阻断离子通道影响人喉鳞癌Hep-2细胞增殖、凋亡及RNA编辑酶ADAR1表达的体内外实验研究
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摘要
喉癌发生发展机制复杂,至今未完全明了。细胞恶性变与细胞信号传导的过程紧密相关,信号传导的异常在恶性肿瘤的发生发展过程中扮演着重要角色,而细胞信号传导与多种因素如蛋白质代谢、离子通道功能状态等密切相关。
首先,细胞信号传导过程中伴随着大量的蛋白质代谢,而蛋白质代谢与RNA编辑密切相关。RNA编辑是转录后mRNA和tRNA碱基变化的现象,是一种对基因编码的mRNA进行重新修饰的过程,RNA编辑改变可使得RNA所携带的遗传信息发生改变,导致最终的蛋白序列、结构和功能的变化。对RNA发生编辑作用的酶称为RNA编辑酶,在从原虫到哺乳动物的体内广泛存在,近来越来越多的研究表明RNA编辑酶的异常与肿瘤发生发展密切相关。
其次,细胞信号传导过程中伴随着细胞膜离子通道功能状态的改变。
钾离子通道广泛分布在生物细胞中,其中延迟整流钾通道属于电压门控钾离子通道,除在可兴奋细胞起决定动作电位的复极相及维持不应期的作用外,还与肿瘤细胞的恶性增殖有关。氯离子是生物体内最多的阴离子,在维持静息膜电位,调节细胞内钙、pH值、细胞增生和分化中起着重要的作用,研究表明氯离子通道和细胞增殖及凋亡密切相关。钙离子通道是一种极为常见的信号传递通路,钙离子通道的变化可影响肿瘤细胞的生长,其阻滞剂能通过抑制钙离子的内流与释放影响肿瘤细胞的生长增殖。
为探讨喉癌发生发展与离子通道、RNA编辑酶ADAR1表达的关系,本研究在体外培养Hep-2细胞的基础上,采用穿孔膜片钳全细胞记录法、MTT比色法、结晶紫比色法、流式细胞术、TUNEL染色法、Western blot及逆转录-聚合酶链反应(RT-PCR)等方法观察Hep-2细胞离子通道特性、喉癌组织RNA编辑酶ADAR1的表达、阻断离子通道对Hep-2细胞增殖、凋亡、ERK1/2、AKT1蛋白磷酸化水平及RNA编辑酶ADAR1表达的影响及其相关性。
为进一步明确离子通道阻断剂在体内代谢后对肿瘤的影响,本研究以Hep-2细胞株建立移植瘤模型,应用不同浓度的氯离子通道阻断剂(NPPB)分组进行治疗实验,观察NPPB的抑瘤作用,并探讨其可能机制。本研究分为以下三部分:
第一部分Hep-2细胞离子通道特性及喉癌组织RNA编辑酶ADAR1的表达
方法
1.采用穿孔膜片钳全细胞记录法研究人喉鳞癌Hep-2细胞钾离子、氯离子及钙离子通道特性;
2.采用半定量逆转录-聚合酶链反应检测人喉鳞癌Hep-2细胞、喉癌组织、癌旁组织及其他喉疾病组织RNA编辑酶ADAR1 mRNA的表达;
3.统计学处理:以SPSS13.0统计软件作统计学处理,RT-PCR结果采用均数±标准差((?)±s)的形式表示,进行t检验及方差分析,P＜0.05表示有统计学意义。
结果
1.Hep-2细胞膜的静息膜电位为(-29.8±1.9)mV,Hep-2细胞膜钾离子电流具有电压依赖性、外向整流特性的特点,可被四乙胺(TEA)阻断;
2.Hep-2细胞单通道氯电流具有明显容积敏感性、外向整流特性及电压依赖性,可被5-硝基-2-(3苯丙氨基)苯甲酸(NPPB)阻断;
3.在阻断Hep-2细胞膜上钠、钾电流后,钳制电压为-90 mV时,Hep-2细胞膜上可连续记录到钙离子电流,以10 mV为阶跃电压去极化,在-60 mV获得钙离子通道的反转电流,属于低电压激活钙通道,可被尼莫地平(ND)阻断;
4.喉癌及其癌旁组织ADAR1 mRNA均有表达,两者相对表达量间存在显著性差异(P＜0.001);不同临床分期组、不同病理分级组、有无淋巴结转移组组间比较,差异均无统计学意义(P均＞0.05);
5.Hep-2细胞ADAR1 mRNA有表达,相对表达量为2.852±0.735,显著高于对照之正常喉粘膜组织(P＜0.05)。
第二部分阻断离子通道对人喉鳞癌Hep-2细胞增殖、凋亡、ERK1/2、AKT1蛋白磷酸化及RNA编辑酶ADAR1表达的影响
方法
1.采用MTT比色法及结晶紫比色法测定阻断钾、氯及钙离子通道对Hep-2细胞增殖及生长的影响;
2.采用TUNEL染色法检测阻断离子通道对Hep-2细胞凋亡的影响;
3.采用流式细胞术测定阻断离子通道前后Hep-2细胞凋亡率及细胞周期分布的变化;
4.采用Western blot检测阻断离子通道前后Hep-2细胞ERK1/2和AKT1蛋白磷酸化水平变化情况;
5.采用逆转录-聚合酶链反应检测阻断离子通道对Hep-2细胞RNA编辑酶ADAR1mRNA表达的影响;
6.统计学处理:应用SPSS13.0统计软件作统计学处理,结果采用方差分析加LSD法两两比较,相关性用spearman相关性分析,P＜0.05表示有统计学意义。
结果
1.与对照组比较,5、10、15、20mM TEA明显抑制Hep-2细胞增殖、诱导凋亡,具有明显的时间和剂量依赖性,对Hep-2细胞的G0/G1期有阻滞作用;
2.与对照组比较,50、100、150 NPPB浓度依赖性地抑制了Hep-2细胞增殖,作用12h后均可诱导Hep-2细胞凋亡,作用后G0/G1期细胞比例较作用前明显增加,S期细胞比例较作用前明显减少,具有时间依赖性;
3.5、10、50、100μM ND量-效依赖性地抑制Hep-2细胞增殖,作用12h时G0/G1期前可见亚二倍体凋亡峰,24、48h亚二倍体峰逐渐增高,随作用时间的延长,凋亡率逐渐增加,作用后G0/G1期细胞比例较作用前明显增加,S期细胞比例较作用前明显减少;
4.阻断钾、氯、钙离子通道没有明显改变Hep-2细胞ERK1/2和AKT1总蛋白水平,但显著降低了Hep-2细胞ERK1/2和AKT1蛋白磷酸化水平;
5.TEA(5、10、15、20mM)阻断Hep-2细胞钾通道、NPPB(50、100、150μM)阻断氯通道、ND(5、10、50、100μM)阻断钙通道30min、1h、2h后ADAR1 mRNA相对表达量降低,与阻断前比较存在显著性差异,具有时间浓度依赖性;
6.阻断钾、氯、钙离子通道抑制Hep-2细胞增殖与ERK1/2和AKT1蛋白磷酸化水平的降低、RNA编辑酶ADAR1mRNA相对表达量的下调密切相关。
第三部分氯离子通道阻滞剂NPPB对人喉癌裸鼠移植瘤的抑制作用
方法
1.以Hep-2细胞建立人喉癌裸鼠皮下移植瘤模型,待成瘤后分为5组,对照组注射PBS,治疗组分别注射10、50、100、150μM NPPB;
2.定期测定裸小鼠体质量及移植瘤体积用以绘制移植瘤生长变化的曲线及评价NPPB毒性。治疗结束后称重剥除之移植瘤,计算NPPB抑瘤率;
3.以TUNEL法测定移植瘤细胞凋亡的情况及其细胞凋亡指数;
4.应用Western blot检测移植瘤ERK1/2和AKT1蛋白磷酸化水平;
5.以逆转录-聚合酶链反应(RT-PCR)检测经NPPB阻断氯离子通道后移植瘤ADAR1mRNA表达情况;
6.统计学处理:应用SPSS13.0统计软件作统计学处理,采用t检验,P＜0.05表示有统计学意义。
结果
1.与对照组比较,实验组(50、100、150μM NPPB组)肿瘤生长较慢,肿瘤体积小于对照组,且随治疗时间延长,这种抑制效果更加明显,治疗结束时,实验组(50、100、150μM NPPB)瘤体积减小(P均＜0.05)、瘤质量降低(P均＜0.01);
2.实验组(50、100、150μM NPPB组)肿瘤组织中凋亡细胞数明显增多,与对照组比较,均有显著性差异(P均＜0.05);
3.实验组ERK1/2和AKT1总蛋白水平与对照组比较无显著性差别(P均＞0.05)。与对照组比较,实验组(50、100、150μM NPPB组)ERK1/2和AKT1蛋白磷酸化水平降低(P均＜0.05);
4.与对照组比较,实验组(50、100、150μM NPPB组)RNA编辑酶ADAR1mRNA相对表达量降低(P均＜0.05);
5.治疗过程中,裸鼠未见明显不良反应,各组末体质量/初体质量均＞0.8,表明各实验组无毒性反应。
结论
本文采用穿孔膜片钳全细胞记录法、MTT比色法、结晶紫比色法、流式细胞术、TUNEL染色法、Western blot及逆转录-聚合酶链反应等方法观察了Hep-2细胞离子通道特性、喉癌组织RNA编辑酶ADAR1 mRNA的表达、阻断离子通道对Hep-2细胞增殖、凋亡、ERK1/2和AKT1蛋白磷酸化水平及RNA编辑酶ADAR1 mRNA的影响,并探讨了体内阻断氯通道对人喉癌裸鼠移植瘤的抑制作用及其可能机制,得出如下结论:
1.Hep-2细胞具有电压依赖、外向整流特性的钾离子通道及容积敏感性、外向整流特性及电压依赖性的氯离子通道,具有低电压激活的钙离子通道,即T-型钙通道;
2.Hep-2细胞、喉癌及其喉旁组织ADAR1 mRNA均有表达,RNA编辑酶ADAR1与喉癌的发生有关,但与临床分期、病理分级及有无淋巴结转移无关;
3.阻断钾、氯、钙通道可抑制Hep-2细胞增殖和诱导凋亡,使细胞阻滞于G0/G1期,阻断离子通道抑制Hep-2细胞增殖和诱导凋亡可能是通过改变细胞周期而实现的,呈时间剂量依赖关系;
4.阻断钾、氯、钙通道可浓度依赖性地下调Hep-2细胞ERK1/2和AKT1蛋白磷酸化水平的表达,ERK1/2和AKT1蛋白磷酸化水平的降低与离子通道阻断剂抑制Hep-2细胞增殖密切相关;
5.阻断钾、氯、钙通道浓度依赖性下调Hep-2细胞RNA编辑酶ADAR1mRNA的相对表达,RNA编辑酶ADAR1mRNA相对表达量与离子通道阻断剂抑制Hep-2细胞增殖密切相关;
6.成功构建了人喉癌Hep-2细胞裸鼠皮下移植瘤模型,证实NPPB(50、100和150μM)能抑制喉癌移植瘤生长,对机体无明显毒副作用;
7.与体外实验结果相似,阻断氯通道下调喉癌裸鼠移植瘤ERK1/2和AKT1蛋白磷酸化水平的表达;同时下调RNA编辑酶ADAR1 mRNA表达;体内证实了阻断氯通道对人喉癌裸鼠移植瘤的生长抑制作用,其机制可能与下调ERK1/2和AKT1蛋白磷酸化水平及RNA编辑酶ADAR1 mRNA表达密切相关。
The mechanism of the occurrence and development of laryngeal carcinoma is complex.It is not clear today.Cellular malignant change is closely related to cellular signal transmitting.Abnormal cell signal transmitting plays an important role in the occurrence and development of malignancy.Cellular signal transmitting is effected by many facts,such as protein metabolism and ion channels.
Firstly,there is plenty of protein metabolism during cell signal transmitting.RNA editing is the phenomenon of nucleotide variation of post-transcriptional mRNA and tRNA.It is closely related to protein metabolism.RNA editing is a process that modifies gene coding mRNA.If RNA editing changes,genetic information carried by RNA will be changed,protein sequence,structure and function also will be changed.Enzyme which effects RNA editing is RNA-dependent adenosine deaminase(ADAR).Studies discovered that abnormity expression of ADAR1 was closely related to proliferation of carcinoma cell.
Secondly,ion channels of cell membrane changes in the process of cell signal transmitting.One side,potassium(K) ion channels exists widely in eukaryote cell from protozoa to mammalian.Delayed rectifier potassium channel is one of voltage-gated potassium channels.Not only in excitable cell does it play an important decisive role in repolarization phase of action potential and maintenance of refractory period,but also it is closely related to proliferation of carcinoma cell.On the other side,Cl~- is the most common anion.It is necessary for the cell biosynthesis,signal transduction,and function of membrane normal receptors.Recently,some reports showed that cell C1C plays a critical role in cell proliferation and differentiation.Another side,calcium ion channels are important in the signal transmitting access.The changes of intracellular calcium ion concentration can cause tumor cell growth.Calcium channel blockers can inhibit the proliferation of carcinoma cell by controlling influx and release of calcium ion.Although it is clear that cellular ion transport proteins is an important part of carcinoma cell,there is no data from literature reports about ion channels in laryngeal carcinoma in China and abroad nowadays.
Therefore,in this study,perforated patch-clamp whole-cell recording technique, MTT colorimetric assay,crystal violet method,flow cytometry,TUNEL Staining, Western blot and reverse transcription-polymerase chain reaction(RT-PCR) were used to study the characteristics of ion channels in Hep-2 cells,phosphorylated ERK1/2,AKT1 protein level and the expression of ADAR1 mRNA,the relationships among blocking ion channels,apoptosis and proliferation in Hep-2 cells,to study the possible mechanism of its occurring and developing.
To study the effection of chloride ion channels' blocker on tumor in vivo,Hep-2 cells were used to establish laryngeal carcinoma nude mice xenograft modes.Different groups were treated with different concentration of NPPB.Our studies were divided into three subsections.
First part The characteristics of ion channels in Hep-2 cells and the expression of ADAR1 mRNA in human laryngeal carcinoma
Methods
1.Potassium ion channels,chloride ion channels and calcium ion channels were measured by perforated patch-clamp whole-cell recording technique.
2.The expression of ADAR1 mRNA in laryngeal carcinoma tissues,tumor-adjacent tissues and Hep-2 cells were detected by using RT-PCR.
3.Statistical analysis:Statistical analyses were treated with the SPSS 13.0.t test and ANOVA analysis of variance was employed.The significant level was P＜0.05.
Results
1.Resting membrane potential of Hep-2 cell was(-29.8±1.9)mV.A sort of voltage-dependent outwardly rectifying transmembrane current was recorded.This current could be blocked by TEA.
2.Single channel chloride current of Hep-2 cell was outwardly rectifying, voltage-dependent and volume-sensitive.The chloride current could be blocked by NPPB.
3.When sodium current and potassium current were blocked,voltage of clamp was -90 mV;calcium current of Hep-2 cell membrane was recorded continuously.When depolarized by10 mV step,calcium reverse current was recorded in -60 mV.Calcium current of Hep-2 cell membrane was low-voltage activated calcium channels.The calcium current could be blocked by ND.
4.ADAR1 mRNA expressed in laryngeal carcinoma and corresponding para-carcinoma tissues.There were not significant differences among different clinic stages,different classes and metastasis of lymph(P＞0.05).
5.ADAR1 mRNA expressed in Hep-2 cell.The relative expression of ADAR1 mRNA was 2.852±0.735.There was significant differences between Hep-2 cell and control group(P＜0.05).
Second part The effects of blocking ion channels on Hep-2 cell proliferation, apoptosis,phosphorylated ERK1/2,AKT1 protein levels and the expression of ADAR1 mRNA
Methods
1.MTT colorimetric assay and crystal violet was used to determine Hep-2 cells proliferation before and after blocking ion channels.
2.TUNEL staining was used to determine Hep-2 cells apoptosis before and after blocking ion channels.
3.Cell cycle,apoptosis of Hep-2 cells was determined by flow cytometry.
4.The total and phosphorylated ERK1/2 and AKT1 protein levels of Hep-2 cells were detected by Western blot before and after blocking ion channels.
5.The expressions of ADAR1 mRNA were examined by RT-PCR before and after ion channels blocked in Hep-2 cells.
6.Statistical analysis:Statistical analyses were treated with the SPSS 13.0.The differences among the groups were compared with the ANOVA.The relationships among the inhibited Hep-2 cells proliferation,phosphorylated ERK1/2 and AKT1 protein levels and expressions of ADAR1 mRNA were studed by Spearman analysis.The significant level was P＜0.05.
Results
1.Compared with control group,the blocker of potassium ion channel(5,10,15, 20mM TEA) could significantly inhibit the proliferation of Hep-2 cell and induce the apoptosis dose-effect dependently,time-effect dependently.Blocking potassium ion channel could increase the cell ratio of G0/G1.
2.The blocker of chloride ion channel(50,100,150μM NPPB) suppressed proliferation of Hep-2 cell concentration dependently.When Hep-2 cell was affected by NPPB for 12h,cell apoptosis was induced,which showed obvious time-effect relationship. After affected by NPPB,the cell ratio of G0/G1 increased,the cell ratio of S decreased, which showed obvious time-effect and dose-effect relationship.
3.The blocker of calcium ion channel(5、10、50、100μM ND) suppressed proliferation of Hep-2 cell.When ND effected for 12h,24h and 48h,the cell number in sub-G0/G1 peak(apoptotic peak) was increased gradually as time went on,the cell ratio of S decreased.
4.The total protein level of ERK1/2 and AKT1 did not significantly change before and after potassium,chloride and calcium ion channel was blocked,but blocking potassium,chloride and calcium ion channel decreased phosphorylated protein level of ERK1/2 and AKT1,which showed obvious dose-effect relationship.Compared with control group,there were significant differences(p＜0.05).
5.When TEA(5,10,15,20mM) blocked potassium ion channel,NPPB(50,100, 150μM) blocked chloride ion channel,ND(5,10,50,100μM) blocked calcium ion channel for 30min、1h and 2h.The relative expression of ADAR1 mRNA were decreased respectively time-effect and dose-effect dependently.
6.Obvious correlation was found among Hep-2 cell proliferation suppressed by the blockers of ion channels(TEA,NPPB,ND),expression of phosphorylated protein level of ERK1/2,AKT1 and expression of ADAR1 mRNA.
Third part The effects of blocking chloride ion channels on human laryngeal carcinoma xenograft tumors in nude mice
Methods
1.Hep-2 cells were used to establish laryngeal carcinoma nude mice xenograft models.The mice were divided into 5 groups after the neoplasm formed.The control group was treated with PBS,the treated groups with NPPB at different doses(10,50,100, 150μM).
2.The nude mice's body mass and the tumors' size were determined termly to the painting growth curves of the xenograft models.After the treatment was ended,we stripped out the xenograft tumors and weighted.The inhibitory rate of xenograft tumors growth was figured out.
3.TUNEL method was used to detecte the AI of the xenograft tumors.
4.Western blot was used to detecte the phosphorylation levels of ERK1/2 and Akt1 protein in the xenograft tumors.
5.The mRNA expression of ADAR1 in the xenograft tumors was analyzed by using RT-PCR.
6.Statistical analysis:Statistical analyses were treated with the SPSS 13.0.t-test was used.The significant level was P＜0.05.
Results
1.When the treatment was ended,the tumor mass and the tumor size of the experimental groups(50,100,150μM NPPB) were significantly decreased(P＜0.05) comparing with the contrast group.
2.The number of apoptotic cells in the experimental groups(50,100,150μM NPPB) were much more than that in the control group(P＜0.05).
3.The expression of total protein of ERK1/2 and Akt1 in the xenograft tumors in the experimental groups(50,100,150μM NPPB) were not significantly(P＞0.05) changed. Compared with the contrast group,the phosphorylation levels of ERK1/2 and AKT1 in the experimental groups(50,100,150μM NPPB) were not significantly(P＜0.05) decreased.
4.The mRNA expression level of ADAR1 in the experimental groups(50,100, 150μM NPPB) was lower than that in the control group(P＜0.05).
5.In the course of treatment,no adverse effects of NPPB were observed in the mice. The values of final body weight(FBW)/initial body weight(IBW) in five groups were more than 0.8 and it indicated that there was no toxic reaction in the mice.
Conclusions
For the first time,the effects of blockers of ion channels(TEA,NPPB and ND) on proliferation,apoptosis,cell cycle,and the phosphorylated ERK1/2,AKT1 protein levels,mRNA expression of ADAR1 in human laryngeal carcinoma Hep-2 cells were investigated by MTT,FCM,TUNEL,Western blot and RT-PCR in this study,and human laryngeal carcinoma xenograft tumors were also studied.Our conclusions were listed below:
1.Hep-2 cell has voltage-dependent outwardly rectifying transmembrane potassium ion current.Chloride current of Hep-2 cell was outwardly rectifying,voltage-dependent and volume-sensitive.Calcium current of Hep-2 cell membrane was low-voltage activated.
2.ADAR1 mRNA expressed in laryngeal carcinoma and corresponding para-carcinoma tissues.The expression of ADAR1 mRNA was irrespective with clinical stages,pathologic grade and metastasis of lymph.
3.Blockers of potassium,chloride and calcium ion channels(TEA,NPPB and ND) could inhibit the proliferation,induce apoptosis of Hep-2 cells and this inhibitory effect showed time and dose dependence.The proliferation inhibition and apoptosis induction effects of TEA,NPPB and ND might be related to the cell cycle arrest at G0/G1 stage.
4.Blockers of potassium,chloride and calcium ion channels(TEA,NPPB and ND) could down regulate the phosphorylation levels of ERK1/2 and AKT1 protein in Hep-2 cells in a time-dependent manner,which was relative with the proliferation of Hep-2 cells inhibited by TEA,NPPB and ND.
5.Blockers of potassium,chloride and calcium ion channels(TEA,NPPB and ND) could inhibit the mRNA expression of ADAR1 in Hep-2 cells in a time-dependent manner. Down regulation of ADAR1mRNA expression was relative with inhibitory effect of TEA, NPPB and ND in the proliferation of Hep-2 cells.
6.We established laryngeal carcinoma xenograft models successfully in nude mice,confirmed that NPPB(50,100 and 150μM) could inhibit the growth of xenograft tumors in dose-dependent manner.
7.NPPB could down phosphorylation levels of ERK1/2 and AKT1 protein,induce cell apoptosis,and down regulating the mRNA expression of ADAR1 in laryngeal carcinoma xenograft tumors in nude mice.The mechanism of the effects might be related to down regulation of ERK1/2 and AKT1 protein,down regulation of ADAR1 mRNA expression.
首先,细胞信号传导过程中伴随着大量的蛋白质代谢,而蛋白质代谢与RNA编辑密切相关。RNA编辑是转录后mRNA和tRNA碱基变化的现象,是一种对基因编码的mRNA进行重新修饰的过程,RNA编辑改变可使得RNA所携带的遗传信息发生改变,导致最终的蛋白序列、结构和功能的变化。对RNA发生编辑作用的酶称为RNA编辑酶,在从原虫到哺乳动物的体内广泛存在,近来越来越多的研究表明RNA编辑酶的异常与肿瘤发生发展密切相关。
其次,细胞信号传导过程中伴随着细胞膜离子通道功能状态的改变。
钾离子通道广泛分布在生物细胞中,其中延迟整流钾通道属于电压门控钾离子通道,除在可兴奋细胞起决定动作电位的复极相及维持不应期的作用外,还与肿瘤细胞的恶性增殖有关。氯离子是生物体内最多的阴离子,在维持静息膜电位,调节细胞内钙、pH值、细胞增生和分化中起着重要的作用,研究表明氯离子通道和细胞增殖及凋亡密切相关。钙离子通道是一种极为常见的信号传递通路,钙离子通道的变化可影响肿瘤细胞的生长,其阻滞剂能通过抑制钙离子的内流与释放影响肿瘤细胞的生长增殖。
为探讨喉癌发生发展与离子通道、RNA编辑酶ADAR1表达的关系,本研究在体外培养Hep-2细胞的基础上,采用穿孔膜片钳全细胞记录法、MTT比色法、结晶紫比色法、流式细胞术、TUNEL染色法、Western blot及逆转录-聚合酶链反应(RT-PCR)等方法观察Hep-2细胞离子通道特性、喉癌组织RNA编辑酶ADAR1的表达、阻断离子通道对Hep-2细胞增殖、凋亡、ERK1/2、AKT1蛋白磷酸化水平及RNA编辑酶ADAR1表达的影响及其相关性。
为进一步明确离子通道阻断剂在体内代谢后对肿瘤的影响,本研究以Hep-2细胞株建立移植瘤模型,应用不同浓度的氯离子通道阻断剂(NPPB)分组进行治疗实验,观察NPPB的抑瘤作用,并探讨其可能机制。本研究分为以下三部分:
第一部分Hep-2细胞离子通道特性及喉癌组织RNA编辑酶ADAR1的表达
方法
1.采用穿孔膜片钳全细胞记录法研究人喉鳞癌Hep-2细胞钾离子、氯离子及钙离子通道特性;
2.采用半定量逆转录-聚合酶链反应检测人喉鳞癌Hep-2细胞、喉癌组织、癌旁组织及其他喉疾病组织RNA编辑酶ADAR1 mRNA的表达;
3.统计学处理:以SPSS13.0统计软件作统计学处理,RT-PCR结果采用均数±标准差((?)±s)的形式表示,进行t检验及方差分析,P＜0.05表示有统计学意义。
结果
1.Hep-2细胞膜的静息膜电位为(-29.8±1.9)mV,Hep-2细胞膜钾离子电流具有电压依赖性、外向整流特性的特点,可被四乙胺(TEA)阻断;
2.Hep-2细胞单通道氯电流具有明显容积敏感性、外向整流特性及电压依赖性,可被5-硝基-2-(3苯丙氨基)苯甲酸(NPPB)阻断;
3.在阻断Hep-2细胞膜上钠、钾电流后,钳制电压为-90 mV时,Hep-2细胞膜上可连续记录到钙离子电流,以10 mV为阶跃电压去极化,在-60 mV获得钙离子通道的反转电流,属于低电压激活钙通道,可被尼莫地平(ND)阻断;
4.喉癌及其癌旁组织ADAR1 mRNA均有表达,两者相对表达量间存在显著性差异(P＜0.001);不同临床分期组、不同病理分级组、有无淋巴结转移组组间比较,差异均无统计学意义(P均＞0.05);
5.Hep-2细胞ADAR1 mRNA有表达,相对表达量为2.852±0.735,显著高于对照之正常喉粘膜组织(P＜0.05)。
第二部分阻断离子通道对人喉鳞癌Hep-2细胞增殖、凋亡、ERK1/2、AKT1蛋白磷酸化及RNA编辑酶ADAR1表达的影响
方法
1.采用MTT比色法及结晶紫比色法测定阻断钾、氯及钙离子通道对Hep-2细胞增殖及生长的影响;
2.采用TUNEL染色法检测阻断离子通道对Hep-2细胞凋亡的影响;
3.采用流式细胞术测定阻断离子通道前后Hep-2细胞凋亡率及细胞周期分布的变化;
4.采用Western blot检测阻断离子通道前后Hep-2细胞ERK1/2和AKT1蛋白磷酸化水平变化情况;
5.采用逆转录-聚合酶链反应检测阻断离子通道对Hep-2细胞RNA编辑酶ADAR1mRNA表达的影响;
6.统计学处理:应用SPSS13.0统计软件作统计学处理,结果采用方差分析加LSD法两两比较,相关性用spearman相关性分析,P＜0.05表示有统计学意义。
结果
1.与对照组比较,5、10、15、20mM TEA明显抑制Hep-2细胞增殖、诱导凋亡,具有明显的时间和剂量依赖性,对Hep-2细胞的G0/G1期有阻滞作用;
2.与对照组比较,50、100、150 NPPB浓度依赖性地抑制了Hep-2细胞增殖,作用12h后均可诱导Hep-2细胞凋亡,作用后G0/G1期细胞比例较作用前明显增加,S期细胞比例较作用前明显减少,具有时间依赖性;
3.5、10、50、100μM ND量-效依赖性地抑制Hep-2细胞增殖,作用12h时G0/G1期前可见亚二倍体凋亡峰,24、48h亚二倍体峰逐渐增高,随作用时间的延长,凋亡率逐渐增加,作用后G0/G1期细胞比例较作用前明显增加,S期细胞比例较作用前明显减少;
4.阻断钾、氯、钙离子通道没有明显改变Hep-2细胞ERK1/2和AKT1总蛋白水平,但显著降低了Hep-2细胞ERK1/2和AKT1蛋白磷酸化水平;
5.TEA(5、10、15、20mM)阻断Hep-2细胞钾通道、NPPB(50、100、150μM)阻断氯通道、ND(5、10、50、100μM)阻断钙通道30min、1h、2h后ADAR1 mRNA相对表达量降低,与阻断前比较存在显著性差异,具有时间浓度依赖性;
6.阻断钾、氯、钙离子通道抑制Hep-2细胞增殖与ERK1/2和AKT1蛋白磷酸化水平的降低、RNA编辑酶ADAR1mRNA相对表达量的下调密切相关。
第三部分氯离子通道阻滞剂NPPB对人喉癌裸鼠移植瘤的抑制作用
方法
1.以Hep-2细胞建立人喉癌裸鼠皮下移植瘤模型,待成瘤后分为5组,对照组注射PBS,治疗组分别注射10、50、100、150μM NPPB;
2.定期测定裸小鼠体质量及移植瘤体积用以绘制移植瘤生长变化的曲线及评价NPPB毒性。治疗结束后称重剥除之移植瘤,计算NPPB抑瘤率;
3.以TUNEL法测定移植瘤细胞凋亡的情况及其细胞凋亡指数;
4.应用Western blot检测移植瘤ERK1/2和AKT1蛋白磷酸化水平;
5.以逆转录-聚合酶链反应(RT-PCR)检测经NPPB阻断氯离子通道后移植瘤ADAR1mRNA表达情况;
6.统计学处理:应用SPSS13.0统计软件作统计学处理,采用t检验,P＜0.05表示有统计学意义。
结果
1.与对照组比较,实验组(50、100、150μM NPPB组)肿瘤生长较慢,肿瘤体积小于对照组,且随治疗时间延长,这种抑制效果更加明显,治疗结束时,实验组(50、100、150μM NPPB)瘤体积减小(P均＜0.05)、瘤质量降低(P均＜0.01);
2.实验组(50、100、150μM NPPB组)肿瘤组织中凋亡细胞数明显增多,与对照组比较,均有显著性差异(P均＜0.05);
3.实验组ERK1/2和AKT1总蛋白水平与对照组比较无显著性差别(P均＞0.05)。与对照组比较,实验组(50、100、150μM NPPB组)ERK1/2和AKT1蛋白磷酸化水平降低(P均＜0.05);
4.与对照组比较,实验组(50、100、150μM NPPB组)RNA编辑酶ADAR1mRNA相对表达量降低(P均＜0.05);
5.治疗过程中,裸鼠未见明显不良反应,各组末体质量/初体质量均＞0.8,表明各实验组无毒性反应。
结论
本文采用穿孔膜片钳全细胞记录法、MTT比色法、结晶紫比色法、流式细胞术、TUNEL染色法、Western blot及逆转录-聚合酶链反应等方法观察了Hep-2细胞离子通道特性、喉癌组织RNA编辑酶ADAR1 mRNA的表达、阻断离子通道对Hep-2细胞增殖、凋亡、ERK1/2和AKT1蛋白磷酸化水平及RNA编辑酶ADAR1 mRNA的影响,并探讨了体内阻断氯通道对人喉癌裸鼠移植瘤的抑制作用及其可能机制,得出如下结论:
1.Hep-2细胞具有电压依赖、外向整流特性的钾离子通道及容积敏感性、外向整流特性及电压依赖性的氯离子通道,具有低电压激活的钙离子通道,即T-型钙通道;
2.Hep-2细胞、喉癌及其喉旁组织ADAR1 mRNA均有表达,RNA编辑酶ADAR1与喉癌的发生有关,但与临床分期、病理分级及有无淋巴结转移无关;
3.阻断钾、氯、钙通道可抑制Hep-2细胞增殖和诱导凋亡,使细胞阻滞于G0/G1期,阻断离子通道抑制Hep-2细胞增殖和诱导凋亡可能是通过改变细胞周期而实现的,呈时间剂量依赖关系;
4.阻断钾、氯、钙通道可浓度依赖性地下调Hep-2细胞ERK1/2和AKT1蛋白磷酸化水平的表达,ERK1/2和AKT1蛋白磷酸化水平的降低与离子通道阻断剂抑制Hep-2细胞增殖密切相关;
5.阻断钾、氯、钙通道浓度依赖性下调Hep-2细胞RNA编辑酶ADAR1mRNA的相对表达,RNA编辑酶ADAR1mRNA相对表达量与离子通道阻断剂抑制Hep-2细胞增殖密切相关;
6.成功构建了人喉癌Hep-2细胞裸鼠皮下移植瘤模型,证实NPPB(50、100和150μM)能抑制喉癌移植瘤生长,对机体无明显毒副作用;
7.与体外实验结果相似,阻断氯通道下调喉癌裸鼠移植瘤ERK1/2和AKT1蛋白磷酸化水平的表达;同时下调RNA编辑酶ADAR1 mRNA表达;体内证实了阻断氯通道对人喉癌裸鼠移植瘤的生长抑制作用,其机制可能与下调ERK1/2和AKT1蛋白磷酸化水平及RNA编辑酶ADAR1 mRNA表达密切相关。
The mechanism of the occurrence and development of laryngeal carcinoma is complex.It is not clear today.Cellular malignant change is closely related to cellular signal transmitting.Abnormal cell signal transmitting plays an important role in the occurrence and development of malignancy.Cellular signal transmitting is effected by many facts,such as protein metabolism and ion channels.
Firstly,there is plenty of protein metabolism during cell signal transmitting.RNA editing is the phenomenon of nucleotide variation of post-transcriptional mRNA and tRNA.It is closely related to protein metabolism.RNA editing is a process that modifies gene coding mRNA.If RNA editing changes,genetic information carried by RNA will be changed,protein sequence,structure and function also will be changed.Enzyme which effects RNA editing is RNA-dependent adenosine deaminase(ADAR).Studies discovered that abnormity expression of ADAR1 was closely related to proliferation of carcinoma cell.
Secondly,ion channels of cell membrane changes in the process of cell signal transmitting.One side,potassium(K) ion channels exists widely in eukaryote cell from protozoa to mammalian.Delayed rectifier potassium channel is one of voltage-gated potassium channels.Not only in excitable cell does it play an important decisive role in repolarization phase of action potential and maintenance of refractory period,but also it is closely related to proliferation of carcinoma cell.On the other side,Cl~- is the most common anion.It is necessary for the cell biosynthesis,signal transduction,and function of membrane normal receptors.Recently,some reports showed that cell C1C plays a critical role in cell proliferation and differentiation.Another side,calcium ion channels are important in the signal transmitting access.The changes of intracellular calcium ion concentration can cause tumor cell growth.Calcium channel blockers can inhibit the proliferation of carcinoma cell by controlling influx and release of calcium ion.Although it is clear that cellular ion transport proteins is an important part of carcinoma cell,there is no data from literature reports about ion channels in laryngeal carcinoma in China and abroad nowadays.
Therefore,in this study,perforated patch-clamp whole-cell recording technique, MTT colorimetric assay,crystal violet method,flow cytometry,TUNEL Staining, Western blot and reverse transcription-polymerase chain reaction(RT-PCR) were used to study the characteristics of ion channels in Hep-2 cells,phosphorylated ERK1/2,AKT1 protein level and the expression of ADAR1 mRNA,the relationships among blocking ion channels,apoptosis and proliferation in Hep-2 cells,to study the possible mechanism of its occurring and developing.
To study the effection of chloride ion channels' blocker on tumor in vivo,Hep-2 cells were used to establish laryngeal carcinoma nude mice xenograft modes.Different groups were treated with different concentration of NPPB.Our studies were divided into three subsections.
First part The characteristics of ion channels in Hep-2 cells and the expression of ADAR1 mRNA in human laryngeal carcinoma
Methods
1.Potassium ion channels,chloride ion channels and calcium ion channels were measured by perforated patch-clamp whole-cell recording technique.
2.The expression of ADAR1 mRNA in laryngeal carcinoma tissues,tumor-adjacent tissues and Hep-2 cells were detected by using RT-PCR.
3.Statistical analysis:Statistical analyses were treated with the SPSS 13.0.t test and ANOVA analysis of variance was employed.The significant level was P＜0.05.
Results
1.Resting membrane potential of Hep-2 cell was(-29.8±1.9)mV.A sort of voltage-dependent outwardly rectifying transmembrane current was recorded.This current could be blocked by TEA.
2.Single channel chloride current of Hep-2 cell was outwardly rectifying, voltage-dependent and volume-sensitive.The chloride current could be blocked by NPPB.
3.When sodium current and potassium current were blocked,voltage of clamp was -90 mV;calcium current of Hep-2 cell membrane was recorded continuously.When depolarized by10 mV step,calcium reverse current was recorded in -60 mV.Calcium current of Hep-2 cell membrane was low-voltage activated calcium channels.The calcium current could be blocked by ND.
4.ADAR1 mRNA expressed in laryngeal carcinoma and corresponding para-carcinoma tissues.There were not significant differences among different clinic stages,different classes and metastasis of lymph(P＞0.05).
5.ADAR1 mRNA expressed in Hep-2 cell.The relative expression of ADAR1 mRNA was 2.852±0.735.There was significant differences between Hep-2 cell and control group(P＜0.05).
Second part The effects of blocking ion channels on Hep-2 cell proliferation, apoptosis,phosphorylated ERK1/2,AKT1 protein levels and the expression of ADAR1 mRNA
Methods
1.MTT colorimetric assay and crystal violet was used to determine Hep-2 cells proliferation before and after blocking ion channels.
2.TUNEL staining was used to determine Hep-2 cells apoptosis before and after blocking ion channels.
3.Cell cycle,apoptosis of Hep-2 cells was determined by flow cytometry.
4.The total and phosphorylated ERK1/2 and AKT1 protein levels of Hep-2 cells were detected by Western blot before and after blocking ion channels.
5.The expressions of ADAR1 mRNA were examined by RT-PCR before and after ion channels blocked in Hep-2 cells.
6.Statistical analysis:Statistical analyses were treated with the SPSS 13.0.The differences among the groups were compared with the ANOVA.The relationships among the inhibited Hep-2 cells proliferation,phosphorylated ERK1/2 and AKT1 protein levels and expressions of ADAR1 mRNA were studed by Spearman analysis.The significant level was P＜0.05.
Results
1.Compared with control group,the blocker of potassium ion channel(5,10,15, 20mM TEA) could significantly inhibit the proliferation of Hep-2 cell and induce the apoptosis dose-effect dependently,time-effect dependently.Blocking potassium ion channel could increase the cell ratio of G0/G1.
2.The blocker of chloride ion channel(50,100,150μM NPPB) suppressed proliferation of Hep-2 cell concentration dependently.When Hep-2 cell was affected by NPPB for 12h,cell apoptosis was induced,which showed obvious time-effect relationship. After affected by NPPB,the cell ratio of G0/G1 increased,the cell ratio of S decreased, which showed obvious time-effect and dose-effect relationship.
3.The blocker of calcium ion channel(5、10、50、100μM ND) suppressed proliferation of Hep-2 cell.When ND effected for 12h,24h and 48h,the cell number in sub-G0/G1 peak(apoptotic peak) was increased gradually as time went on,the cell ratio of S decreased.
4.The total protein level of ERK1/2 and AKT1 did not significantly change before and after potassium,chloride and calcium ion channel was blocked,but blocking potassium,chloride and calcium ion channel decreased phosphorylated protein level of ERK1/2 and AKT1,which showed obvious dose-effect relationship.Compared with control group,there were significant differences(p＜0.05).
5.When TEA(5,10,15,20mM) blocked potassium ion channel,NPPB(50,100, 150μM) blocked chloride ion channel,ND(5,10,50,100μM) blocked calcium ion channel for 30min、1h and 2h.The relative expression of ADAR1 mRNA were decreased respectively time-effect and dose-effect dependently.
6.Obvious correlation was found among Hep-2 cell proliferation suppressed by the blockers of ion channels(TEA,NPPB,ND),expression of phosphorylated protein level of ERK1/2,AKT1 and expression of ADAR1 mRNA.
Third part The effects of blocking chloride ion channels on human laryngeal carcinoma xenograft tumors in nude mice
Methods
1.Hep-2 cells were used to establish laryngeal carcinoma nude mice xenograft models.The mice were divided into 5 groups after the neoplasm formed.The control group was treated with PBS,the treated groups with NPPB at different doses(10,50,100, 150μM).
2.The nude mice's body mass and the tumors' size were determined termly to the painting growth curves of the xenograft models.After the treatment was ended,we stripped out the xenograft tumors and weighted.The inhibitory rate of xenograft tumors growth was figured out.
3.TUNEL method was used to detecte the AI of the xenograft tumors.
4.Western blot was used to detecte the phosphorylation levels of ERK1/2 and Akt1 protein in the xenograft tumors.
5.The mRNA expression of ADAR1 in the xenograft tumors was analyzed by using RT-PCR.
6.Statistical analysis:Statistical analyses were treated with the SPSS 13.0.t-test was used.The significant level was P＜0.05.
Results
1.When the treatment was ended,the tumor mass and the tumor size of the experimental groups(50,100,150μM NPPB) were significantly decreased(P＜0.05) comparing with the contrast group.
2.The number of apoptotic cells in the experimental groups(50,100,150μM NPPB) were much more than that in the control group(P＜0.05).
3.The expression of total protein of ERK1/2 and Akt1 in the xenograft tumors in the experimental groups(50,100,150μM NPPB) were not significantly(P＞0.05) changed. Compared with the contrast group,the phosphorylation levels of ERK1/2 and AKT1 in the experimental groups(50,100,150μM NPPB) were not significantly(P＜0.05) decreased.
4.The mRNA expression level of ADAR1 in the experimental groups(50,100, 150μM NPPB) was lower than that in the control group(P＜0.05).
5.In the course of treatment,no adverse effects of NPPB were observed in the mice. The values of final body weight(FBW)/initial body weight(IBW) in five groups were more than 0.8 and it indicated that there was no toxic reaction in the mice.
Conclusions
For the first time,the effects of blockers of ion channels(TEA,NPPB and ND) on proliferation,apoptosis,cell cycle,and the phosphorylated ERK1/2,AKT1 protein levels,mRNA expression of ADAR1 in human laryngeal carcinoma Hep-2 cells were investigated by MTT,FCM,TUNEL,Western blot and RT-PCR in this study,and human laryngeal carcinoma xenograft tumors were also studied.Our conclusions were listed below:
1.Hep-2 cell has voltage-dependent outwardly rectifying transmembrane potassium ion current.Chloride current of Hep-2 cell was outwardly rectifying,voltage-dependent and volume-sensitive.Calcium current of Hep-2 cell membrane was low-voltage activated.
2.ADAR1 mRNA expressed in laryngeal carcinoma and corresponding para-carcinoma tissues.The expression of ADAR1 mRNA was irrespective with clinical stages,pathologic grade and metastasis of lymph.
3.Blockers of potassium,chloride and calcium ion channels(TEA,NPPB and ND) could inhibit the proliferation,induce apoptosis of Hep-2 cells and this inhibitory effect showed time and dose dependence.The proliferation inhibition and apoptosis induction effects of TEA,NPPB and ND might be related to the cell cycle arrest at G0/G1 stage.
4.Blockers of potassium,chloride and calcium ion channels(TEA,NPPB and ND) could down regulate the phosphorylation levels of ERK1/2 and AKT1 protein in Hep-2 cells in a time-dependent manner,which was relative with the proliferation of Hep-2 cells inhibited by TEA,NPPB and ND.
5.Blockers of potassium,chloride and calcium ion channels(TEA,NPPB and ND) could inhibit the mRNA expression of ADAR1 in Hep-2 cells in a time-dependent manner. Down regulation of ADAR1mRNA expression was relative with inhibitory effect of TEA, NPPB and ND in the proliferation of Hep-2 cells.
6.We established laryngeal carcinoma xenograft models successfully in nude mice,confirmed that NPPB(50,100 and 150μM) could inhibit the growth of xenograft tumors in dose-dependent manner.
7.NPPB could down phosphorylation levels of ERK1/2 and AKT1 protein,induce cell apoptosis,and down regulating the mRNA expression of ADAR1 in laryngeal carcinoma xenograft tumors in nude mice.The mechanism of the effects might be related to down regulation of ERK1/2 and AKT1 protein,down regulation of ADAR1 mRNA expression.
引文
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