STAT3及ErbB2 RNA干涉结合放疗治疗脑胶质瘤的实验研究
摘要
脑肿瘤又称颅内肿瘤,是一种起病缓慢并逐渐加重的脑部疾病。原发性颅内肿瘤以胶质瘤最为多见,约占总数的40%。目前临床脑肿瘤的治疗手段主要包括手术治疗、放射治疗和化学药物治疗。由于采用单个治疗方法往往达不到理想的疗效,因此,在治疗时常常联合应用手术及放、化疗。而术后的放化疗效果将对患者的预后起着至关重要的影响,因此如何增加放疗化疗的敏感性以增强放化疗效果是目前研究的焦点。此外,在常规手段治疗脑肿瘤的过程中,肿瘤组织周围的正常组织在治疗时不可避免地会遭受损伤,最理想的治疗方法是只杀伤肿瘤细胞,而对正常产生较小的影响。
RNAi具有高度的序列专一性,可以特异地使特定基因沉默,因此在治疗由于某个基因表达异常增高引起的疾病中非常有用。肿瘤的发生常常与原癌基因的表达异常增高相关,此时,利用RNAi技术下调这些异常高表达的基因,可特异性的影响肿瘤细胞,而对正常细胞无损伤,在实际研究过程中,找到符合要求的靶基因是获得理想治疗效果的关键。
STAT3(信号转导子和转录激活子3)属于STATS蛋白家族。STATs是一类存在于胞浆中的由细胞因子、生长因子等多肽类配体激活的转录因子,共有七个同源家族成员。STAT3在多种肿瘤组织中呈现持续性激活状态,在肿瘤的演变和恶化的过程中起着重要的作用。此外,STAT3还对人肺腺癌和直肠癌的放化疗敏感性有影响。ErbB2是位于细胞表面的受体酪氨酸激酶ErbB家族成员。ErbB家族还包括EGF-R(ErbB1),ErbB3和ErbB4。目前研究表明,许多肿瘤细胞系中的ErbB2呈高表达状态,并且ErbB2已经被用来作为乳腺癌病理分级指标参数和预后指标。Ricardo等还发现,ErbB2表达下调可以提高肿瘤细胞的辐射敏感性。众所周知,细胞增殖力增强将使肿瘤体积增大,细胞凋亡增强则将减小肿瘤体积,因此肿瘤体积改变的实质是细胞增殖和凋亡的动态平衡的最后外在体现。已有研究表明,STAT3和ErbB2对多种肿瘤细胞增殖力的维持具有重要作用。基于二者在肿瘤细胞中特异性高表达并且具有促进细胞增殖的功能,以及二者与肿瘤细胞放化疗敏感性的关系,因此本研究拟以STAT3和ErbB2为靶点应用RNAi技术、联合放疗治疗探索治疗恶性脑胶质瘤的新途径。
在本论文中,我们主要进行了以下四个方面的研究:
一.STAT3和ErbB2干涉载体的构建、筛选和干涉效果的确定
设计STAT3和ErbB2干涉的靶位点以及GFP对照靶位点,利用pSilence2.1-U6-H1载体,构建pSilence2.1-U6-STAT3,pSilence2.1-U6-ErbB2,pSilence2.1-U6-GFP质粒,转染人脑胶质瘤U251细胞。经Western Blot和Real-Time PCR实验验证表明:与对照组相比,实验组细胞靶蛋白和靶mRNA表达水平都降低了8～14倍。
二.STAT3 RNAi和ErbB2 RNAi抑制细胞增殖的协同作用及其与放疗的联合效果
克隆形成率是衡量细胞状态和增殖能力的金标准。在研究细胞辐射敏感性时,也通常要求使用克隆形成率来进行判断。为了确定本研究中使用的放疗剂量,我们首先用不同剂量~(60)Coγ射线照射U251细胞,随后对其进行克隆形成率实验,根据实验结果,确定2Gy为治疗用照射剂量。同时使用MTT方法检测不同剂量~(60)Coγ射线照射后U251细胞的增殖能力,结果与克隆形成率实验一致。我们将实验分为control组、pSilence2.1-GFP组、pSilence2.1-STAT3组、pSilence2.1-ErbB2组、pSilence2.1-STAT3+pSilence2.1-ErbB2组、control+2Gy组、pSilence2.1-GFP+2Gy组、pSilence2.1-STAT3+2Gy组、pSilence2.1-ErbB2+2Gy组以及pSilence2.1-STAT3+pSilence2.1-ErbB2+2Gy组,共10组,用克隆形成率实验检测各种处理对细胞增殖能力的影响。结果表明:
(1)STAT3 RNAi和ErbB2 RNAi均能抑制U251细胞增殖,且二者具有协同作用;
(2)STAT3 RNAi和ErbB2 RNAi均能增加U251细胞的辐射敏感性,将二者同时进行干涉时,U251细胞的辐射敏感性进一步增强。
三.STAT3 RNAi和ErbB2 RNAi对U251细胞凋亡及细胞周期的影响
肿瘤发生的主要原因是细胞周期失调后导致的细胞无限制增殖。那么,转染pSilence2.1-STAT3和pSilence2.1-ErbB2后,什么原因致使星形胶质瘤细胞U251的增殖得到控制了呢?二者对正常星形胶质细胞的增殖是否有同样的影响呢?为了帮助弄清这两个问题,使用出生24小时内大鼠,分离培养原代星形胶质细胞(命名为NA细胞),供研究使用。接下来,利用Hoechst 33258染色,观察到U251细胞在转染pSilence2.1-STAT3或pSilence2.1-ErbB2后24h,即出现了细胞凋亡特异的核浓缩现象,并随时间推移而加重,同时出现DNA lader现象。NA细胞在转染pSilence2.1-STAT3或pSilence2.1-ErbB2后无核浓缩现象。同时我们使用Annexin-V试剂盒和流式细胞仪定量检测了凋亡的发生,结果表明:U251细胞在转染pSilence2.1-STAT3后出现明显的凋亡,并且细胞凋亡呈时间依赖性,在48h凋亡细胞数量达最高峰,约为总数的50%。而在转染pSilence2.1-ErbB2后,U251细胞在12h和24h时都无显著凋亡发生,而在48h时出现非常显著的细胞凋亡。NA细胞在转染pSilence2.1-STAT3或pSilence2.1-ErbB2后,48h内没有出现显著的细胞凋亡。流式细胞仪细胞周期检测结果表明:转染pSilence2.1-STAT3或pSilence2.1-ErbB2 12h时,细胞都出现S期和G2-M期阻滞,24h时细胞都出现G0-G1期阻滞,36h时,二者都为S期阻滞,并且G2-M期细胞百分比数都为0,可见细胞已停止分裂。
四.STAT3 RNAi和ErbB2 RNAi诱导U251细胞凋亡机制的研究
为了进一步确定STAT3 RNAi和ErbB2 RNAi诱导凋亡的机制,进一步测定了转染后不同时间点U251细胞以及NA细胞的caspase3/7、caspase8和caspase9的水平;Caspase抑制剂Z-VAD-FMK以及Caspase3/7抑制剂AC-DEVD-CHO加入后,凋亡发生的水平;利用JC-1探针测定线粒体膜电位水平,Caspase抑制剂Z-VAD-fmk以及caspase3/7抑制剂AC-DEVD-CHO加入后,线粒体膜电位水平;DCF探针测定ROS水平;MDA试剂盒测定丙二醛水平;彗星电泳实验测定DNA双链断裂;Western Blot检测凋亡相关蛋白水平。研究结果证明:(1)STAT3 RNAi和ErbB2 RNAi诱导U251细胞凋亡进程中激活caspase3/7,并能够引起线粒体膜电位明显降低。Caspase抑制剂Z-VAD-fmk可明显抑制caspase3/7活化,并抑制大部分细胞凋亡,可减弱线粒体膜电位降低的水平,这些都提示STAT3 RNAi和ErbB2 RNAi诱导的细胞凋亡为caspase依赖性细胞凋亡。(2)进一步发现STAT3 RNAi和ErbB2 RNAi后,同时存在线粒体途径凋亡和死亡受体途径凋亡,caspase8和caspase9的水平均有增高,且均能被Z-VAD和AC-DEVD-CHO大部分回复。(3)发现STAT3 RNAi诱导U251细胞凋亡过程中,伴随有ROS升高,ErbB2 RNAi诱导U251细胞凋亡过程中,伴随有MDA水平升高,均出现DNA双链断裂现象,提示凋亡与DNA损伤、ROS升高和膜损伤可能有关。(4)发现STAT3 RNAi和ErbB2 RNAi诱导U251细胞凋亡过程中,bcl2蛋白以及survivin蛋白表达降低。
本研究的创新之处在于:利用肿瘤组织高表达,而正常组织表达较低的核转录因子STAT3和膜受体酪氨酸激酶ErbB2为靶点治疗,结合放疗,提高U251辐射敏感性的复合治疗方法,比起单一方法治疗,更有针对性,可见到更明显的治疗效果。
Cerebral tumor,also known as intracranial tumor,often occurs slowly and aggravates gradually.The most common type of primary intracranial tumor is glioma(40%).At present,the major approaches to treat the disease are surgery, radiation therapy and chemotherapy.Since single method usually could not obtain an ideal therapeutic effect,combination of surgery,radiation therapy and chemotherapy is a routine choice.And the effect of radiation therapy and chemotherapy will influence greatly on the prognosis of the patient.So how to increase cell sensitivity to radiation and chemotherapy is a research focus.Since normal tissue surrounding the tumor will be damaged unavoidably during the course of routine treatment,an ideal approach to treat cerebral tumor is to selectively kill tumor cell with little effect on normal tissue.
RNAi can silence specific gene expression in highly sequence-specific manner. RNAi may have promising therapeutic effect on diseases caused by upregulation of certain gene.Tumorigenesis usually correlate with the overexpression of some protoncogenes,RNAi targeting these genes may specificly affect tumor cells with no harm to normal cells.Determination of eligible target gene is key to ideal therapeutic effect.
STAT3(signal transducers and activator of transcription 3)belongs to STATs protein family.STATs are a family of 7 transcription factors activated by polypeptide ligand,such as cytokine and growth factor.STAT3 is consistly activated in many tumors with important roles in development and aggravation of tumor.In addition, STAT3 can affect therapeutic effects of radiotherapy and chemotherapy on human pulmonary adenoma and rectal carcinoma.ErbB2 is a member of the ErbB receptor tyrosine kinase family.ErbB family are located on cell surface and also includes EGF-R(ErbB1),ErbB3 and ErbB4.Many studies have suggested that ErbB2 was highly expressed in tumor cells,and its expression level is used as pathological classification parameter and prognostic indicator of breast cancer.Ricardo et al suggested that downregulation of ErbB2 could increase radiosensitivity of tumar cells. It is known that tumor growth result from increased cell proliferation ability and cell apoptosis result in decreased tumor mass.Changes in tumor volume reflect dynamic equilibrium between cell proliferation and apoptosis.It has been shown that STAT3 and ErbB2 could maintain cell proliferation of many tumors.Based on tumor specific expression of STAT3 and ErbB2,their facilitation of cell proliferation,and their relationship with tumor radiotherapy and chemotherapy,STAT3 and ErbB2 were selected as targets of RNAi combined with radiotherapy for the experimental treatment of glioma.
Data are as follows:
1.To construct and screen RNAi plasmid vector,and to verify the silencing effect
We designed the target sequence of STAT3,ErbB2 and GFP control and constructed pSilence2.1-U6-STAT3,pSilence2.1-U6-ErbB2 and pSilence2.1-U6-GFP plasmids using pSilence2.1-U6-H1 vector,and then transfected them into U251 cell,a kind of human brain glioma cell line.The results of western blot assay and Real-Time PCR assay suggested that there is a 8-fold to 14-fold decrease in STAT3 and ErbB2 protein and mRNA expression.
2.The synergism effect of STAT3 RNAi and ErbB2 RNAi and the combining effect of RNAi with radiotherapy
Colony formation assay is the golden standard to assess the survival state and reproductive activity of cells.Cloning efficiency assay is often used for study of cell radiosensitivity.In this study,to determine the dose ofγ-ray used in this study,the cloning efficiency of cells was firstly analyzed after the cells were exposed to ~(60)Coγ-ray of different doses.According to the result of the cloning efficiency assay,the dose of 2Gy was defined as the dose of treatment.The result of MTT which was conducted to assess the ability of cell proliferation after exposure of ~(60)Coγ-ray of different dose further verified the result of cloning efficiency assay.Next,the study was divided into 10 groups,including the following:control,pSilence2.1-GFP,pSilence2.1-STAT3, pSilence2.1-ErbB2,pSilence2.1-STAT3+pSilence2.1-ErbB2,control+2Gy, pSilence2.1-GFP+2Gy,pSilence2.1-STAT3+2Gy,pSilence2.1-ErbB2+2Gy and pSilence2.1-STAT3+pSilence2.1-ErbB2+2Gy group.The cloning efficiency assay was conducted to determine the effect of different treatments on cell proliferation ability. The Result of the study including the following:
(1)Both STAT3 RNAi and ErbB2 RNAi could inhibit the proliferation of U251 cell, and they could take effect synergistically
(2)Both STAT3 RNAi and ErbB2 RNAi could enhance the radiosensitivity of U251 cell,and the U251 became more radiosensitive when STAT3 RNAi and ErbB2 RNAi was used simultaneously.
3.The effect of STAT3 RNAi and ErbB2 RNAi on the U251 apoptosis and cell cycle
It is well known that the tumorigenesis is mainly due to unlimited cell proliferation following cell cycle disturbance.Then,what was the cause that led the proliferation of U251 cell under control after transfecting the pSilence2.1-STAT3 and pSilence2.1-ErbB2 into U251 cell? Would there be the same effect after transfecting the plasmids into normal astrocyte cell? To clarify the two problems,the primary astrocyte cell(named NA cell)was isolated from the brain mantle of new born(within 24h)rat,and cultured to prepare for study.Firstly,Hoechst 33258 stain assay was conducted,and apoptosis-specific nuclear condense phenomenon was observed in the study after the U251 cell was transfected with pSilence2.1-STAT3 or pSilence2.1-ErbB2,and the phenomenon became more aggravated in a time-dependent manner.But the nuclear condense phenomenon didn't appear after the plasmids were tansfected into NA cell.The DNA ladder phenomenon was also observed after transfection.At the same time,the Annexin-V kit was used for quantifying the cell apoptosis.The result suggested that:U251 cell appeared significant apoptosis phenomenon in a time-dependent manner after it was transfected with pSilence2.1-STAT3,and rate of the apoptosis cell achieved the peak 48 hours after transfection with a rate of about 50%.The U251 cell transfected with pSilence2.1-ErbB2 didn't appear significant cell apoptosis until 48 hours after transfection.Any plasmid couldn't induce the NA cell apoptosis at any time after transfection.The cell cycle was also examined by flow cytometry.The result suggested that:the U251 cell was arrested in S and G2-M phase at 12 hours point after the cell was transfected with pSilence2.1-STAT3 or pSilence2.1-ErbB2;the U251 cell was arrested in G0-G1 phase at 24 hours point after the cell was transfected with pSilence2.1-STAT3 or pSilence2.1-ErbB2;the U251 cell was arrested in s phase at 36 hours point after the cell was transfected with pSilence2.1-STAT3 or pSilence2.1-ErbB2,Interestingly,there was no cell existing in G2-M phase at this time point after transfecting pSilence2.1-STAT3 or pSilence2.1-ErbB2 which indicated the U251 cell stopped dividing.
4.The research of STAT3 RNAi and ErbB2 RNAi apoptosis mechanism
To clarify the apoptosis mechanism,the levels of caspase3/7,caspase8 and caspase9 in U251 cell and NA cell were examined at different time point after transfection.The effect of Z-VAD-FMK(broad-spectrum caspase inhibitor)and AC-DEVD-CHO(caspase3/7-specific inhibitor)was also observed after adding them to the transfected cell.Mitochondria membrane potential was examined with or without Z-VAD-FMK and AC-DEVD-CHO after transfetion.ROS and MDA levels were also examined after transfection.Comet assay was conducted to detect the DNA double-strand breaks after transfection.The apoptosis-related protein was examined by western blot.The data of these studies were as fllowing:(1)Caspase3/7 were activated during the cause of apoptosis induced by STAT3 RNAi and ErbB2 RNAi, and Mitochondria membrane potential declined at the same time;Z-VAD-FMK could inhibit the activation of caspase3/7 significantly and most of the cell apoptosis,and it could weaken the decline of mitochondria membrane potential.All of the above result suggested that the apoptosis induced by STAT3 RNAi and ErbB2 RNAi was caspase-dependent.(2)Mitochondria apoptosis pathway and death receptor pathway were all involved in the apoptosis induced by STAT3 RNAi and ErbB2 RNAi.The level of caspase8 and caspase9 increased after transfection and the increase could be inhibited mostly by Z-VAD-FMK and AC-DEVD-CHO.(3)STAT3 RNAi could induce the increase of ROS level,and ErbB2 RNAi could induce the increase of MDA level.Not only STAT3 RNAi but also ErbB2 RNAi could induce DNA double-strain breaks.These results indicated that ROS and DNA double-strain breaks were involved in the cell apoptosis induced by RNAi.(4)The down regulation of bcl2 and survivin protein was observed after pSilence2.1-STAT3 and pSilence2.1-ErbB2 transfection.
The innovation of the study include the following:knockdown of 2 target genes, STAT3 and ErbB2 whose expression were high in tumour cell but low in normal cell,combined with radiotherapy to enhance the radiosensitivity of U251 cell,which was a combining therapy and more targeted and effective than single therapy.
RNAi具有高度的序列专一性,可以特异地使特定基因沉默,因此在治疗由于某个基因表达异常增高引起的疾病中非常有用。肿瘤的发生常常与原癌基因的表达异常增高相关,此时,利用RNAi技术下调这些异常高表达的基因,可特异性的影响肿瘤细胞,而对正常细胞无损伤,在实际研究过程中,找到符合要求的靶基因是获得理想治疗效果的关键。
STAT3(信号转导子和转录激活子3)属于STATS蛋白家族。STATs是一类存在于胞浆中的由细胞因子、生长因子等多肽类配体激活的转录因子,共有七个同源家族成员。STAT3在多种肿瘤组织中呈现持续性激活状态,在肿瘤的演变和恶化的过程中起着重要的作用。此外,STAT3还对人肺腺癌和直肠癌的放化疗敏感性有影响。ErbB2是位于细胞表面的受体酪氨酸激酶ErbB家族成员。ErbB家族还包括EGF-R(ErbB1),ErbB3和ErbB4。目前研究表明,许多肿瘤细胞系中的ErbB2呈高表达状态,并且ErbB2已经被用来作为乳腺癌病理分级指标参数和预后指标。Ricardo等还发现,ErbB2表达下调可以提高肿瘤细胞的辐射敏感性。众所周知,细胞增殖力增强将使肿瘤体积增大,细胞凋亡增强则将减小肿瘤体积,因此肿瘤体积改变的实质是细胞增殖和凋亡的动态平衡的最后外在体现。已有研究表明,STAT3和ErbB2对多种肿瘤细胞增殖力的维持具有重要作用。基于二者在肿瘤细胞中特异性高表达并且具有促进细胞增殖的功能,以及二者与肿瘤细胞放化疗敏感性的关系,因此本研究拟以STAT3和ErbB2为靶点应用RNAi技术、联合放疗治疗探索治疗恶性脑胶质瘤的新途径。
在本论文中,我们主要进行了以下四个方面的研究:
一.STAT3和ErbB2干涉载体的构建、筛选和干涉效果的确定
设计STAT3和ErbB2干涉的靶位点以及GFP对照靶位点,利用pSilence2.1-U6-H1载体,构建pSilence2.1-U6-STAT3,pSilence2.1-U6-ErbB2,pSilence2.1-U6-GFP质粒,转染人脑胶质瘤U251细胞。经Western Blot和Real-Time PCR实验验证表明:与对照组相比,实验组细胞靶蛋白和靶mRNA表达水平都降低了8～14倍。
二.STAT3 RNAi和ErbB2 RNAi抑制细胞增殖的协同作用及其与放疗的联合效果
克隆形成率是衡量细胞状态和增殖能力的金标准。在研究细胞辐射敏感性时,也通常要求使用克隆形成率来进行判断。为了确定本研究中使用的放疗剂量,我们首先用不同剂量~(60)Coγ射线照射U251细胞,随后对其进行克隆形成率实验,根据实验结果,确定2Gy为治疗用照射剂量。同时使用MTT方法检测不同剂量~(60)Coγ射线照射后U251细胞的增殖能力,结果与克隆形成率实验一致。我们将实验分为control组、pSilence2.1-GFP组、pSilence2.1-STAT3组、pSilence2.1-ErbB2组、pSilence2.1-STAT3+pSilence2.1-ErbB2组、control+2Gy组、pSilence2.1-GFP+2Gy组、pSilence2.1-STAT3+2Gy组、pSilence2.1-ErbB2+2Gy组以及pSilence2.1-STAT3+pSilence2.1-ErbB2+2Gy组,共10组,用克隆形成率实验检测各种处理对细胞增殖能力的影响。结果表明:
(1)STAT3 RNAi和ErbB2 RNAi均能抑制U251细胞增殖,且二者具有协同作用;
(2)STAT3 RNAi和ErbB2 RNAi均能增加U251细胞的辐射敏感性,将二者同时进行干涉时,U251细胞的辐射敏感性进一步增强。
三.STAT3 RNAi和ErbB2 RNAi对U251细胞凋亡及细胞周期的影响
肿瘤发生的主要原因是细胞周期失调后导致的细胞无限制增殖。那么,转染pSilence2.1-STAT3和pSilence2.1-ErbB2后,什么原因致使星形胶质瘤细胞U251的增殖得到控制了呢?二者对正常星形胶质细胞的增殖是否有同样的影响呢?为了帮助弄清这两个问题,使用出生24小时内大鼠,分离培养原代星形胶质细胞(命名为NA细胞),供研究使用。接下来,利用Hoechst 33258染色,观察到U251细胞在转染pSilence2.1-STAT3或pSilence2.1-ErbB2后24h,即出现了细胞凋亡特异的核浓缩现象,并随时间推移而加重,同时出现DNA lader现象。NA细胞在转染pSilence2.1-STAT3或pSilence2.1-ErbB2后无核浓缩现象。同时我们使用Annexin-V试剂盒和流式细胞仪定量检测了凋亡的发生,结果表明:U251细胞在转染pSilence2.1-STAT3后出现明显的凋亡,并且细胞凋亡呈时间依赖性,在48h凋亡细胞数量达最高峰,约为总数的50%。而在转染pSilence2.1-ErbB2后,U251细胞在12h和24h时都无显著凋亡发生,而在48h时出现非常显著的细胞凋亡。NA细胞在转染pSilence2.1-STAT3或pSilence2.1-ErbB2后,48h内没有出现显著的细胞凋亡。流式细胞仪细胞周期检测结果表明:转染pSilence2.1-STAT3或pSilence2.1-ErbB2 12h时,细胞都出现S期和G2-M期阻滞,24h时细胞都出现G0-G1期阻滞,36h时,二者都为S期阻滞,并且G2-M期细胞百分比数都为0,可见细胞已停止分裂。
四.STAT3 RNAi和ErbB2 RNAi诱导U251细胞凋亡机制的研究
为了进一步确定STAT3 RNAi和ErbB2 RNAi诱导凋亡的机制,进一步测定了转染后不同时间点U251细胞以及NA细胞的caspase3/7、caspase8和caspase9的水平;Caspase抑制剂Z-VAD-FMK以及Caspase3/7抑制剂AC-DEVD-CHO加入后,凋亡发生的水平;利用JC-1探针测定线粒体膜电位水平,Caspase抑制剂Z-VAD-fmk以及caspase3/7抑制剂AC-DEVD-CHO加入后,线粒体膜电位水平;DCF探针测定ROS水平;MDA试剂盒测定丙二醛水平;彗星电泳实验测定DNA双链断裂;Western Blot检测凋亡相关蛋白水平。研究结果证明:(1)STAT3 RNAi和ErbB2 RNAi诱导U251细胞凋亡进程中激活caspase3/7,并能够引起线粒体膜电位明显降低。Caspase抑制剂Z-VAD-fmk可明显抑制caspase3/7活化,并抑制大部分细胞凋亡,可减弱线粒体膜电位降低的水平,这些都提示STAT3 RNAi和ErbB2 RNAi诱导的细胞凋亡为caspase依赖性细胞凋亡。(2)进一步发现STAT3 RNAi和ErbB2 RNAi后,同时存在线粒体途径凋亡和死亡受体途径凋亡,caspase8和caspase9的水平均有增高,且均能被Z-VAD和AC-DEVD-CHO大部分回复。(3)发现STAT3 RNAi诱导U251细胞凋亡过程中,伴随有ROS升高,ErbB2 RNAi诱导U251细胞凋亡过程中,伴随有MDA水平升高,均出现DNA双链断裂现象,提示凋亡与DNA损伤、ROS升高和膜损伤可能有关。(4)发现STAT3 RNAi和ErbB2 RNAi诱导U251细胞凋亡过程中,bcl2蛋白以及survivin蛋白表达降低。
本研究的创新之处在于:利用肿瘤组织高表达,而正常组织表达较低的核转录因子STAT3和膜受体酪氨酸激酶ErbB2为靶点治疗,结合放疗,提高U251辐射敏感性的复合治疗方法,比起单一方法治疗,更有针对性,可见到更明显的治疗效果。
Cerebral tumor,also known as intracranial tumor,often occurs slowly and aggravates gradually.The most common type of primary intracranial tumor is glioma(40%).At present,the major approaches to treat the disease are surgery, radiation therapy and chemotherapy.Since single method usually could not obtain an ideal therapeutic effect,combination of surgery,radiation therapy and chemotherapy is a routine choice.And the effect of radiation therapy and chemotherapy will influence greatly on the prognosis of the patient.So how to increase cell sensitivity to radiation and chemotherapy is a research focus.Since normal tissue surrounding the tumor will be damaged unavoidably during the course of routine treatment,an ideal approach to treat cerebral tumor is to selectively kill tumor cell with little effect on normal tissue.
RNAi can silence specific gene expression in highly sequence-specific manner. RNAi may have promising therapeutic effect on diseases caused by upregulation of certain gene.Tumorigenesis usually correlate with the overexpression of some protoncogenes,RNAi targeting these genes may specificly affect tumor cells with no harm to normal cells.Determination of eligible target gene is key to ideal therapeutic effect.
STAT3(signal transducers and activator of transcription 3)belongs to STATs protein family.STATs are a family of 7 transcription factors activated by polypeptide ligand,such as cytokine and growth factor.STAT3 is consistly activated in many tumors with important roles in development and aggravation of tumor.In addition, STAT3 can affect therapeutic effects of radiotherapy and chemotherapy on human pulmonary adenoma and rectal carcinoma.ErbB2 is a member of the ErbB receptor tyrosine kinase family.ErbB family are located on cell surface and also includes EGF-R(ErbB1),ErbB3 and ErbB4.Many studies have suggested that ErbB2 was highly expressed in tumor cells,and its expression level is used as pathological classification parameter and prognostic indicator of breast cancer.Ricardo et al suggested that downregulation of ErbB2 could increase radiosensitivity of tumar cells. It is known that tumor growth result from increased cell proliferation ability and cell apoptosis result in decreased tumor mass.Changes in tumor volume reflect dynamic equilibrium between cell proliferation and apoptosis.It has been shown that STAT3 and ErbB2 could maintain cell proliferation of many tumors.Based on tumor specific expression of STAT3 and ErbB2,their facilitation of cell proliferation,and their relationship with tumor radiotherapy and chemotherapy,STAT3 and ErbB2 were selected as targets of RNAi combined with radiotherapy for the experimental treatment of glioma.
Data are as follows:
1.To construct and screen RNAi plasmid vector,and to verify the silencing effect
We designed the target sequence of STAT3,ErbB2 and GFP control and constructed pSilence2.1-U6-STAT3,pSilence2.1-U6-ErbB2 and pSilence2.1-U6-GFP plasmids using pSilence2.1-U6-H1 vector,and then transfected them into U251 cell,a kind of human brain glioma cell line.The results of western blot assay and Real-Time PCR assay suggested that there is a 8-fold to 14-fold decrease in STAT3 and ErbB2 protein and mRNA expression.
2.The synergism effect of STAT3 RNAi and ErbB2 RNAi and the combining effect of RNAi with radiotherapy
Colony formation assay is the golden standard to assess the survival state and reproductive activity of cells.Cloning efficiency assay is often used for study of cell radiosensitivity.In this study,to determine the dose ofγ-ray used in this study,the cloning efficiency of cells was firstly analyzed after the cells were exposed to ~(60)Coγ-ray of different doses.According to the result of the cloning efficiency assay,the dose of 2Gy was defined as the dose of treatment.The result of MTT which was conducted to assess the ability of cell proliferation after exposure of ~(60)Coγ-ray of different dose further verified the result of cloning efficiency assay.Next,the study was divided into 10 groups,including the following:control,pSilence2.1-GFP,pSilence2.1-STAT3, pSilence2.1-ErbB2,pSilence2.1-STAT3+pSilence2.1-ErbB2,control+2Gy, pSilence2.1-GFP+2Gy,pSilence2.1-STAT3+2Gy,pSilence2.1-ErbB2+2Gy and pSilence2.1-STAT3+pSilence2.1-ErbB2+2Gy group.The cloning efficiency assay was conducted to determine the effect of different treatments on cell proliferation ability. The Result of the study including the following:
(1)Both STAT3 RNAi and ErbB2 RNAi could inhibit the proliferation of U251 cell, and they could take effect synergistically
(2)Both STAT3 RNAi and ErbB2 RNAi could enhance the radiosensitivity of U251 cell,and the U251 became more radiosensitive when STAT3 RNAi and ErbB2 RNAi was used simultaneously.
3.The effect of STAT3 RNAi and ErbB2 RNAi on the U251 apoptosis and cell cycle
It is well known that the tumorigenesis is mainly due to unlimited cell proliferation following cell cycle disturbance.Then,what was the cause that led the proliferation of U251 cell under control after transfecting the pSilence2.1-STAT3 and pSilence2.1-ErbB2 into U251 cell? Would there be the same effect after transfecting the plasmids into normal astrocyte cell? To clarify the two problems,the primary astrocyte cell(named NA cell)was isolated from the brain mantle of new born(within 24h)rat,and cultured to prepare for study.Firstly,Hoechst 33258 stain assay was conducted,and apoptosis-specific nuclear condense phenomenon was observed in the study after the U251 cell was transfected with pSilence2.1-STAT3 or pSilence2.1-ErbB2,and the phenomenon became more aggravated in a time-dependent manner.But the nuclear condense phenomenon didn't appear after the plasmids were tansfected into NA cell.The DNA ladder phenomenon was also observed after transfection.At the same time,the Annexin-V kit was used for quantifying the cell apoptosis.The result suggested that:U251 cell appeared significant apoptosis phenomenon in a time-dependent manner after it was transfected with pSilence2.1-STAT3,and rate of the apoptosis cell achieved the peak 48 hours after transfection with a rate of about 50%.The U251 cell transfected with pSilence2.1-ErbB2 didn't appear significant cell apoptosis until 48 hours after transfection.Any plasmid couldn't induce the NA cell apoptosis at any time after transfection.The cell cycle was also examined by flow cytometry.The result suggested that:the U251 cell was arrested in S and G2-M phase at 12 hours point after the cell was transfected with pSilence2.1-STAT3 or pSilence2.1-ErbB2;the U251 cell was arrested in G0-G1 phase at 24 hours point after the cell was transfected with pSilence2.1-STAT3 or pSilence2.1-ErbB2;the U251 cell was arrested in s phase at 36 hours point after the cell was transfected with pSilence2.1-STAT3 or pSilence2.1-ErbB2,Interestingly,there was no cell existing in G2-M phase at this time point after transfecting pSilence2.1-STAT3 or pSilence2.1-ErbB2 which indicated the U251 cell stopped dividing.
4.The research of STAT3 RNAi and ErbB2 RNAi apoptosis mechanism
To clarify the apoptosis mechanism,the levels of caspase3/7,caspase8 and caspase9 in U251 cell and NA cell were examined at different time point after transfection.The effect of Z-VAD-FMK(broad-spectrum caspase inhibitor)and AC-DEVD-CHO(caspase3/7-specific inhibitor)was also observed after adding them to the transfected cell.Mitochondria membrane potential was examined with or without Z-VAD-FMK and AC-DEVD-CHO after transfetion.ROS and MDA levels were also examined after transfection.Comet assay was conducted to detect the DNA double-strand breaks after transfection.The apoptosis-related protein was examined by western blot.The data of these studies were as fllowing:(1)Caspase3/7 were activated during the cause of apoptosis induced by STAT3 RNAi and ErbB2 RNAi, and Mitochondria membrane potential declined at the same time;Z-VAD-FMK could inhibit the activation of caspase3/7 significantly and most of the cell apoptosis,and it could weaken the decline of mitochondria membrane potential.All of the above result suggested that the apoptosis induced by STAT3 RNAi and ErbB2 RNAi was caspase-dependent.(2)Mitochondria apoptosis pathway and death receptor pathway were all involved in the apoptosis induced by STAT3 RNAi and ErbB2 RNAi.The level of caspase8 and caspase9 increased after transfection and the increase could be inhibited mostly by Z-VAD-FMK and AC-DEVD-CHO.(3)STAT3 RNAi could induce the increase of ROS level,and ErbB2 RNAi could induce the increase of MDA level.Not only STAT3 RNAi but also ErbB2 RNAi could induce DNA double-strain breaks.These results indicated that ROS and DNA double-strain breaks were involved in the cell apoptosis induced by RNAi.(4)The down regulation of bcl2 and survivin protein was observed after pSilence2.1-STAT3 and pSilence2.1-ErbB2 transfection.
The innovation of the study include the following:knockdown of 2 target genes, STAT3 and ErbB2 whose expression were high in tumour cell but low in normal cell,combined with radiotherapy to enhance the radiosensitivity of U251 cell,which was a combining therapy and more targeted and effective than single therapy.
引文
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6.马向涛,王杉,杜如昱等,STAT3信号传导通路对结肠癌细胞G1～S期的调控[J],北京大学学报(医学版),2003,35(1):50-53.
7.Menendez JA,Vellon L,Mehrni I,et al,Inhibition of fatty acid synthase(FAS)suppresses HER2/neu(erbB-2)oncogene overexpression in cancer cells,PNAS,2004,101(29):10715-20.
8.Yen L,Cao Z,Wu X,et al,Loss of Nrdpl enhances ErbB2/ErbB3-dependent breast tumor cell growth,Cancer Res,2006,66(23):11279-86.
9.Liu B,Ordonez-Ercan D,Fan Z,et al,DownregμLation of erbB3 abrogates erbB2-mediated tamoxifen resistance in breast cancer cells,Int J Cancer,2007,120(9):1874-82.
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12. Lee SO, Lou W, Qureshi KM, et al, RNA interference targeting STAT3 inhibits growth and induces apoptosis of human prostate cancer cells [J], Prostate, 2004, 60(4):303-309.
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