CUGBP1在人星形细胞瘤中的表达及其敲减后对U251细胞的影响机制研究
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摘要
近年来,肿瘤发生的机制已经成为国内外学者关注的焦点,而从分子生物学角度阐述肿瘤发生、发展等越来越受到重视。在我国,神经胶质瘤是神经系统的第一大肿瘤,具有发病率高、进展快、预后差等特点,而且目前发病有上升趋势。作为RNA结合蛋白质CELF家族的一员,CUGBP1在细胞核内通过刺激非连续外显子的保留或删除而调节一些mRNA前体的选择性剪接;在细胞质中,通过结合到靶向mRNA的特定序列而调节一些mRNA的翻译或降解;还可通过RNA编辑酶而调节一些mRNA的编辑。CUGBP1不仅在胚胎及心脏发育、骨骼肌及脂肪组织分化、生殖细胞形成等方面具有重要的影响,而且在肝脏细胞及乳腺上皮细胞增殖甚至肿瘤形成以及一些肿瘤的恶化中发挥作用。但其在人胶质瘤细胞中是否异常表达以及改变这种表达后对胶质瘤有何影响,国内外尚未见相关报道。
研究意义
旨在分析CUGBP1在胶质瘤细胞中的表达水平,明确其与人胶质瘤的关系;同时,从细胞增殖、细胞凋亡的现象及相关基因的变化解释CUGBP1与胶质瘤发生、发展的关系。
研究目的
通过CUGBP1在不同胶质瘤细胞株、胶质瘤脑组织以及正常脑组织中的表达检测和观察CUGBP1基因敲减后细胞增殖、细胞凋亡变化以及相关基因表达改变,分析CUGBP1与胶质瘤发生的相关性,以阐明CUGBP1在胶质瘤发生、发展中的作用及机制。
实验方法
1、表达检测选择RT-PCR和实时定量RT-PCR方法从mRNA水平上观察并比较CUGBP1在不同胶质瘤细胞株、胶质瘤脑组织以及正常脑组织中的表达。
半定量RT-PCR法检测CUGBP1在不同胶质瘤细胞株的表达,同时以管家基因GAPDH作为系统阳性对照。
实时定量RT-PCR法检测CUGBP1在不同病理级别胶质瘤脑组织的表达水平,并以正常脑组织中的水平作为参照标准,同时以GAPDH作为系统阳性对照。
2、RNA干扰慢病毒构建及其敲减效率检测为进一步探索CUGBP1与胶质瘤的相关性及其机制,构建CUGBP1-shRNA慢病毒,转染胶质瘤细胞株U251,并检测其转染及敲减效率。
2.1 RNA干扰慢病毒构建设计干扰靶点序列,连接到慢病毒载体,包装成病毒颗粒。
2.2细胞计数运用普通光学显微镜和荧光显微镜观察和细胞计数,评估转染效率。
2.3实时定量RT-PCR法检测CUGBP1-shRNA慢病毒转染胶质瘤细胞的敲减效果。
3、细胞增殖CUGBP1在某些病理情况下与肝脏及乳腺上皮细胞的增殖密切相关,是否在胶质瘤发生中也有类似现象,我们通过CUGBP1-shRNA慢病毒转染胶质瘤细胞而敲减CUGBP1表达以揭示其与细胞增殖的关系。
3.1细胞计数,绘制CUGBP1-shRNA慢病毒转染组及Scr-shRNA慢病毒转染组两组细胞生长曲线,分析敲减对细胞生长的影响。
3.2双抗体夹心酶标免疫分析法测定转染CUGBP1-shRNA慢病毒的人胶质瘤细胞BrdU掺入的OD值,同时以Scr-shRNA慢病毒转染细胞作为阴性对照,分析敲减对DNA合成及细胞增殖的影响。
4、细胞周期分布及细胞凋亡CUGBP1在强直性肌营养不良成肌细胞中使某些与细胞周期退出相关的蛋白质如P21等表达或活性改变,进而使细胞周期退出障碍,从而发生突变或异常增生的几率增加。这种现象是否也出现在胶质瘤细胞,我们通过CUGBP1-shRNA慢病毒转染胶质瘤细胞而敲减CUGBP1表达以揭示其与细胞周期分布及细胞凋亡的关系。
4.1运用FACS检测CUGBP1-shRNA慢病毒转染胶质瘤细胞后的细胞周期分布比例,同时以转染Scr-shRNA慢病毒细胞作为阴性对照,分析敲减对细胞周期分布的影响。
4.2运用FACS检测CUGBP1-shRNA慢病毒转染胶质瘤细胞后的细胞凋亡比例,同时以转染Scr-shRNA慢病毒细胞作为阴性对照,分析敲减对细胞凋亡的影响
5、细胞周期及细胞凋亡相关基因检测为进一步探究CUGBP1与胶质瘤细胞增殖和凋亡相关的机制,我们检测了敲减后相关基因的表达。
5.1实时定量RT-PCR法检测敲减后凋亡抑制基因BCL2的表达并与阴性对照比较,分析敲减对BCL2的影响。
5.2实时定量RT-PCR法检测敲减后细胞周期抑制基因CDKN1A的表达并与阴性对照比较,分析敲减对CDKN1A的影响。
5.3实时定量RT-PCR法检测敲减后DNA合成相关基因PCNA的表达并与阴性对照比较,分析敲减对PCNA的影响。
5.4实时定量RT-PCR法检测敲减后DNA合成相关基因MCM2-7的表达并与阴性对照比较,分析敲减对MCM2-7的影响。
实验结果
1、表达检测
1.1以管家基因GAPDH作为参照,RT-PCR显示CUGBP1 mRNA在不同胶质瘤细胞株中均有表达。
1.2以管家基因GAPDH作为内参,实时定量RT-PCR显示在人胶质瘤组织中CUGBP1 mRNA的表达量较正常脑组织明显增高(约2.96倍),并且随着肿瘤分级的增加,其表达量有增高的趋势,胶质瘤Ⅰ级增高约2.26倍、胶质瘤Ⅲ级增高约2.36倍、胶质瘤Ⅳ级增高约4.27倍。
2、敲减效率检测
2.1运用普通光学显微镜和荧光显微镜观察和计数CUGBP1-shRNA慢病毒转染组及Scr-shRNA慢病毒转染组两组转染后72小时荧光阳性细胞占所有胶质瘤细胞的比例,显示均大于95%,提示高效转染。
2.2以管家基因GAPDH作为内参,实时定量RT-PCR显示CUGBP1-shRNA慢病毒转染胶质瘤细胞中CUGBP1 mRNA的表达量较阴性对照组降低,最高达约92%,提示CUGBP1-shRNA慢病毒能高效转染胶质瘤细胞株,并使CUGBP基因在mRNA水平上受到明显抑制,是一种可靠的基因沉默工具。
3、细胞增殖
3.1以Scr-shRNA慢病毒转染细胞作为阴性对照,借助CELLOMICS对转染CUGBP1-shRNA慢病毒人胶质瘤细胞每天检测读板一次并拍照,连续5天。结果显示对照组细胞随着培养天数的增加细胞数目也逐渐增加,第5天时细胞数目增加了3倍;而CUGBP1-shRNA组的细胞数目没有明显变化,提示CUGBP1基因敲减使人胶质瘤细胞的增殖受到显著抑制。
3.2以Scr-shRNA慢病毒转染细胞作为阴性对照,利用双抗体夹心酶标免疫分析法测定转染CUGBP1-shRNA慢病毒胶质瘤细胞BrdU掺入的OD值,结果显示CUGBP1-shRNA组胶质瘤细胞的ODBrdU在第一天和第三天均比阴性对照组减少,第三天的减少更显著,提示CUGBP1基因敲减抑制了DNA合成,进而抑制胶质瘤细胞的增殖能力。
4、细胞周期及细胞凋亡
4.1以Scr-shRNA慢病毒转染细胞作阴性对照,利用FACS对CUGBP1-shRNA慢病毒转染人胶质瘤细胞进行细胞周期检测,显示CUGBP1-shRNA转染胶质瘤细胞处于S期的细胞比例显著增加,达67.71%,而处于G0//G1期和G2/M期的细胞比例明显减少,提示CUGBP1基因敲减使胶质瘤细胞的细胞周期分布发生改变,S期细胞比例增多,表明发生S期阻滞。
4.2以Scr-shRNA慢病毒转染细胞作阴性对照,利用FACS对CUGBP1-shRNA慢
病毒转染人胶质瘤细胞进行细胞凋亡检测,显示CUGBP1-shRNA转染胶质瘤细胞的细胞凋亡比例与对照组比较有明显的增加,达73.8%,提示CUGBP1基因敲减使胶质瘤细胞株的细胞(早期)凋亡明显增加,表明该基因有抑制胶质瘤细胞凋亡的作用。
5、细胞周期及细胞凋亡相关基因表达
5.1以管家基因GAPDH作为内参,采用实时定量RT-PCR方法对CUGBP1-shRNA慢病毒感染胶质瘤细胞的BCL2 mRNA的表达进行检测,同时以Scr-shRNA慢病毒感染细胞作为阴性对照,结果显示CUGBP1-shRNA组胶质瘤细胞BCL2 mRNA的表达量较阴性对照组显著降低。提示CUGBP 1基因敲减下调了BCL2基因的表达,因而CUGBP1基因具有上调BCL2的表达进而抑制细胞凋亡,促进肿瘤细胞生长的作用。
5.2以管家基因GAPDH作为内参,采用Real-time定量RT-PCR的方法对CUGBP1-shRNA慢病毒感染胶质瘤细胞CDKN1A mRNA的表达进行检测,同时以Scr-shRNA慢病毒感染细胞作为阴性对照,结果显示CUGBP1-shRNA组胶质瘤细胞CDKN1A mRNA的表达量较阴性对照组显著升高,提示CUGBP1基因敲减上调了CDKN1A基因的表达,因而CUGBP1基因具有下调CDKN1A基因的表达进而解除其对细胞周期的抑制,促进肿瘤细胞生长的作用。
5.3以管家基因GAPDH作为内参,采用Real-time定量RT-PCR方法对CUGBP1-shRNA慢病毒感染胶质瘤细胞PCNA mRNA的表达进行检测,同时以Scr-shRNA慢病毒感染细胞作为阴性对照,结果显示CUGBP1-shRNA组胶质瘤细胞的PCNA mRNA表达量较阴性对照组显著降低,提示CUGBP1基因敲减下调了PCNA基因的表达,因而CUGBP1基因具有上调PCNA基因表达进而促进DNA合成、促进胶质瘤细胞生长的作用。
5.4以管家基因GAPDH作为内参,采用Real-time定量RT-PCR方法对CUGBP1-shRNA慢病毒感染胶质瘤细胞MCM2-7 mRNA的表达进行检测,同时以Scr-shRNA慢病毒感染细胞作为阴性对照,结果显示CUGBP1-shRNA组胶质瘤细胞的MCM2-7 mRNA的表达量除MCM6-7轻度降低外,其它均较阴性对照组显著降低,提示CUGBP1基因敲减下调了MCM2-7基因的表达,因而CUGBP1基因具有上调MCM2-5基因表达进而促进细胞DNA合成、促进胶质瘤细胞生长的作用。
结论:
1、本研究首次发现CUGBP1在不同胶质瘤细胞株细胞及人胶质瘤组织中高表达,而且其表达有随胶质瘤病理分级的增加而增加的趋势。
2、CUGBP1-shRNA慢病毒能够高效转染恶性胶质瘤细胞株U251细胞并稳定敲减其CUGBP1基因表达。
3、CUGBP1基因敲减能够抑制恶性胶质瘤U251细胞增殖及DNA合成功能,因而抑制U251细胞的生长。
4、CUGBP1基因敲减能够引起恶性胶质瘤U251细胞S期阻滞,抑制细胞周期进程;CUGBP1基因敲减还能够诱导U251细胞(早期)凋亡。
5、CUGBP1基因敲减能够下调细胞凋亡抑制基因BCL2表达,上调细胞周期抑制基因CDKN1A表达,还能够下调DNA合成相关基因PCNA及MCM2-7表达。以上结论提示CUGBP1敲减可能通过抑制细胞增殖及DNA合成、抑制细胞周期进程及诱导细胞凋亡而抑制胶质瘤的发生和发展;这间接证明CUGBP1具有促进细胞增殖及DNA合成,促进细胞周期进程及抑制细胞凋亡,进而促进胶质瘤发生发展的癌基因样作用。CUGBP1可以作为恶性胶质瘤的预测指标之一并可作为恶性胶质瘤基因治疗的潜在分子靶点。
Recently, the mechanisms of tumorigenesis have been the focus of scholars at home and abroad, and it has been given increasing attention to elucidate tumor genesis and metastasis from the perspective of tumor molecular biology. In China, the neurogliomas is the first neoplasm of nervous system with high incidence, rapid process, and poor prognosis, and the morbidity tends to rise at present. As a member of RNA binding protein CELF family, CUGBP1 can regulate the alternative splicing by stimulating inclusion or exclusion of the non-consecutive exons in the nucleus pre- mRNA. In the cytoplasm, it can adjust their translation or degradation by binding to the specific sequence in certain target mRNAs. It can also influence the mRNA editing through RNA editing enzyme complex. CUGBP1 is involved in embryonic and cardiac development, skeletal muscle and adipose tissue differentiation and germ cell formation, and also play a great role in the liver cells and mammary epithelial cell proliferation, tumor genesis, as well as the deterioration of certain tumors. Whether CUGBP1 is abnormally expressed in human glioma cells or tisses and involved in the glioma genesis and metastasis has not been documented at home and abroad.
Significance
We have found CUGBP1 is highly expressed in glioma cells and its expression level is related with the pathological classes of human glioma. At the same time, our research has explained the correlation between CUGBP1 and genesis and metastasis of neoroglioma from the point of cell proliferation, apoptosis and the related genes alteration at their mRNA expression.
Objective
CUGBP1 is expressed in different glioma cell lines, glioma brain tissue and normal brain tissue.Furthermore, cell proliferation, apoptosis and related gene expression are observed to have changed after CUGBP1 gene knockdown. We aim to analyze the correlation between CUGBJP1 and glioma genesis so as to elucidate the mechanism and effect of CUGBP1 in glioma genesis and metastasis.
Methods
Detection of CUGBP1mRNA
RT-PCR and real-time quantitative RT-PCR were used to observed and compare the expression of CUGBP1 at the mRNA level in different glioma cell lines, neuroglioma and normal brain tissue.
By contrast of the housekeeping gene GAPDH, we detected the expression of CUGBP1 in different glioma cell lines through semi-quantitative RT-PCR.
We detected the expression of CUGBP1 in different pathological class of neuroglioma by real-time quantitative RT-PCR, by contrast of normal brain tissue and GAPDH.
Construction of RNA interfering lentivirus & Knockdown efficiency
To further explorate the correlation between CUGBJP1 and glioma genesis and its mechanism, we congstructed CUGBP1-shRNA lentivirus and transfected them into human glioma cell line of U251, by contrast of Scr-shRNA lentivirus vector transfected U251 cells. We also identified their efficiency of transfection and knockdown.
Interferring target sequences were designed, then connected to lentiviral vector and packaged into virus particles.
The results of transfection were assessed through observation and cell counting by common microscopy and fluorescence microscopy.
The effects of knockdown on CUGBP1 gene expression were detected in transfected human glioma cells by real-time quantitative RT-PCR.
Cell proliferation
CUGBP1 was closely related with proliferation of in liver cells and mammary epithelial cell in certain pathological contexts. We wondered whether there existed similar phenomenon in human neuroglioma. We knocked down CUGBP1 gene through transfecting CUGBP1-shRNA lentivirus vector into glioma cells to reveal its relationship with cell proliferation.
Cell counting was performed and taken down for CUGBP1-shRNA lentivirus transfected glioma cells after transtection at different time point (1d, 2d, 3d, 4d and 5d) through CELLOMICS, by contrast of scr-shRNA lentivirus transfected cell. Their growth curves were drawed and the effects of knockdown on glioma cell growth were analyzed.
BrdU were added into the culture medium of CUGBP1-shRNA lentivirus vector transfected glioma cells 2-24 hours before detecting, and the OD values of BrdU incorporation were detected by pairs antibody sandwich enzyme immunoassay, by contrast of Scr-shRNA lentiviral vector transfected cells. Further more, the effects of knockdown on DNA synthesis and cell proliferation of neuroglioma were analyzed.
Cell cycle and cell apoptosis CUGBP1 was related with the expression or activity of certain proteins such as P21, which were associated with cell cycle withdrawal and resulted in that the risk of mutation or dysplasia was increased in myoblast of myotonic dystrophy. We wondered whether there existed similar phenomenon in human neuroglioma. We knocked down CUGBP1 gene through transfecting CUGBP1-shRNA lentivirus vector into glioma cells to discover its relationship with cell cycle and cell apoptosis.
PI were added into the CUGBP1-shRNA lentivirus vector transfected glioma cells, subsequently, the proportion of cell cycle distribution was detected in these cells through FACS assay, by contrast of Scr-shRNA transfected cells. The effects of knockdown on cell cycle distribution were analyzed.
Annexin V-APC was added into the CUGBP1-shRNA lentivirus vector transfected glioma cells. The ratio of cell apoptosis was detected in these cells through FACS assay, by contrast of Scr-shRNA transfected cells. Further more, the impacts of knockdown on cell apoptosis were analyzed.
The expression of Cell cycle and apoptosis related gene To further explore the correlations and its mechanisms between CUGBP1 and glioma cell proliferation and apoptosis-related downstream genes, we examined the expression of these genes after knockdown of CUGBP1 gene.
The expression of apoptosis repression related genes BCL2 was examined in CUGBP1-shRNA lentivirus vector transfected glioma cells through real-time quantitative PCR, by contrast of Scr-shRNA transfected cells. The impact of the knockdown on expression of BCL2 in neuroglioma cells was analyzed.
The expression of cell cycle suppressor genes CDKN1A was examined in CUGBP1-shRNA lentivirus vector transfected glioma cells through real-time quantitative PCR, by contrast of Scr-shRNA transfected cells. The effect of the knockdown on expression of CDKN1A in neuroglioma cells was analyzed.
The expression of DNA synthesis related gene PCNA was examined in CUGBP1-shRNA lentivirus vector transfected glioma cells through real-time quantitative PCR, by contrast of Scr-shRNA transfected cells. The effect of the knockdown on expression of PCNA in neuroglioma cells was analyzed.
The expression of DNA synthesis related genes MCM2-7 were examined in CUGBP1-shRNA lentivirus vector transfected glioma cells through real-time quantitative PCR, by contrast of Scr-shRNA transfected cells. The effect of the knockdown on expression of MCM2-7 in neuroglioma cells was analyzed.
Results
Detection of CUGBP1mRNA
By contrast of the housekeeping gene GAPDH, the CUGBP1 were high expressed at mRNA level in human glioma cell lines U87, U251, U373 and human glioblastoma cell line A172 through RT-PCR assay.
The expression of CUGBP1 mRNA in human neuroglioma increased as 2.96 times much as that in normal brain tissue via real-time quantitative RT-PCR assay, by contrast of housekeeping gene GAPDH. Further more, while the tumor grade increases, CUGBP1 mRNA in neurogliomas tends to increase, with about 2.26 fold increase in glioma grade one, 2.36 fold in grade three and 4.27 fold in grade four.
Knockdown efficiency
Fluorescence-positive cells accounted for more than 95% among CUGBP1-shRNA lentivirus or Scr-shRNA lentivirus transfected glioma cells 72 hours after transfection by common microscopy and fluorescence microscopy, indicating successful transfection. By contrast of GAPDH, the expression of CUGBP1mRNA in CUGBP1-shRNA lentivirus transfected glioma cells, compared with that in control cells, decreased by about 92% through real-time quantitative RT-PCR assay, suggesting that CUGBP1-shRNA lentivirus effectively transfected glioma cell lines and inhibited expression of CUGBP1mRNA significantly as a reliable tool for gene silencing.
Cell proliferation
We examined CUGBP1-shRNA and Scr-siRNA lentiviral vector transfected cells once a day for 5 days and took pictures by CELLOMICS. While Scr-shRNA lentiviral vector transfected cells tended to gradually increase and up to 3 times by the fifth day, CUGBP1-shRNA lentiviral vector transfected cells remained no change and even decrease on fifth day, suggesting that the knockdown of CUGBP1 gene inhibited proliferation of glioma cells. By contrast of Scr-shRNA lentivirus transfected cells, we detected OD values of
BrdU incorporation in CUGBP1-shRNA lentivirus transfected glioma cells through double-antibody sandwich enzyme immunoassay. OD value in CUGBP1-shRNA lentivirus transfected cells decreased on first and third day and even much lessen on third day, compared with that in control cells, suggesting that the knockdown of CUGBP1 gene inhibited DNA synthesis, thereby inhibiting proliferation of glioma cells.
Cell cycle and apoptosis
By contrast of Scr-siRNA lentivirus transfected cells, we examined the cell cycle of CUGBP1-shRNA lentivirus transfected glioma cells through FACS assay. The cell ratio in cell cycle S phase among CUGBP1-shRNA lentivirus transfected glioma cells increased significantly, reaching 67.71%, while the cell ratio in the G0 / G1 and G2 / M phase was significantly reduced, suggesting that the knockdown of CUGBP1 gene resulted in abnormal cell cycle distribution of glioma cells. The cells in S phase increased, implying that the S phase arrest occurred.
By contrast of Scr-shRNA lentivirus transfected cells, we examined the cell apoptosis of CUGBP1-shRNA lentivirus transfected glioma cells through FACS assay. The apoptosis ratio among CUGBP1-shRNA lentivirus transfected glioma cells increased significantly, reaching 73.8%, suggesting that the knockdown of CUGBP1 gene in the glioma cell line significantly increased apoptosis, thereby indicating that the gene can inhibit glioma cell apoptosis.
The expression of Cell cycle and apoptosis related gene.
By contrast of housekeeping gene GAPDH, we detected the expression of BCL2 mRNA in CUGBP1-shRNA lentivirus and Scr-siRNA lentivirus infected glioma cells through real-time quantitative RT-PCR. The expression of BCL2 mRNA significantly decreased in CUGBP1-shRNA lentivirus infected glioma cells, compared with that in control cells, suggesting that the knockdown of CUGBP1 gene in the glioma cell line significantly inhibited the expression of BCL2 gene, thereby indicating that CUGBP1 gene can promote the expression of BCL2, thus inhibit apoptosis and promote tumor cell growth.
By contrast of housekeeping gene GAPDH, we detected the expression of CDKN1A mRNA in CUGBP1-shRNA lentivirus and Scr-shRNA lentivirus infected glioma cells through real-time quantitative RT-PCR. The expression of CDKN1A mRNA significantly increased in CUGBP1-shRNA lentivirus infected glioma cells, compared with that in control cells, suggesting that the knockdown of CUGBP1 gene in the glioma cell line significantly promoted the expression of CDKN1A gene, thereby indicating that CUGBP1 gene can inhibit the expression of CDKN1A, thus deinhibit cell cycle and promote tumor cell growth.
By contrast of housekeeping gene GAPDH, we detected the expression of PCNA mRNA in CUGBP1-shRNA lentivirus and Scr-shRNA lentivirus infected glioma cells through real-time quantitative RT-PCR. The expression of PCNA mRNA significantly decreased in CUGBP1-shRNA lentivirus infected glioma cells, compared with that in control cells, suggesting that the knockdown of CUGBP1 gene in the glioma cell line significantly inhibited the expression of PCNA gene, thereby indicating that CUGBP1 gene can promote the expression of PCNA , thus promote DNA synthesis and tumor cell growth.
By contrast of housekeeping gene GAPDH, we detected the expression of MCM2-7 mRNA in CUGBP1-shRNA lentivirus and Scr-shRNA lentivirus infected glioma cells through real-time quantitative RT-PCR. The expression of MCM2-7 mRNA significantly decreased in CUGBP1-shRNA lentivirus infected glioma cells, compared with that in control cells, suggesting that the knockdown of CUGBP1 gene in the glioma cell line significantly inhibited the expression of MCM2-7 gene, thereby indicating that CUGBP1 gene can promote the expression of MCM2-7, thus promote cell DNA synthesis and tumor cell growth.
Conclusion:
Firstly, our study innovatively discovers that CUGBP1 is highly expressed in different glioma cell lines and human neuroglioma. Furthermore, its expression tends to increase with the glioma pathological grade.
Secondly, CUGBP1-shRNA lentiviral vector can efficiently transfect human glioma cell lines and stably knock down CUGBP gene.
Thirdly, the knockdown of CUGBP1 gene can inhibit DNA synthesis for glioma cell, thus inhibiting the growth of glioma cells.
Forthly, the knockdown of CUGBP1 gene may cause S phase arrest and inhibit cell cycle process. Moreover, its knockdown also significantly increases the apoptosisl ratio of neuroglioma cells.
Last but not least, the knockdown of CUGBP1 gene can downregulate the expression of apoptosis inhibitor gene BCL2 and DNA synthesis related genes PCNA and MCM2-7, also upregulate the expression of cell cycle inhibitative gene CDKN1A.
CUGBP1 may promote cell DNA synthesis, promote cell cycle process and inhibit glioma cell apoptosis, thus promote development of glioma cells. Additionly, CUGBP1 may serve as one of predicting indicators or a potential gene therapeutic target for treatment of malignant glioma.
研究意义
旨在分析CUGBP1在胶质瘤细胞中的表达水平,明确其与人胶质瘤的关系;同时,从细胞增殖、细胞凋亡的现象及相关基因的变化解释CUGBP1与胶质瘤发生、发展的关系。
研究目的
通过CUGBP1在不同胶质瘤细胞株、胶质瘤脑组织以及正常脑组织中的表达检测和观察CUGBP1基因敲减后细胞增殖、细胞凋亡变化以及相关基因表达改变,分析CUGBP1与胶质瘤发生的相关性,以阐明CUGBP1在胶质瘤发生、发展中的作用及机制。
实验方法
1、表达检测选择RT-PCR和实时定量RT-PCR方法从mRNA水平上观察并比较CUGBP1在不同胶质瘤细胞株、胶质瘤脑组织以及正常脑组织中的表达。
半定量RT-PCR法检测CUGBP1在不同胶质瘤细胞株的表达,同时以管家基因GAPDH作为系统阳性对照。
实时定量RT-PCR法检测CUGBP1在不同病理级别胶质瘤脑组织的表达水平,并以正常脑组织中的水平作为参照标准,同时以GAPDH作为系统阳性对照。
2、RNA干扰慢病毒构建及其敲减效率检测为进一步探索CUGBP1与胶质瘤的相关性及其机制,构建CUGBP1-shRNA慢病毒,转染胶质瘤细胞株U251,并检测其转染及敲减效率。
2.1 RNA干扰慢病毒构建设计干扰靶点序列,连接到慢病毒载体,包装成病毒颗粒。
2.2细胞计数运用普通光学显微镜和荧光显微镜观察和细胞计数,评估转染效率。
2.3实时定量RT-PCR法检测CUGBP1-shRNA慢病毒转染胶质瘤细胞的敲减效果。
3、细胞增殖CUGBP1在某些病理情况下与肝脏及乳腺上皮细胞的增殖密切相关,是否在胶质瘤发生中也有类似现象,我们通过CUGBP1-shRNA慢病毒转染胶质瘤细胞而敲减CUGBP1表达以揭示其与细胞增殖的关系。
3.1细胞计数,绘制CUGBP1-shRNA慢病毒转染组及Scr-shRNA慢病毒转染组两组细胞生长曲线,分析敲减对细胞生长的影响。
3.2双抗体夹心酶标免疫分析法测定转染CUGBP1-shRNA慢病毒的人胶质瘤细胞BrdU掺入的OD值,同时以Scr-shRNA慢病毒转染细胞作为阴性对照,分析敲减对DNA合成及细胞增殖的影响。
4、细胞周期分布及细胞凋亡CUGBP1在强直性肌营养不良成肌细胞中使某些与细胞周期退出相关的蛋白质如P21等表达或活性改变,进而使细胞周期退出障碍,从而发生突变或异常增生的几率增加。这种现象是否也出现在胶质瘤细胞,我们通过CUGBP1-shRNA慢病毒转染胶质瘤细胞而敲减CUGBP1表达以揭示其与细胞周期分布及细胞凋亡的关系。
4.1运用FACS检测CUGBP1-shRNA慢病毒转染胶质瘤细胞后的细胞周期分布比例,同时以转染Scr-shRNA慢病毒细胞作为阴性对照,分析敲减对细胞周期分布的影响。
4.2运用FACS检测CUGBP1-shRNA慢病毒转染胶质瘤细胞后的细胞凋亡比例,同时以转染Scr-shRNA慢病毒细胞作为阴性对照,分析敲减对细胞凋亡的影响
5、细胞周期及细胞凋亡相关基因检测为进一步探究CUGBP1与胶质瘤细胞增殖和凋亡相关的机制,我们检测了敲减后相关基因的表达。
5.1实时定量RT-PCR法检测敲减后凋亡抑制基因BCL2的表达并与阴性对照比较,分析敲减对BCL2的影响。
5.2实时定量RT-PCR法检测敲减后细胞周期抑制基因CDKN1A的表达并与阴性对照比较,分析敲减对CDKN1A的影响。
5.3实时定量RT-PCR法检测敲减后DNA合成相关基因PCNA的表达并与阴性对照比较,分析敲减对PCNA的影响。
5.4实时定量RT-PCR法检测敲减后DNA合成相关基因MCM2-7的表达并与阴性对照比较,分析敲减对MCM2-7的影响。
实验结果
1、表达检测
1.1以管家基因GAPDH作为参照,RT-PCR显示CUGBP1 mRNA在不同胶质瘤细胞株中均有表达。
1.2以管家基因GAPDH作为内参,实时定量RT-PCR显示在人胶质瘤组织中CUGBP1 mRNA的表达量较正常脑组织明显增高(约2.96倍),并且随着肿瘤分级的增加,其表达量有增高的趋势,胶质瘤Ⅰ级增高约2.26倍、胶质瘤Ⅲ级增高约2.36倍、胶质瘤Ⅳ级增高约4.27倍。
2、敲减效率检测
2.1运用普通光学显微镜和荧光显微镜观察和计数CUGBP1-shRNA慢病毒转染组及Scr-shRNA慢病毒转染组两组转染后72小时荧光阳性细胞占所有胶质瘤细胞的比例,显示均大于95%,提示高效转染。
2.2以管家基因GAPDH作为内参,实时定量RT-PCR显示CUGBP1-shRNA慢病毒转染胶质瘤细胞中CUGBP1 mRNA的表达量较阴性对照组降低,最高达约92%,提示CUGBP1-shRNA慢病毒能高效转染胶质瘤细胞株,并使CUGBP基因在mRNA水平上受到明显抑制,是一种可靠的基因沉默工具。
3、细胞增殖
3.1以Scr-shRNA慢病毒转染细胞作为阴性对照,借助CELLOMICS对转染CUGBP1-shRNA慢病毒人胶质瘤细胞每天检测读板一次并拍照,连续5天。结果显示对照组细胞随着培养天数的增加细胞数目也逐渐增加,第5天时细胞数目增加了3倍;而CUGBP1-shRNA组的细胞数目没有明显变化,提示CUGBP1基因敲减使人胶质瘤细胞的增殖受到显著抑制。
3.2以Scr-shRNA慢病毒转染细胞作为阴性对照,利用双抗体夹心酶标免疫分析法测定转染CUGBP1-shRNA慢病毒胶质瘤细胞BrdU掺入的OD值,结果显示CUGBP1-shRNA组胶质瘤细胞的ODBrdU在第一天和第三天均比阴性对照组减少,第三天的减少更显著,提示CUGBP1基因敲减抑制了DNA合成,进而抑制胶质瘤细胞的增殖能力。
4、细胞周期及细胞凋亡
4.1以Scr-shRNA慢病毒转染细胞作阴性对照,利用FACS对CUGBP1-shRNA慢病毒转染人胶质瘤细胞进行细胞周期检测,显示CUGBP1-shRNA转染胶质瘤细胞处于S期的细胞比例显著增加,达67.71%,而处于G0//G1期和G2/M期的细胞比例明显减少,提示CUGBP1基因敲减使胶质瘤细胞的细胞周期分布发生改变,S期细胞比例增多,表明发生S期阻滞。
4.2以Scr-shRNA慢病毒转染细胞作阴性对照,利用FACS对CUGBP1-shRNA慢
病毒转染人胶质瘤细胞进行细胞凋亡检测,显示CUGBP1-shRNA转染胶质瘤细胞的细胞凋亡比例与对照组比较有明显的增加,达73.8%,提示CUGBP1基因敲减使胶质瘤细胞株的细胞(早期)凋亡明显增加,表明该基因有抑制胶质瘤细胞凋亡的作用。
5、细胞周期及细胞凋亡相关基因表达
5.1以管家基因GAPDH作为内参,采用实时定量RT-PCR方法对CUGBP1-shRNA慢病毒感染胶质瘤细胞的BCL2 mRNA的表达进行检测,同时以Scr-shRNA慢病毒感染细胞作为阴性对照,结果显示CUGBP1-shRNA组胶质瘤细胞BCL2 mRNA的表达量较阴性对照组显著降低。提示CUGBP 1基因敲减下调了BCL2基因的表达,因而CUGBP1基因具有上调BCL2的表达进而抑制细胞凋亡,促进肿瘤细胞生长的作用。
5.2以管家基因GAPDH作为内参,采用Real-time定量RT-PCR的方法对CUGBP1-shRNA慢病毒感染胶质瘤细胞CDKN1A mRNA的表达进行检测,同时以Scr-shRNA慢病毒感染细胞作为阴性对照,结果显示CUGBP1-shRNA组胶质瘤细胞CDKN1A mRNA的表达量较阴性对照组显著升高,提示CUGBP1基因敲减上调了CDKN1A基因的表达,因而CUGBP1基因具有下调CDKN1A基因的表达进而解除其对细胞周期的抑制,促进肿瘤细胞生长的作用。
5.3以管家基因GAPDH作为内参,采用Real-time定量RT-PCR方法对CUGBP1-shRNA慢病毒感染胶质瘤细胞PCNA mRNA的表达进行检测,同时以Scr-shRNA慢病毒感染细胞作为阴性对照,结果显示CUGBP1-shRNA组胶质瘤细胞的PCNA mRNA表达量较阴性对照组显著降低,提示CUGBP1基因敲减下调了PCNA基因的表达,因而CUGBP1基因具有上调PCNA基因表达进而促进DNA合成、促进胶质瘤细胞生长的作用。
5.4以管家基因GAPDH作为内参,采用Real-time定量RT-PCR方法对CUGBP1-shRNA慢病毒感染胶质瘤细胞MCM2-7 mRNA的表达进行检测,同时以Scr-shRNA慢病毒感染细胞作为阴性对照,结果显示CUGBP1-shRNA组胶质瘤细胞的MCM2-7 mRNA的表达量除MCM6-7轻度降低外,其它均较阴性对照组显著降低,提示CUGBP1基因敲减下调了MCM2-7基因的表达,因而CUGBP1基因具有上调MCM2-5基因表达进而促进细胞DNA合成、促进胶质瘤细胞生长的作用。
结论:
1、本研究首次发现CUGBP1在不同胶质瘤细胞株细胞及人胶质瘤组织中高表达,而且其表达有随胶质瘤病理分级的增加而增加的趋势。
2、CUGBP1-shRNA慢病毒能够高效转染恶性胶质瘤细胞株U251细胞并稳定敲减其CUGBP1基因表达。
3、CUGBP1基因敲减能够抑制恶性胶质瘤U251细胞增殖及DNA合成功能,因而抑制U251细胞的生长。
4、CUGBP1基因敲减能够引起恶性胶质瘤U251细胞S期阻滞,抑制细胞周期进程;CUGBP1基因敲减还能够诱导U251细胞(早期)凋亡。
5、CUGBP1基因敲减能够下调细胞凋亡抑制基因BCL2表达,上调细胞周期抑制基因CDKN1A表达,还能够下调DNA合成相关基因PCNA及MCM2-7表达。以上结论提示CUGBP1敲减可能通过抑制细胞增殖及DNA合成、抑制细胞周期进程及诱导细胞凋亡而抑制胶质瘤的发生和发展;这间接证明CUGBP1具有促进细胞增殖及DNA合成,促进细胞周期进程及抑制细胞凋亡,进而促进胶质瘤发生发展的癌基因样作用。CUGBP1可以作为恶性胶质瘤的预测指标之一并可作为恶性胶质瘤基因治疗的潜在分子靶点。
Recently, the mechanisms of tumorigenesis have been the focus of scholars at home and abroad, and it has been given increasing attention to elucidate tumor genesis and metastasis from the perspective of tumor molecular biology. In China, the neurogliomas is the first neoplasm of nervous system with high incidence, rapid process, and poor prognosis, and the morbidity tends to rise at present. As a member of RNA binding protein CELF family, CUGBP1 can regulate the alternative splicing by stimulating inclusion or exclusion of the non-consecutive exons in the nucleus pre- mRNA. In the cytoplasm, it can adjust their translation or degradation by binding to the specific sequence in certain target mRNAs. It can also influence the mRNA editing through RNA editing enzyme complex. CUGBP1 is involved in embryonic and cardiac development, skeletal muscle and adipose tissue differentiation and germ cell formation, and also play a great role in the liver cells and mammary epithelial cell proliferation, tumor genesis, as well as the deterioration of certain tumors. Whether CUGBP1 is abnormally expressed in human glioma cells or tisses and involved in the glioma genesis and metastasis has not been documented at home and abroad.
Significance
We have found CUGBP1 is highly expressed in glioma cells and its expression level is related with the pathological classes of human glioma. At the same time, our research has explained the correlation between CUGBP1 and genesis and metastasis of neoroglioma from the point of cell proliferation, apoptosis and the related genes alteration at their mRNA expression.
Objective
CUGBP1 is expressed in different glioma cell lines, glioma brain tissue and normal brain tissue.Furthermore, cell proliferation, apoptosis and related gene expression are observed to have changed after CUGBP1 gene knockdown. We aim to analyze the correlation between CUGBJP1 and glioma genesis so as to elucidate the mechanism and effect of CUGBP1 in glioma genesis and metastasis.
Methods
Detection of CUGBP1mRNA
RT-PCR and real-time quantitative RT-PCR were used to observed and compare the expression of CUGBP1 at the mRNA level in different glioma cell lines, neuroglioma and normal brain tissue.
By contrast of the housekeeping gene GAPDH, we detected the expression of CUGBP1 in different glioma cell lines through semi-quantitative RT-PCR.
We detected the expression of CUGBP1 in different pathological class of neuroglioma by real-time quantitative RT-PCR, by contrast of normal brain tissue and GAPDH.
Construction of RNA interfering lentivirus & Knockdown efficiency
To further explorate the correlation between CUGBJP1 and glioma genesis and its mechanism, we congstructed CUGBP1-shRNA lentivirus and transfected them into human glioma cell line of U251, by contrast of Scr-shRNA lentivirus vector transfected U251 cells. We also identified their efficiency of transfection and knockdown.
Interferring target sequences were designed, then connected to lentiviral vector and packaged into virus particles.
The results of transfection were assessed through observation and cell counting by common microscopy and fluorescence microscopy.
The effects of knockdown on CUGBP1 gene expression were detected in transfected human glioma cells by real-time quantitative RT-PCR.
Cell proliferation
CUGBP1 was closely related with proliferation of in liver cells and mammary epithelial cell in certain pathological contexts. We wondered whether there existed similar phenomenon in human neuroglioma. We knocked down CUGBP1 gene through transfecting CUGBP1-shRNA lentivirus vector into glioma cells to reveal its relationship with cell proliferation.
Cell counting was performed and taken down for CUGBP1-shRNA lentivirus transfected glioma cells after transtection at different time point (1d, 2d, 3d, 4d and 5d) through CELLOMICS, by contrast of scr-shRNA lentivirus transfected cell. Their growth curves were drawed and the effects of knockdown on glioma cell growth were analyzed.
BrdU were added into the culture medium of CUGBP1-shRNA lentivirus vector transfected glioma cells 2-24 hours before detecting, and the OD values of BrdU incorporation were detected by pairs antibody sandwich enzyme immunoassay, by contrast of Scr-shRNA lentiviral vector transfected cells. Further more, the effects of knockdown on DNA synthesis and cell proliferation of neuroglioma were analyzed.
Cell cycle and cell apoptosis CUGBP1 was related with the expression or activity of certain proteins such as P21, which were associated with cell cycle withdrawal and resulted in that the risk of mutation or dysplasia was increased in myoblast of myotonic dystrophy. We wondered whether there existed similar phenomenon in human neuroglioma. We knocked down CUGBP1 gene through transfecting CUGBP1-shRNA lentivirus vector into glioma cells to discover its relationship with cell cycle and cell apoptosis.
PI were added into the CUGBP1-shRNA lentivirus vector transfected glioma cells, subsequently, the proportion of cell cycle distribution was detected in these cells through FACS assay, by contrast of Scr-shRNA transfected cells. The effects of knockdown on cell cycle distribution were analyzed.
Annexin V-APC was added into the CUGBP1-shRNA lentivirus vector transfected glioma cells. The ratio of cell apoptosis was detected in these cells through FACS assay, by contrast of Scr-shRNA transfected cells. Further more, the impacts of knockdown on cell apoptosis were analyzed.
The expression of Cell cycle and apoptosis related gene To further explore the correlations and its mechanisms between CUGBP1 and glioma cell proliferation and apoptosis-related downstream genes, we examined the expression of these genes after knockdown of CUGBP1 gene.
The expression of apoptosis repression related genes BCL2 was examined in CUGBP1-shRNA lentivirus vector transfected glioma cells through real-time quantitative PCR, by contrast of Scr-shRNA transfected cells. The impact of the knockdown on expression of BCL2 in neuroglioma cells was analyzed.
The expression of cell cycle suppressor genes CDKN1A was examined in CUGBP1-shRNA lentivirus vector transfected glioma cells through real-time quantitative PCR, by contrast of Scr-shRNA transfected cells. The effect of the knockdown on expression of CDKN1A in neuroglioma cells was analyzed.
The expression of DNA synthesis related gene PCNA was examined in CUGBP1-shRNA lentivirus vector transfected glioma cells through real-time quantitative PCR, by contrast of Scr-shRNA transfected cells. The effect of the knockdown on expression of PCNA in neuroglioma cells was analyzed.
The expression of DNA synthesis related genes MCM2-7 were examined in CUGBP1-shRNA lentivirus vector transfected glioma cells through real-time quantitative PCR, by contrast of Scr-shRNA transfected cells. The effect of the knockdown on expression of MCM2-7 in neuroglioma cells was analyzed.
Results
Detection of CUGBP1mRNA
By contrast of the housekeeping gene GAPDH, the CUGBP1 were high expressed at mRNA level in human glioma cell lines U87, U251, U373 and human glioblastoma cell line A172 through RT-PCR assay.
The expression of CUGBP1 mRNA in human neuroglioma increased as 2.96 times much as that in normal brain tissue via real-time quantitative RT-PCR assay, by contrast of housekeeping gene GAPDH. Further more, while the tumor grade increases, CUGBP1 mRNA in neurogliomas tends to increase, with about 2.26 fold increase in glioma grade one, 2.36 fold in grade three and 4.27 fold in grade four.
Knockdown efficiency
Fluorescence-positive cells accounted for more than 95% among CUGBP1-shRNA lentivirus or Scr-shRNA lentivirus transfected glioma cells 72 hours after transfection by common microscopy and fluorescence microscopy, indicating successful transfection. By contrast of GAPDH, the expression of CUGBP1mRNA in CUGBP1-shRNA lentivirus transfected glioma cells, compared with that in control cells, decreased by about 92% through real-time quantitative RT-PCR assay, suggesting that CUGBP1-shRNA lentivirus effectively transfected glioma cell lines and inhibited expression of CUGBP1mRNA significantly as a reliable tool for gene silencing.
Cell proliferation
We examined CUGBP1-shRNA and Scr-siRNA lentiviral vector transfected cells once a day for 5 days and took pictures by CELLOMICS. While Scr-shRNA lentiviral vector transfected cells tended to gradually increase and up to 3 times by the fifth day, CUGBP1-shRNA lentiviral vector transfected cells remained no change and even decrease on fifth day, suggesting that the knockdown of CUGBP1 gene inhibited proliferation of glioma cells. By contrast of Scr-shRNA lentivirus transfected cells, we detected OD values of
BrdU incorporation in CUGBP1-shRNA lentivirus transfected glioma cells through double-antibody sandwich enzyme immunoassay. OD value in CUGBP1-shRNA lentivirus transfected cells decreased on first and third day and even much lessen on third day, compared with that in control cells, suggesting that the knockdown of CUGBP1 gene inhibited DNA synthesis, thereby inhibiting proliferation of glioma cells.
Cell cycle and apoptosis
By contrast of Scr-siRNA lentivirus transfected cells, we examined the cell cycle of CUGBP1-shRNA lentivirus transfected glioma cells through FACS assay. The cell ratio in cell cycle S phase among CUGBP1-shRNA lentivirus transfected glioma cells increased significantly, reaching 67.71%, while the cell ratio in the G0 / G1 and G2 / M phase was significantly reduced, suggesting that the knockdown of CUGBP1 gene resulted in abnormal cell cycle distribution of glioma cells. The cells in S phase increased, implying that the S phase arrest occurred.
By contrast of Scr-shRNA lentivirus transfected cells, we examined the cell apoptosis of CUGBP1-shRNA lentivirus transfected glioma cells through FACS assay. The apoptosis ratio among CUGBP1-shRNA lentivirus transfected glioma cells increased significantly, reaching 73.8%, suggesting that the knockdown of CUGBP1 gene in the glioma cell line significantly increased apoptosis, thereby indicating that the gene can inhibit glioma cell apoptosis.
The expression of Cell cycle and apoptosis related gene.
By contrast of housekeeping gene GAPDH, we detected the expression of BCL2 mRNA in CUGBP1-shRNA lentivirus and Scr-siRNA lentivirus infected glioma cells through real-time quantitative RT-PCR. The expression of BCL2 mRNA significantly decreased in CUGBP1-shRNA lentivirus infected glioma cells, compared with that in control cells, suggesting that the knockdown of CUGBP1 gene in the glioma cell line significantly inhibited the expression of BCL2 gene, thereby indicating that CUGBP1 gene can promote the expression of BCL2, thus inhibit apoptosis and promote tumor cell growth.
By contrast of housekeeping gene GAPDH, we detected the expression of CDKN1A mRNA in CUGBP1-shRNA lentivirus and Scr-shRNA lentivirus infected glioma cells through real-time quantitative RT-PCR. The expression of CDKN1A mRNA significantly increased in CUGBP1-shRNA lentivirus infected glioma cells, compared with that in control cells, suggesting that the knockdown of CUGBP1 gene in the glioma cell line significantly promoted the expression of CDKN1A gene, thereby indicating that CUGBP1 gene can inhibit the expression of CDKN1A, thus deinhibit cell cycle and promote tumor cell growth.
By contrast of housekeeping gene GAPDH, we detected the expression of PCNA mRNA in CUGBP1-shRNA lentivirus and Scr-shRNA lentivirus infected glioma cells through real-time quantitative RT-PCR. The expression of PCNA mRNA significantly decreased in CUGBP1-shRNA lentivirus infected glioma cells, compared with that in control cells, suggesting that the knockdown of CUGBP1 gene in the glioma cell line significantly inhibited the expression of PCNA gene, thereby indicating that CUGBP1 gene can promote the expression of PCNA , thus promote DNA synthesis and tumor cell growth.
By contrast of housekeeping gene GAPDH, we detected the expression of MCM2-7 mRNA in CUGBP1-shRNA lentivirus and Scr-shRNA lentivirus infected glioma cells through real-time quantitative RT-PCR. The expression of MCM2-7 mRNA significantly decreased in CUGBP1-shRNA lentivirus infected glioma cells, compared with that in control cells, suggesting that the knockdown of CUGBP1 gene in the glioma cell line significantly inhibited the expression of MCM2-7 gene, thereby indicating that CUGBP1 gene can promote the expression of MCM2-7, thus promote cell DNA synthesis and tumor cell growth.
Conclusion:
Firstly, our study innovatively discovers that CUGBP1 is highly expressed in different glioma cell lines and human neuroglioma. Furthermore, its expression tends to increase with the glioma pathological grade.
Secondly, CUGBP1-shRNA lentiviral vector can efficiently transfect human glioma cell lines and stably knock down CUGBP gene.
Thirdly, the knockdown of CUGBP1 gene can inhibit DNA synthesis for glioma cell, thus inhibiting the growth of glioma cells.
Forthly, the knockdown of CUGBP1 gene may cause S phase arrest and inhibit cell cycle process. Moreover, its knockdown also significantly increases the apoptosisl ratio of neuroglioma cells.
Last but not least, the knockdown of CUGBP1 gene can downregulate the expression of apoptosis inhibitor gene BCL2 and DNA synthesis related genes PCNA and MCM2-7, also upregulate the expression of cell cycle inhibitative gene CDKN1A.
CUGBP1 may promote cell DNA synthesis, promote cell cycle process and inhibit glioma cell apoptosis, thus promote development of glioma cells. Additionly, CUGBP1 may serve as one of predicting indicators or a potential gene therapeutic target for treatment of malignant glioma.
引文
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