胞和肝癌细胞中p21活性的结果一致。

综上,LincRNA-p21上调p21水平,通过抑制cyclin D蛋白表达诱导食管癌细胞G1/S期阻滞,抑制食管癌细胞周期进程,使得细胞DNA合成受阻,从而抑制食管癌细胞增殖。目前,LincRNA-p21在肿瘤中的调控机制仍存在争论。本研究仅用了1株癌细胞进行细胞增殖、细胞周期等表型实验,实验结果存在一定的局限性。本课题组将进一步在其他食管癌细胞株中验证LincRNA-p21的表达和表型实验,并确定关键作用靶点,以期完善整个实验研究,为食管癌相关生物标志应用提供思路。此外,关于LincRNA-p21p21的调控,如果在过表达LincRNA-p21的基础上,降低LincRNA-p21的水平,p21的水平也随之降低,并且细胞功能也相应改变,那么LincRNA-p21p21的调控的特异性将增强,后续应补充这部分实验。


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