CPEB4在人脑胶质瘤中的表达及其对胶质瘤细胞株增殖、凋亡和侵袭力的影响
发布时间：2018-07-15 09:20
【摘要】：脑胶质瘤是中枢神经系统最常见的原发恶性肿瘤，约占成人颅内肿瘤的35.26%-60.96%(平均44.69%)。由于其浸润性生长，在肿瘤与正常组织之间常无明显的边界，因此手术治疗难以彻底切除肿瘤组织。尽管我们努力尝试各种治疗方法，但是脑胶质瘤仍然无法治愈。这就要求我们为胶质瘤的治疗寻找新的策略。近年来，随着基因工程技术的发展，人们开始从分子生物学的角度探求脑胶质瘤的发病机制，脑胶质瘤的基因治疗逐渐受到关注。CPEB4是新近发现的胞质多聚腺苷酸化元件结合蛋白（CPEBs）家族中的一员，是一种具有序列特异性的RNA结合蛋白，含有一个RNA识别基序和一个锌指结构，位于人染色体5q21区域，定位于细胞质，长度为7769bp。胞质多聚腺苷酸化元件结合蛋白（CPEBs）于大约二十年前被首次发现在卵母细胞成熟过程中，其在已确定的信使RNA的翻译过程中起着重要的调控作用，人们在早期做了很多关于CPEB控制胞质多聚腺苷酸化和翻译的工作，发现CPEB通过调控mRNA这一活动而影响诸多进程如生殖细胞的发育、细胞分裂与细胞衰老、突触可塑性以及学习与记忆等。然而，关于近年CPEBs的一些热点研究表明它不仅在体细胞组织表达，而且在控制成年有机体时间和空间的基因表达方面具有重要的功能。这些新发现的功能包括调节衰老和增殖之间的平衡，及肿瘤的发生等。CPEBs在各种组织和肿瘤中高度表达，且有部分重叠。最近的一项新研究表明，CPEB4在胰腺癌和胶质母细胞瘤中高度表达，这与肿瘤的生长、恶性度的增加及血管生成等有关。然而，CPEB4在脑胶质瘤中的表达没有被详细描述。 鉴于以上研究背景，我们通过施行免疫组织化学、蛋白免疫印迹以及实时荧光定量PCR等实验方法，检测了CPEB4在正常脑组织和不同级别脑胶质瘤的蛋白及RNA水平的表达情况。此外，应用RNA干扰技术敲减胶质瘤细胞系中CPEB4的表达，来探究其在胶质瘤细胞增殖、凋亡和侵袭中的作用。本实验研究包括以下三个部分： 第一部分: CPEB4在人脑胶质瘤组织中的表达及其临床意义 目的：探讨CPEB4在正常脑组织中和各级别脑胶质瘤组织中的表达情况。 方法： 1收集样本:收集2011年10月至2012年11月期间在河北医科大学第二医院住院，经行手术切除的63例脑胶质瘤组织及9例正常脑组织。肿瘤标本均由神经病理医师确诊，正常脑组织取自脑出血或脑外伤需行内减压术的患者。每例患者分两部分留取标本，一部分置于10%福尔马林液中固定，另一部分保存于2ml无菌冻存管并立即投至液氮中速冻。 2应用免疫组织化学法检测:9例正常脑组织及63例胶质细胞瘤组织中CPEB4蛋白的表达情况，并比较其表达差异，分析其与脑胶质瘤病理级别的相关性。 3应用实时荧光定量聚合酶链反应（Real-time qPCR）测定CPEB4mRNA的表达水平。 4应用蛋白质免疫印迹Western blot检测正常脑组织及胶质瘤组织中CPEB4蛋白的表达。 结果： 1免疫组织化学结果：CPEB4在胶质细胞瘤组织中的总阳性表达率为88.89%(56/63)，CPEB4在9例正常脑组织中表达呈阴性或很弱，脑胶质瘤组织的CPEB4阳性表达率明显高于正常脑组织，具有显著的统计学意义（P＜0.01）；低级别（I、II级）和高级别（III、IV级）胶质瘤组织阳性表达率分别为75%（15/20）和95.4%（41/43），差异具有统计学意义（P＜0.05）。 2Western blot结果：CPEB4蛋白在脑胶质瘤组织中的表达明显高于正常脑组织。 CPEB4蛋白在正常脑组织及I、II、III、 IV级脑胶质瘤中相对表达量分别为0.070±0.012,0.417±0.044,0.495±0.043,0.709±0.080,0.713±0.027。CPEB4蛋白在低级别（I、II级）脑胶质瘤中的表达量低于高级别（III、IV级）（P＜0.05）。 3Real-time qPCR结果：CPEB4mRNA在正常脑组织、低级别及高级别中相对表达没有统计学差异（P＞0.05）。 结论： 1免疫组化与Western blot检测均得出CPEB4蛋白在人脑胶质瘤组织中高度表达，并且其表达随着胶质瘤病理级别的增高而增加，III、IV级脑胶质瘤中CPEB4蛋白表达要高于I、II级脑胶质瘤，差异显著；但是，在mRNA水平，CPEB4的表达却无明显差异。对于这种蛋白水平与RNA水平表达不一致的情况，我们分析可能是与此RNA的稳定性差、易降解有关。 2CPEB4的高表达与胶质瘤级别的升高呈显著的正相关，可作为脑胶质瘤临床分级的重要指标。 第二部分: CPEB4在人脑胶质瘤细胞株U87、U251中的表达 目的：检测CPEB4在人脑胶质瘤细胞株U87和U251中的表达情况，选出CPEB4表达量较高的细胞株以进行进一步的生物学功能试验。 方法： 1应用蛋白质免疫印迹Western blot检测U87和U251细胞中CPEB4蛋白表达情况。 2应用实时荧光定量聚合酶链反应（Real-time qPCR）测定U87和U251细胞中CPEB4mRNA的表达。 结果：人脑胶质瘤细胞株U87的CPEB4蛋白和mRNA均高于U251（P＜0.05）。 结论：U87细胞株可被选择作为CPEB4的优势表达株，进一步行以CPEB4为研究靶点的生物学功能试验第三部分CPEB4慢病毒载体感染人脑胶质瘤细胞株U87及其对U87细胞增殖、凋亡和侵袭力的影响研究 目的：构建CPEB4干扰慢病毒载体，以构建好的CPEB4siRNA干扰慢病毒载体感染U87细胞株，探讨RNA干扰(RNAinierference，RNAi)对胶质瘤细胞株U87中CPEB4表达的抑制作用及其对U87细胞增殖、凋亡和侵袭力的影响。 方法： 1构建CPEB4干扰慢病毒载体：针对目的基因CPEB4的序列信息，严格按照RNA干扰序列设计原则，设计3个RNA干扰靶点序列，合成SiRNA并向U87细胞分别转染3种si RNA，利用western blot筛选干扰效果最佳的siRNA，根据此siRNA序列，合成双链DNA Oligo，，两端带有BamHI和ECORI酶切位点粘性末端，退火后得到的双链产物无需酶切可直接连入BamHI和ECORI线性化的pSRL-SIH1-H1-GFP载体。将连接产物转入细菌感受态细胞，提取细菌培养物中的质粒，对长出的克隆抽提质粒并酶切鉴定，然后进行测序比对,鉴定阳性克隆即为构建成功的CPEB4RNA干扰慢病毒载体。 2在体外应用RNA干扰技术以siCPEB4(CPEB4SiRNA)靶向感染胶质瘤细胞株U87，分为转染组、阴性对照组和空白对照组； 3应用Western blot和Real time qPCR分别检测CPEB4-siRNA慢病毒感染U87细胞后1天、2天、3天、4天、5天细胞中CPEB4蛋白和mRNA相对表达量变化； 4应用流式细胞技术（FCM）检测CPEB4SiRNA慢病毒感染U87细胞后的细胞周期变化，并检测分析转染前后细胞凋亡水平变化； 5应用MTT比色实验检测分析感染CPEB4-SiRNA慢病毒1天、2天、3天、4天、5天、6天及7天后U87细胞的增殖情况； 6应用Transwell侵袭实验检测分析转染前后细胞体外侵袭能力的变化。 结果： 1成功构建并包装了携带CPEB4基因的慢病毒表达载体，经过阳性克隆测序证实插入序列完全正确； 2Western blot和Real time qPCR结果显示，慢病毒感染组的CPEB4蛋白和mRNA相对表达量明显低于空白对照组与阴性对照组，差异具有统计学意义(P0.05)；空白对照组与阴性对照组间表达无统计学差异(P0.05)； 3流式细胞仪检测细胞周期结果显示，相对于阴性对照组细胞以及空白对照组细胞，转染CPEB4siRNA慢病毒后U87细胞阻滞于G1期，S期细胞数相对较少；阴性对照组和空白对照组的U87细胞的细胞状态和细胞周期没有变化；流式细胞仪分析各组细胞的凋亡水平显示，相对于空白对照组细胞(0.13±0.08)%和阴性对照组细胞(0.47±0.12)%，U87细胞感染CPEB4si-RNA慢病毒后早期凋亡率增加，达(19.16±3.85)%，具有统计学差异(P0.01)； 4MTT检测细胞活性显示，从第三天起，与阴性对照组（0.476±0.048）和未转染组（0.495±0.031）相比，慢病毒感染组（0.42±0.05）细胞生长速度开始减慢，并具有统计学差异(P＜0.05)，且随感染时间的延长，抑制程度越来越明显。而未转染组与阴性对照组组之间无明显差异(P0.05)。说明CPEB4siRNA对U87细胞生长增殖具有显著抑制作用； 5Transwell侵袭试验结果显示，经CPEB4siRNA慢病毒感染后，U87细胞穿膜数量明显减少，低于阴性对照组及空白对照组，差异具有统计学意义。换句话说, CPEB4能够促进胶质瘤细胞的体外侵袭力。 结论： CPEB4靶向转染能够有效敲减这个基因在人脑胶质瘤U87细胞系内的表达，使肿瘤细胞增殖和侵袭能力受到显著抑制，阻滞细胞周期，并促进其凋亡增加，为脑胶质瘤基因治疗提供了新的靶点。
[Abstract]:Human glioma is one of the most common primary malignant tumors in the central nervous system , accounting for 35.26 % - 60.96 % ( mean 44.69 % ) of intracranial tumors .
In view of the above background , we examined the expression of CPEB4 in normal brain tissue and brain glioma at different levels by performing immunohistochemistry , Western blotting and real - time fluorescence quantitative PCR .
The expression of CPEB4 in human glioma tissues and its clinical significance
Objective : To investigate the expression of CPEB4 in normal brain tissue and brain glioma at all levels .
Method :
Samples were collected : 63 cases of brain glioma and 9 normal brain tissues were collected from the Second Affiliated Hospital of Hebei Medical University between October 2011 and November 2012 . All the tumor specimens were confirmed by neurologists . Normal brain tissues were taken from patients with intracerebral hemorrhage or traumatic brain trauma . The samples were collected in two parts . One part was fixed in 10 % formalin solution , and the other part was stored in 2 ml of sterile frozen tubes and immediately frozen in liquid nitrogen .
The expression of CPEB4 protein in 9 normal brain tissues and 63 cases of gliocytoma were detected by immunohistochemical method . The correlation between the expression of CPEB4 and the pathological grade of glioma was analyzed .
3 . Real - time fluorescence quantitative polymerase chain reaction ( Real - time qPCR ) was used to determine the expression level of CPEB4mRNA .
4 . Western blot was used to detect the expression of CPEB4 protein in normal brain tissues and glioma tissues .
Results :
Results : The expression rate of CPEB4 was 88.89 % ( 56 / 63 ) . CPEB4 was negative or weak in 9 normal brain tissues , and the positive rate of CPEB4 was significantly higher than that of normal brain tissue ( P < 0.01 ) .
The positive expression rates of low - grade ( I , II ) and high - level ( III , IV ) glioma tissues were 75 % ( 15 / 20 ) and 95.4 % ( 41 / 43 ) , respectively . The difference was statistically significant ( P < 0.05 ) .
2 Western blot showed that the expression of CPEB4 protein in brain glioma was significantly higher than that of normal brain tissue . The relative expression levels of CPEB4 protein in normal brain tissue and I , II , III and IV gliomas were 0.070 卤 0.012 , 0.417 卤 0.044 , 0.495 卤 0.043 , 0.709 卤 0.080 , 0.713 卤 0.027 . CPEB4 protein was lower in low grade gliomas ( grade I , II ) than in high level ( grade III , IV ) ( P < 0.05 ) .
3 Real - time qPCR results showed that the relative expression of CPEB4mRNA in normal brain tissue , low grade and high level was not significantly different ( P > 0.05 ) .
Conclusion :
1 Immunohistochemical and Western blot analysis showed that CPEB4 protein was highly expressed in human glioma tissues , and the expression of CPEB4 increased with the pathological grade of glioma .
However , in the case of mRNA levels , there was no significant difference in the expression of CPEB4 . In the case of non - uniform expression of this protein level with the level of RNA , we analyzed the possibility of poor stability and easy degradation of this RNA .
The high expression of 2CPEB4 is positively correlated with the increase of glioma grade , which can be used as an important index for clinical grading of glioma .
The second part : the expression of CPEB4 in human glioma cell line U87 and U251
Objective : To detect the expression of CPEB4 in human glioma cell lines U87 and U251 , and to select the cell line with high expression of CPEB4 for further biological function test .
Method :
1 . Western blot was used to detect the expression of CPEB4 protein in U87 and U251 cells .
2 . Real - time quantitative polymerase chain reaction ( Real - time qPCR ) was used to determine the expression of CPEB4mRNA in U87 and U251 cells .
Results : The CPEB4 protein and mRNA of human glioma cell line U87 were higher than U251 ( P < 0.05 ) .
Conclusion : U87 cell line can be selected as the dominant expression strain of CPEB4 , and further study the effect of CPEB4 on the biological function test of target site . The third part CPEB4 lentivirus vector is infected with human glioma cell line U87 and its effect on proliferation , apoptosis and invasion of U87 cell line .
Objective : To construct CPEB4 interfering slow virus vector , construct good CPEB4siRNA interfering slow virus vector to infect U87 cell line , investigate the inhibitory effect of RNA interference ( RNAi ) on the expression of CPEB4 in human glioma cell line U87 and its effect on proliferation , apoptosis and invasion of U87 cell line .
Method :
1 . Construction of CPEB4 interfering lentivirus vector : According to the sequence information of the target gene CPEB4 , three RNA interference target sequences were designed strictly according to the design principles of RNA interference sequence . Three kinds of siRNA were synthesized by Western blot . Two - stranded DNA oligo - stranded DNA Oligo was synthesized . Two - stranded DNA oligo - stranded DNA was synthesized and digested with BamHI and ECORI .
2 In vitro RNA interference technique was used to target cell line U87 with siCPEB4 ( CPEB4SiRNA ) , which was divided into transfection group , negative control group and blank control group .
3 . Western blot and Real time qPCR were used to detect the changes of CPEB4 protein and mRNA relative expression in the cells of CPEB4 - siRNA infected with CPEB4 - siRNA for 1 day , 2 days , 3 days , 4 days and 5 days respectively .
4 . Flow cytometry ( FCM ) was used to detect the changes of cell cycle after CPEB4SiRNA virus infection .
5 . MTT colorimetric assay was used to detect the proliferation of U87 cells infected with CPEB4 - SiRNA in 1 day , 2 days , 3 days , 4 days , 5 days , 6 days and 7 days .
6 . Transwell invasion assay was used to detect the changes of invasion ability of cells in vitro and after transfection .
Results :
1 . The lentivirus expression vector carrying CPEB4 gene was successfully constructed and packaged , and the insertion sequence was confirmed by positive clone sequencing .
2 Western blot and Real time qPCR showed that the relative expression of CPEB4 protein and mRNA in chronic viral infection group was significantly lower than that in control group and negative control group ( P0.05 ) .
There was no statistical difference between the blank control group and the negative control group ( P0.05 ) .
The results showed that the number of cells transfected with CPEB4siRNA was lower than that of negative control group and blank control group .
Cell status and cell cycle of U87 cells in the negative control group and the blank control group were not changed ;
Flow cytometry showed that the apoptosis rate of cells in each group was significantly higher than that of control group ( 0.13 卤 0.08 ) % and negative control group ( 0.47 卤 0.12 ) % , and the early apoptotic rate of U87 cells was increased by ( 19.16 卤 3.85 ) % , which was statistically significant ( P0.01 ) .
Compared with the negative control group ( 0.476 卤 0.048 ) and the untransfected group ( 0.495 卤 0.031 ) , the growth rate of the slow virus infection group ( 0.42 卤 0.05 ) was slower than that of the negative control group ( P < 0.05 ) .
5Transwell invasion assay showed that the number of U87 cells decreased significantly after CPEB4siRNA was infected with slow virus infection , which was lower than that of negative control group and blank control group . In other words , CPEB4 could promote the invasion of glioma cells in vitro .
Conclusion :
CPEB4 targeted transfection can effectively reduce the expression of the gene in human glioma U87 cell line , inhibit the proliferation and invasion ability of tumor cells , block the cell cycle , and promote the increase of apoptosis , thus providing a new target for gene therapy of glioma .
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.41
本文编号：2123578
[Abstract]:Human glioma is one of the most common primary malignant tumors in the central nervous system , accounting for 35.26 % - 60.96 % ( mean 44.69 % ) of intracranial tumors .
In view of the above background , we examined the expression of CPEB4 in normal brain tissue and brain glioma at different levels by performing immunohistochemistry , Western blotting and real - time fluorescence quantitative PCR .
The expression of CPEB4 in human glioma tissues and its clinical significance
Objective : To investigate the expression of CPEB4 in normal brain tissue and brain glioma at all levels .
Method :
Samples were collected : 63 cases of brain glioma and 9 normal brain tissues were collected from the Second Affiliated Hospital of Hebei Medical University between October 2011 and November 2012 . All the tumor specimens were confirmed by neurologists . Normal brain tissues were taken from patients with intracerebral hemorrhage or traumatic brain trauma . The samples were collected in two parts . One part was fixed in 10 % formalin solution , and the other part was stored in 2 ml of sterile frozen tubes and immediately frozen in liquid nitrogen .
The expression of CPEB4 protein in 9 normal brain tissues and 63 cases of gliocytoma were detected by immunohistochemical method . The correlation between the expression of CPEB4 and the pathological grade of glioma was analyzed .
3 . Real - time fluorescence quantitative polymerase chain reaction ( Real - time qPCR ) was used to determine the expression level of CPEB4mRNA .
4 . Western blot was used to detect the expression of CPEB4 protein in normal brain tissues and glioma tissues .
Results :
Results : The expression rate of CPEB4 was 88.89 % ( 56 / 63 ) . CPEB4 was negative or weak in 9 normal brain tissues , and the positive rate of CPEB4 was significantly higher than that of normal brain tissue ( P < 0.01 ) .
The positive expression rates of low - grade ( I , II ) and high - level ( III , IV ) glioma tissues were 75 % ( 15 / 20 ) and 95.4 % ( 41 / 43 ) , respectively . The difference was statistically significant ( P < 0.05 ) .
2 Western blot showed that the expression of CPEB4 protein in brain glioma was significantly higher than that of normal brain tissue . The relative expression levels of CPEB4 protein in normal brain tissue and I , II , III and IV gliomas were 0.070 卤 0.012 , 0.417 卤 0.044 , 0.495 卤 0.043 , 0.709 卤 0.080 , 0.713 卤 0.027 . CPEB4 protein was lower in low grade gliomas ( grade I , II ) than in high level ( grade III , IV ) ( P < 0.05 ) .
3 Real - time qPCR results showed that the relative expression of CPEB4mRNA in normal brain tissue , low grade and high level was not significantly different ( P > 0.05 ) .
Conclusion :
1 Immunohistochemical and Western blot analysis showed that CPEB4 protein was highly expressed in human glioma tissues , and the expression of CPEB4 increased with the pathological grade of glioma .
However , in the case of mRNA levels , there was no significant difference in the expression of CPEB4 . In the case of non - uniform expression of this protein level with the level of RNA , we analyzed the possibility of poor stability and easy degradation of this RNA .
The high expression of 2CPEB4 is positively correlated with the increase of glioma grade , which can be used as an important index for clinical grading of glioma .
The second part : the expression of CPEB4 in human glioma cell line U87 and U251
Objective : To detect the expression of CPEB4 in human glioma cell lines U87 and U251 , and to select the cell line with high expression of CPEB4 for further biological function test .
Method :
1 . Western blot was used to detect the expression of CPEB4 protein in U87 and U251 cells .
2 . Real - time quantitative polymerase chain reaction ( Real - time qPCR ) was used to determine the expression of CPEB4mRNA in U87 and U251 cells .
Results : The CPEB4 protein and mRNA of human glioma cell line U87 were higher than U251 ( P < 0.05 ) .
Conclusion : U87 cell line can be selected as the dominant expression strain of CPEB4 , and further study the effect of CPEB4 on the biological function test of target site . The third part CPEB4 lentivirus vector is infected with human glioma cell line U87 and its effect on proliferation , apoptosis and invasion of U87 cell line .
Objective : To construct CPEB4 interfering slow virus vector , construct good CPEB4siRNA interfering slow virus vector to infect U87 cell line , investigate the inhibitory effect of RNA interference ( RNAi ) on the expression of CPEB4 in human glioma cell line U87 and its effect on proliferation , apoptosis and invasion of U87 cell line .
Method :
1 . Construction of CPEB4 interfering lentivirus vector : According to the sequence information of the target gene CPEB4 , three RNA interference target sequences were designed strictly according to the design principles of RNA interference sequence . Three kinds of siRNA were synthesized by Western blot . Two - stranded DNA oligo - stranded DNA Oligo was synthesized . Two - stranded DNA oligo - stranded DNA was synthesized and digested with BamHI and ECORI .
2 In vitro RNA interference technique was used to target cell line U87 with siCPEB4 ( CPEB4SiRNA ) , which was divided into transfection group , negative control group and blank control group .
3 . Western blot and Real time qPCR were used to detect the changes of CPEB4 protein and mRNA relative expression in the cells of CPEB4 - siRNA infected with CPEB4 - siRNA for 1 day , 2 days , 3 days , 4 days and 5 days respectively .
4 . Flow cytometry ( FCM ) was used to detect the changes of cell cycle after CPEB4SiRNA virus infection .
5 . MTT colorimetric assay was used to detect the proliferation of U87 cells infected with CPEB4 - SiRNA in 1 day , 2 days , 3 days , 4 days , 5 days , 6 days and 7 days .
6 . Transwell invasion assay was used to detect the changes of invasion ability of cells in vitro and after transfection .
Results :
1 . The lentivirus expression vector carrying CPEB4 gene was successfully constructed and packaged , and the insertion sequence was confirmed by positive clone sequencing .
2 Western blot and Real time qPCR showed that the relative expression of CPEB4 protein and mRNA in chronic viral infection group was significantly lower than that in control group and negative control group ( P0.05 ) .
There was no statistical difference between the blank control group and the negative control group ( P0.05 ) .
The results showed that the number of cells transfected with CPEB4siRNA was lower than that of negative control group and blank control group .
Cell status and cell cycle of U87 cells in the negative control group and the blank control group were not changed ;
Flow cytometry showed that the apoptosis rate of cells in each group was significantly higher than that of control group ( 0.13 卤 0.08 ) % and negative control group ( 0.47 卤 0.12 ) % , and the early apoptotic rate of U87 cells was increased by ( 19.16 卤 3.85 ) % , which was statistically significant ( P0.01 ) .
Compared with the negative control group ( 0.476 卤 0.048 ) and the untransfected group ( 0.495 卤 0.031 ) , the growth rate of the slow virus infection group ( 0.42 卤 0.05 ) was slower than that of the negative control group ( P < 0.05 ) .
5Transwell invasion assay showed that the number of U87 cells decreased significantly after CPEB4siRNA was infected with slow virus infection , which was lower than that of negative control group and blank control group . In other words , CPEB4 could promote the invasion of glioma cells in vitro .
Conclusion :
CPEB4 targeted transfection can effectively reduce the expression of the gene in human glioma U87 cell line , inhibit the proliferation and invasion ability of tumor cells , block the cell cycle , and promote the increase of apoptosis , thus providing a new target for gene therapy of glioma .
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.41
【引证文献】
相关期刊论文 前1条
1 张亚飞;;不同级别脑胶质瘤及正常脑组织中MGMT与XRCC1基因的表达及相关性研究[J];海南医学院学报;2015年09期
本文编号：2123578
本文链接：https://www.wllwen.com/yixuelunwen/shenjingyixue/2123578.html
最近更新
教材专著
