胶质瘤不同病理级别差异miRNA/mRNA动态表达谱构建及其对接研究
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摘要
[研究背景]
胶质瘤是颅内最常见的恶性肿瘤,以星形细胞胶质瘤最为多见,其次为胶质母细胞瘤。研究表明,胶质瘤的发生发展常涉及p53、p16、RB、PTEN、LRRC4等抑癌基因的失活以及c-Myc、HRAS、EGFR、MDM2等基因的过表达,而这些基因在不同病理级别的胶质瘤中具有特定的表达变化模式,如p53突变与过表达、MDM2过表达常发生于各级别胶质瘤,其表达水平与病理分级之间无明显关联,然而亦有文献报道,p53突变与过表达在高级别胶质瘤中表现更为明显；p16、LRRC4氐表达亦常发生于各级别胶质瘤,且在高级别胶质瘤中改变更为明显；PTEN突变,RB失活,c-Myc、HRAS、EGFR过表达则发生于高级别胶质瘤。基因表达受DNA水平、转录水平、转录后水平及蛋白水平等多层面的调控,其中miRNA在转录后水平对基因表达的精细调控近年来倍受关注。研究表明,miRNA在细胞增殖、分化、凋亡以及机体发育、代谢等基本生命活动过程中发挥重要作用,miRNA表达异常与包括胶质瘤在内的多种肿瘤的发生密切相关。研究发现,miR-21、miR-221、miR-10b等在胶质母细胞瘤中表达上调,而原本在正常脑组织中含量丰富的rniR-128、miR-181等则表达下调。进一步研究表明,这些表达异常的miRNA在胶质瘤发生发展过程中可能发挥类似癌基因或抑癌基因的作用。然而,对于miRNA在胶质瘤不同病理级别中的动态表达模式鲜有报道。
基于以上认识,本研究同时采用miRNA芯片和全基因组表达谱芯片探讨miRNA与基因在胶质瘤不同病理级别中的表达变化规律,构建胶质瘤不同病理级别差异miRNA/mRNA动态表达谱,并对筛选的差异miRNA/mRNA进行了对接研究。
[胶质瘤不同病理级别差异miRNA动态表达谱构建]
采用miRNA芯片分析了3例正常脑组织及13例星形细胞胶质瘤组织(Ⅰ级3例、Ⅱ级5例、Ⅲ级5例)中637种miRNA的表达情况,结合非参数检验、方差分析以及t检验获得不同病理级别胶质瘤的差异miRNA。我们对这些差异miRNA在各级别胶质瘤中的表达变化规律进行了总结分析,结果显示,差异miRNA在不同病理级别胶质瘤中的表达模式大致分为8类。其中miR-26a在各级别胶质瘤中均表达上调,尤其以在胶质瘤Ⅲ级中表达上调最为明显,而miR-21、miR-23a则主要在胶质瘤Ⅲ级中表达上调,这些提示miR-26a可能与胶质瘤的发生及恶性进展相关,而miR-21、miR-23a则主要与胶质瘤的恶性进展相关；此外,miR-107、miR-124、miR-128a/128b、miR-138、miR-149、miR-181a、miR-181b、miR-302b在各级别胶质瘤中均表达显著下调,提示这些差异miRNA与胶质瘤的发生密切相关。由此可见,miRNA在不同病理级别胶质瘤中的表达并不是单一的上调或下调,它们存在各自特定的表达变化模式,以此构成胶质瘤不同病理级别特定的差异miRNA表达谱。
[部分差异miRNA在不同病理级别胶质瘤中动态表达模式的验证]
应用Real-time PCR对miR-107、miR-124、miR-138、miR-149、miR-302b. miR-23a在6例正常脑组织及50例胶质瘤组织(Ⅰ级6例、Ⅱ级20例、Ⅲ级9例、Ⅳ级15例)中进行了验证,证实miR-107、miR-124、miR-138. miR-149在各级别胶质瘤中表达均显著下调,且其表达水平与病理分级负相关,该验证结果与芯片结果相符。miR-23a则在胶质瘤Ⅱ、Ⅲ级中表达逐渐上调,以在胶质瘤Ⅲ级中表达水平最高,与芯片结果相符,但在胶质瘤Ⅳ级中几乎检测不到表达。随后我们应用miR-23a寡核苷酸探针在同一批组织样本中进行了原位杂交验证,结果显示,miR-23a在正常脑组织中主要表达于神经元的胞核,而在胶质瘤Ⅱ、Ⅲ级中主要表达于胶质细胞的胞浆,在胶质瘤Ⅳ级中仍未检测到阳性表达。我们推测,miR-23a在神经元胞核中的表达一方面可能是由于原位杂交探针不能区分miRNA前体与成熟体,另一方面不排除miR-23a在胶质瘤发生发展过程中发生表达移位,其具体机制有待于进一步研究。
[miR-124、miR-149在胶质瘤发生发展中可能发挥抑癌功能]
将Lip2000、NC、miR-107、miR-124、miR-138、miR-149 mimics分别转染胶质瘤细胞,通过MTT、细胞划痕实验观察恢复这些miRNA的表达对胶质瘤细胞生物学行为的影响。MTT实验表明转染miR-107、miR-124、miR-138、miR-149 mimics后,胶质瘤细胞SF126、U251的生长受到不同程度的抑制,尤其以miR-124. miR-149的抑制作用最为明显,转染第四天时,与阴性对照组相比,转染miR-124mimics的细胞其增长率降低40%,转染miR-149 mimics的细胞其增长率降低33%；细胞划痕实验表明,miR-124、miR-149能明显抑制SF126、U251细胞的迁移能力,划痕后48hr,其它各组的伤口均已愈合,而转染miR-124、miR-149 mimics的细胞其伤口仍清晰可见。以上提示miR-124、miR-149在胶质瘤发生发展过程中可能发挥抑癌功能。
[胶质瘤不同病理级别差异mRNA动态表达谱构建]
采用全基因组表达谱芯片检测用于miRNA芯片分析的3例正常脑组织及13例胶质瘤组织中30968个基因的表达情况,并运用与miRNA芯片数据分析同样的方法进行差异基因筛选。将筛选的各级别胶质瘤中差异表达基因进行整合,探讨其在不同病理级别胶质瘤中的表达变化规律,结果显示,差异基因在不同病理级别胶质瘤中的表达模式大致分为14类。BMP1、CDK4、DDX5、DEK、HARS、HNRNPA2B1、IKBKB、LAMA4. p53、PCNA、PLAGL2、TLR3、VCAM1等在各级别胶质瘤中均表达上调,其中HARS、HNRNPA2B1、p53、PLAGL2的表达水平与胶质瘤的病理分级无明显关联,BMP1、DDX5、DEK、IKBKB、LAMA4、PCNA、TLR3的表达水平则与病理分级正相关；ANXA1、JAK2、JUN、MMP14、RAP1B、STAT1、VCL、VIM等主要在胶质瘤Ⅱ、Ⅲ级中表达上调；此外,ANK1、APC等在胶质瘤Ⅰ、Ⅱ、Ⅲ级中逐渐表达下调。
[胶质瘤不同病理级别差异mRNA的生物信息学分析]
应用基因功能分析软件DAVID,分别对各级别胶质瘤中表达上调和表达下调的差异基因进行Gene Ontology_BP分析,结果显示：胶质瘤Ⅰ、Ⅱ级中表达上调的基因主要参与转录调控、RNA剪接、核酸代谢、生物大分子组装、细胞应激反应等一系列生物学过程,此外胶质瘤Ⅰ级中表达上调的基因还参与了细胞增殖,胶质瘤Ⅲ级中表达上调的基因则主要参与细胞凋亡、生物大分子组装、细胞粘附、RNA剪接、细胞周期、信号转导等一系列生物学过程；胶质瘤Ⅰ、Ⅱ、Ⅲ级中表达下调的基因主要参与磷酸化代谢、信号转导、蛋白定位、生物大分子运输、神经元分化等一系列生物学过程。由此可见,在不同病理级别胶质瘤中表达下调的基因其生物学功能大致相似,而表达上调的基因其生物学功能重心发生了转移,表现为在胶质瘤Ⅰ级中以参与转录调控、细胞增殖的基因为主,而在胶质瘤Ⅱ、Ⅲ级中参与细胞凋亡、细胞粘附的基因逐渐增多。
结合KEGG数据库,我们发现在胶质瘤中多个信号通路分子,如RAP1B、JUN、FOS、JAK2、STAT1、TCF7L1、IKBKB、TRl3、MYD88等表达上调,而信号通路负调控因子如DUSP8、APC、CYLD等表达下调,提示MAPK、JAK/STAT、WNT、NF-kB、Toll样受体等多条信号通路的活化参与了胶质瘤的发生发展。
[胶质瘤不同病理级别差异ⅠniRNA/mRNA的对接研究]
通过对胶质瘤不同病理级别差异miRNA/mRNA表达谱的对接分析,我们发现,表达上调的miRNA中以miR-23a、miR-26a、miR-338-5p、miR-491-3p对接的靶基因数目较多；表达下调的miRNA中以miR-107、miR-124、miR-128a/128b、miR-138、miR-149、miR-181a、miR-181b、miR-302b对接的靶基因数目较多；差异表达基因中以PLAGL2、HNRNPA2B1、RAP1B、DEK等对接的miRNA数目较多,其中RAP1B与miR-149的负相关表达模式已经证实,提示一种miRNA可调控多个基因的表达,而一个基因的表达又可受多种miRNA调控,同时也说明了上述差异rniRNA与基因在胶质瘤发生发展中可能起重要作用。
综上所述,我们通过对同一批正常脑组织与各级别胶质瘤组织样本中差异miRNA/mRNA动态表达谱的研究及对接分析,初步揭示了差异miRNA与基因在胶质瘤不同病理级别中的表达变化规律,进一步阐明了胶质瘤发生发展的分子机制,为寻找有效的胶质瘤早期诊断及恶性进展的分子标志提供了充分的理论依据。
[Background]
Glioma is the most common intracranial malignant tumor, and astrocytoma is the most common one, followed by glioblastoma. Many researches have shown that the tumorigenesis of gliomas is often correlated with the inactivation of several tumor suppressor genes such as p53, p16, RB, PTEN, LRRC4 and activation of c-Myc, HRAS, EGFR, MDM2. We found that these genes have their specific expression profile in different pathological grades of gliomas. The mutation and overexpression of p53 and MDM2 are common in gliomas and showed no apparent correlation with the tumor grades, however there has also been reported that the mutation and overexpression of p53 is even obvious in high-grade gliomas. p16 and LRRC4 are down-expressed in gliomas and their expression levels are correlated with the glioma grades. The mutation of PTEN, loss of RB and overexpression of c-Myc, HRAS and EGFR are involved in high-grade gliomas.
Gene expression is regulated at the genomic, transcriptomic, post-transcriptomic and proteomic levels. Recently the function of miRNAs in post-transcriptomic regulation are highly been concerned. Many researches have shown that miRNAs are playing important roles in cell proliferation, differentiation, apoptosis, development and metabolism. And it is noticed that the alteration of miRNA expression is common in most type of tumors including glioma. Studies showed that mir-21, mir-221 and mir-lOb are overexpressed, whereas a set of brain-enriched miRNAs such as mir-128 and mir-181 are down-expressed in glioblastomas. Further researches have found that these miRNAs could act as oncogene or tumor suppressors during tumorigenesis. However the miRNA expression profile in different pathological grades of gliomas was little known.
According to the knowledge above, we analyzed simultaneously the expression profiles of miRNA and mRNA in gliomas with different pathological grades by using miRNA and cDNA microarray. Then the dynamics expression profiles of miRNAs and mRNAs were obtained, and docking study was done in gliomas with different pathological grades.
[Construction of miRNA dynamics expression profiles in gliomas with different pathological grades]
The expression levels of 637 miRNAs were detected in 3 normal brain tissue and 13 gliomas (3 grade I,5 grade II and 5 grade III) by miRNA microarray. Multi-methods were used to analyze the differentially expressed miRNAs in different pathological grades. Then the expression profiles of significant miRNAs were analyzed, and further classified into 8 categories. miR-26a is overexpressed in all gliomas especially in grade III whereas miR-21 and miR-23a are overexpressed mainly in grade III, which indicate that miR-26a may contribute to the initiation and progression of gliomas while miR-21 and miR-23a are mainly involved in the progression of gliomas. Additionally, miR-107, miR-124, miR-128a/128b, miR-138, miR-149, miR-181a, miR-181b, miR-302b are significantly down-regulated in all gliomas, and we infer that these miRNAs are correlated with the tumorigenesis of gliomas. These results implied that differentially expressed miRNAs have their specific expression profiles in different pathological grades of glioma.
[Validation of miRNA dynamics expression profiles for six miRNAs in different pathological grades]
The expression profiles of miR-107, miR-124, miR-138, miR-149, miR-302b and miR-23a in 6 normal brain tissue and 50 gliomas (6 grade I,20 grade II,9 grade III and 15 grade IV) were analyzed by Real-time PCR. It has been validated that miR-107, miR-124, miR-138, miR-149 are down-regulated in all gliomas and their expression levels are negatively correlated with pathological grades, which are consistent with the results of microarray. The expression level of miR-23a is increased gradually in gliomas and reaches the highest level in grade III, whereas no expression is detected in grade IV. Then we detected the expression level of miR-23a by in situ hybridization in same samples. The results showed that miR-23a is mainly expressed in nucleus of normal neurons and cytoplasma of glials in glioma gradeⅡandⅢ. We speculated that the expression of miR-23a in nucleus of neurons is possibly due to the inability of miR-23a probe to discriminate the pre-miRNA and mature miRNA. We don't exclude the possibility that there has an expression transition of miR-23a during the progression of gliomas. So, further experimental validation is very necessary.
[The tumor suppressor function of miR-124 and miR-149 in glioma cells lines SF126 and U251]
miR-107, miR-124, miR-138 and miR-149 were transfected into glioma cell lines to observe their cellular biological functions by multiple methods. MTT assay showed that miR-107, miR-124, miR-138 and miR-149 can inhibit the growth of SF126 and U251, and the inhibitory effects of miR-124 and miR-149 are the most significant. At the fourth day after transfection with miR-124 and miR-149, the proliferation rate of SF126 has decreased by 40% and 33% respectively, the similar result was observed in U251. Would healing assay indicated that miR-124 and miR-149 can inhibit the migration ability of SF126 and U251 significantly.48 hours after wounding, all cells have repaired their wounds except those transfected with miR-124 and miR-149 mimics. These results implied a possible tumor suppressor role of miR-124 and miR-149 during the tumorigenesis of gliomas.
[Construction of mRNA dynamics expression profiles in gliomas with different pathological grades]
By cDNA microarray, the expression levels of 30968 genes were detected in the same samples that ever used in miRNA microarray, and the same statistical approach used in miRNA data analysis was also applied. We found the expression profiles of significant genes in gliomas with different pathological grades could be classified into 14 categories. BMP1, CDK4, DDX5, DEK, HARS, HNRNPA2B1, IKBKB, LAMA4, p53, PCNA, PLAGL2, TLR3, VCAM1 are up-regulated in all gliomas. The expression levels of HARS, HNRNPA2B1, p53, PLAGL2 showed no apparent correlation with pathological grades while the expression levels of BMP1, DDX5, DEK, IKBKB, LAMA4, PCNA, TLR3 are positively correlated with pathological grades. ANXA1, JAK2, JUN, MMP14, RAP1B, STAT1, VCL, VIM are down-regulated mainly in glioma grade II and III. Additionally, the expression levels of ANK1 and APC are negatively correlated with glioma grades.
[Bioinformatic analysis of differentially expressed mRNAs]
A comprehensive functional annotation software DAVID was used to analyze the Gene Ontology_BP (biological process) of differentially expressed genes. Analysis showed that the up-regulated genes in glioma grade I are mainly involved in transcription, RNA splicing, cell proliferation and so on. While the up-regulated genes in glioma grade III are mainly involved in apoptosis, biological adhesion, RNA splicing, cell cycle, signal transduction, and so on. The down-regulated genes of glioma grade I, II and III are mainly involved in phosphorus metabolic process, intracellular signaling cascade, protein localization, intracellular transport, neuron differentiation, and so on. These results implied that the biological function of down-regulated genes has no significant difference among the glioma grades, whereas the biological function of up-regulated genes has made an obvious transition from low-grade glioma to high-grade glioma.
Then the signaling pathways were analyzed and found several genes that involved in the regulation of MAPK, JAK/STAT, WNT, NF-κB and Toll-like receptor signaling pathways have altered expression in gliomas. The positive factors in regulation of the signaling pathways mentioned above such as RAP1B, JUN, FOS, JAK2, STAT1, TCF7L1, IKBKB, TRL3, MYD88 are up-regulated in gliomas while the negative factors such as DUSP8, APC, CYLD are down-regulated in gliomas. These results implied that MAPK, JAK/STAT, WNT, NF-κB and Toll-like receptor signaling pathways are activated during the tumorigenesis of gliomas.
[Docking study of differentially expressed miRNAs and mRNAs]
Pearson correlation analysis was used for estimating the correlation between differentially expressed miRNAs and their predicted target mRNAs, and the mRNAs negatively correlated in expression level with their corresponding miRNAs are considered as putative target mRNAs. The negative correlation analysis indicates that miR-23a, miR-26a, miR-338-5p, miR-491-3p, miR-107, miR-124, miR-128a/128b, miR-138, miR-149, miR-181a, miR-181b and miR-302b have more putative target mRNAs than others and PLAGL2, HNRNPA2B1, RAP1B, DEK, etc have more corresponding miRNAs. The negative correlation between RAP IB and miR-149 in expression level has been validated by Real-time PCR. These results implied that one miRNA can regulate several mRNAs while one mRNAs can be regulated by several miRNAs. Taken together, the miRNAs and genes mentioned may play an important role in the initiation and progression of gliomas.
[Conclusion]
We have analyzed simultaneously the expression profiles of differentially expressed miRNAs and mRNAs in gliomas with different pathological grades and obtained some preliminary conclusions, which contribute to the further clarification of the molecular mechanisms involved in the tumorigenesis of gliomas and the finding of diagnostic and deteriorated markers in gliomas.
胶质瘤是颅内最常见的恶性肿瘤,以星形细胞胶质瘤最为多见,其次为胶质母细胞瘤。研究表明,胶质瘤的发生发展常涉及p53、p16、RB、PTEN、LRRC4等抑癌基因的失活以及c-Myc、HRAS、EGFR、MDM2等基因的过表达,而这些基因在不同病理级别的胶质瘤中具有特定的表达变化模式,如p53突变与过表达、MDM2过表达常发生于各级别胶质瘤,其表达水平与病理分级之间无明显关联,然而亦有文献报道,p53突变与过表达在高级别胶质瘤中表现更为明显；p16、LRRC4氐表达亦常发生于各级别胶质瘤,且在高级别胶质瘤中改变更为明显；PTEN突变,RB失活,c-Myc、HRAS、EGFR过表达则发生于高级别胶质瘤。基因表达受DNA水平、转录水平、转录后水平及蛋白水平等多层面的调控,其中miRNA在转录后水平对基因表达的精细调控近年来倍受关注。研究表明,miRNA在细胞增殖、分化、凋亡以及机体发育、代谢等基本生命活动过程中发挥重要作用,miRNA表达异常与包括胶质瘤在内的多种肿瘤的发生密切相关。研究发现,miR-21、miR-221、miR-10b等在胶质母细胞瘤中表达上调,而原本在正常脑组织中含量丰富的rniR-128、miR-181等则表达下调。进一步研究表明,这些表达异常的miRNA在胶质瘤发生发展过程中可能发挥类似癌基因或抑癌基因的作用。然而,对于miRNA在胶质瘤不同病理级别中的动态表达模式鲜有报道。
基于以上认识,本研究同时采用miRNA芯片和全基因组表达谱芯片探讨miRNA与基因在胶质瘤不同病理级别中的表达变化规律,构建胶质瘤不同病理级别差异miRNA/mRNA动态表达谱,并对筛选的差异miRNA/mRNA进行了对接研究。
[胶质瘤不同病理级别差异miRNA动态表达谱构建]
采用miRNA芯片分析了3例正常脑组织及13例星形细胞胶质瘤组织(Ⅰ级3例、Ⅱ级5例、Ⅲ级5例)中637种miRNA的表达情况,结合非参数检验、方差分析以及t检验获得不同病理级别胶质瘤的差异miRNA。我们对这些差异miRNA在各级别胶质瘤中的表达变化规律进行了总结分析,结果显示,差异miRNA在不同病理级别胶质瘤中的表达模式大致分为8类。其中miR-26a在各级别胶质瘤中均表达上调,尤其以在胶质瘤Ⅲ级中表达上调最为明显,而miR-21、miR-23a则主要在胶质瘤Ⅲ级中表达上调,这些提示miR-26a可能与胶质瘤的发生及恶性进展相关,而miR-21、miR-23a则主要与胶质瘤的恶性进展相关；此外,miR-107、miR-124、miR-128a/128b、miR-138、miR-149、miR-181a、miR-181b、miR-302b在各级别胶质瘤中均表达显著下调,提示这些差异miRNA与胶质瘤的发生密切相关。由此可见,miRNA在不同病理级别胶质瘤中的表达并不是单一的上调或下调,它们存在各自特定的表达变化模式,以此构成胶质瘤不同病理级别特定的差异miRNA表达谱。
[部分差异miRNA在不同病理级别胶质瘤中动态表达模式的验证]
应用Real-time PCR对miR-107、miR-124、miR-138、miR-149、miR-302b. miR-23a在6例正常脑组织及50例胶质瘤组织(Ⅰ级6例、Ⅱ级20例、Ⅲ级9例、Ⅳ级15例)中进行了验证,证实miR-107、miR-124、miR-138. miR-149在各级别胶质瘤中表达均显著下调,且其表达水平与病理分级负相关,该验证结果与芯片结果相符。miR-23a则在胶质瘤Ⅱ、Ⅲ级中表达逐渐上调,以在胶质瘤Ⅲ级中表达水平最高,与芯片结果相符,但在胶质瘤Ⅳ级中几乎检测不到表达。随后我们应用miR-23a寡核苷酸探针在同一批组织样本中进行了原位杂交验证,结果显示,miR-23a在正常脑组织中主要表达于神经元的胞核,而在胶质瘤Ⅱ、Ⅲ级中主要表达于胶质细胞的胞浆,在胶质瘤Ⅳ级中仍未检测到阳性表达。我们推测,miR-23a在神经元胞核中的表达一方面可能是由于原位杂交探针不能区分miRNA前体与成熟体,另一方面不排除miR-23a在胶质瘤发生发展过程中发生表达移位,其具体机制有待于进一步研究。
[miR-124、miR-149在胶质瘤发生发展中可能发挥抑癌功能]
将Lip2000、NC、miR-107、miR-124、miR-138、miR-149 mimics分别转染胶质瘤细胞,通过MTT、细胞划痕实验观察恢复这些miRNA的表达对胶质瘤细胞生物学行为的影响。MTT实验表明转染miR-107、miR-124、miR-138、miR-149 mimics后,胶质瘤细胞SF126、U251的生长受到不同程度的抑制,尤其以miR-124. miR-149的抑制作用最为明显,转染第四天时,与阴性对照组相比,转染miR-124mimics的细胞其增长率降低40%,转染miR-149 mimics的细胞其增长率降低33%；细胞划痕实验表明,miR-124、miR-149能明显抑制SF126、U251细胞的迁移能力,划痕后48hr,其它各组的伤口均已愈合,而转染miR-124、miR-149 mimics的细胞其伤口仍清晰可见。以上提示miR-124、miR-149在胶质瘤发生发展过程中可能发挥抑癌功能。
[胶质瘤不同病理级别差异mRNA动态表达谱构建]
采用全基因组表达谱芯片检测用于miRNA芯片分析的3例正常脑组织及13例胶质瘤组织中30968个基因的表达情况,并运用与miRNA芯片数据分析同样的方法进行差异基因筛选。将筛选的各级别胶质瘤中差异表达基因进行整合,探讨其在不同病理级别胶质瘤中的表达变化规律,结果显示,差异基因在不同病理级别胶质瘤中的表达模式大致分为14类。BMP1、CDK4、DDX5、DEK、HARS、HNRNPA2B1、IKBKB、LAMA4. p53、PCNA、PLAGL2、TLR3、VCAM1等在各级别胶质瘤中均表达上调,其中HARS、HNRNPA2B1、p53、PLAGL2的表达水平与胶质瘤的病理分级无明显关联,BMP1、DDX5、DEK、IKBKB、LAMA4、PCNA、TLR3的表达水平则与病理分级正相关；ANXA1、JAK2、JUN、MMP14、RAP1B、STAT1、VCL、VIM等主要在胶质瘤Ⅱ、Ⅲ级中表达上调；此外,ANK1、APC等在胶质瘤Ⅰ、Ⅱ、Ⅲ级中逐渐表达下调。
[胶质瘤不同病理级别差异mRNA的生物信息学分析]
应用基因功能分析软件DAVID,分别对各级别胶质瘤中表达上调和表达下调的差异基因进行Gene Ontology_BP分析,结果显示：胶质瘤Ⅰ、Ⅱ级中表达上调的基因主要参与转录调控、RNA剪接、核酸代谢、生物大分子组装、细胞应激反应等一系列生物学过程,此外胶质瘤Ⅰ级中表达上调的基因还参与了细胞增殖,胶质瘤Ⅲ级中表达上调的基因则主要参与细胞凋亡、生物大分子组装、细胞粘附、RNA剪接、细胞周期、信号转导等一系列生物学过程；胶质瘤Ⅰ、Ⅱ、Ⅲ级中表达下调的基因主要参与磷酸化代谢、信号转导、蛋白定位、生物大分子运输、神经元分化等一系列生物学过程。由此可见,在不同病理级别胶质瘤中表达下调的基因其生物学功能大致相似,而表达上调的基因其生物学功能重心发生了转移,表现为在胶质瘤Ⅰ级中以参与转录调控、细胞增殖的基因为主,而在胶质瘤Ⅱ、Ⅲ级中参与细胞凋亡、细胞粘附的基因逐渐增多。
结合KEGG数据库,我们发现在胶质瘤中多个信号通路分子,如RAP1B、JUN、FOS、JAK2、STAT1、TCF7L1、IKBKB、TRl3、MYD88等表达上调,而信号通路负调控因子如DUSP8、APC、CYLD等表达下调,提示MAPK、JAK/STAT、WNT、NF-kB、Toll样受体等多条信号通路的活化参与了胶质瘤的发生发展。
[胶质瘤不同病理级别差异ⅠniRNA/mRNA的对接研究]
通过对胶质瘤不同病理级别差异miRNA/mRNA表达谱的对接分析,我们发现,表达上调的miRNA中以miR-23a、miR-26a、miR-338-5p、miR-491-3p对接的靶基因数目较多；表达下调的miRNA中以miR-107、miR-124、miR-128a/128b、miR-138、miR-149、miR-181a、miR-181b、miR-302b对接的靶基因数目较多；差异表达基因中以PLAGL2、HNRNPA2B1、RAP1B、DEK等对接的miRNA数目较多,其中RAP1B与miR-149的负相关表达模式已经证实,提示一种miRNA可调控多个基因的表达,而一个基因的表达又可受多种miRNA调控,同时也说明了上述差异rniRNA与基因在胶质瘤发生发展中可能起重要作用。
综上所述,我们通过对同一批正常脑组织与各级别胶质瘤组织样本中差异miRNA/mRNA动态表达谱的研究及对接分析,初步揭示了差异miRNA与基因在胶质瘤不同病理级别中的表达变化规律,进一步阐明了胶质瘤发生发展的分子机制,为寻找有效的胶质瘤早期诊断及恶性进展的分子标志提供了充分的理论依据。
[Background]
Glioma is the most common intracranial malignant tumor, and astrocytoma is the most common one, followed by glioblastoma. Many researches have shown that the tumorigenesis of gliomas is often correlated with the inactivation of several tumor suppressor genes such as p53, p16, RB, PTEN, LRRC4 and activation of c-Myc, HRAS, EGFR, MDM2. We found that these genes have their specific expression profile in different pathological grades of gliomas. The mutation and overexpression of p53 and MDM2 are common in gliomas and showed no apparent correlation with the tumor grades, however there has also been reported that the mutation and overexpression of p53 is even obvious in high-grade gliomas. p16 and LRRC4 are down-expressed in gliomas and their expression levels are correlated with the glioma grades. The mutation of PTEN, loss of RB and overexpression of c-Myc, HRAS and EGFR are involved in high-grade gliomas.
Gene expression is regulated at the genomic, transcriptomic, post-transcriptomic and proteomic levels. Recently the function of miRNAs in post-transcriptomic regulation are highly been concerned. Many researches have shown that miRNAs are playing important roles in cell proliferation, differentiation, apoptosis, development and metabolism. And it is noticed that the alteration of miRNA expression is common in most type of tumors including glioma. Studies showed that mir-21, mir-221 and mir-lOb are overexpressed, whereas a set of brain-enriched miRNAs such as mir-128 and mir-181 are down-expressed in glioblastomas. Further researches have found that these miRNAs could act as oncogene or tumor suppressors during tumorigenesis. However the miRNA expression profile in different pathological grades of gliomas was little known.
According to the knowledge above, we analyzed simultaneously the expression profiles of miRNA and mRNA in gliomas with different pathological grades by using miRNA and cDNA microarray. Then the dynamics expression profiles of miRNAs and mRNAs were obtained, and docking study was done in gliomas with different pathological grades.
[Construction of miRNA dynamics expression profiles in gliomas with different pathological grades]
The expression levels of 637 miRNAs were detected in 3 normal brain tissue and 13 gliomas (3 grade I,5 grade II and 5 grade III) by miRNA microarray. Multi-methods were used to analyze the differentially expressed miRNAs in different pathological grades. Then the expression profiles of significant miRNAs were analyzed, and further classified into 8 categories. miR-26a is overexpressed in all gliomas especially in grade III whereas miR-21 and miR-23a are overexpressed mainly in grade III, which indicate that miR-26a may contribute to the initiation and progression of gliomas while miR-21 and miR-23a are mainly involved in the progression of gliomas. Additionally, miR-107, miR-124, miR-128a/128b, miR-138, miR-149, miR-181a, miR-181b, miR-302b are significantly down-regulated in all gliomas, and we infer that these miRNAs are correlated with the tumorigenesis of gliomas. These results implied that differentially expressed miRNAs have their specific expression profiles in different pathological grades of glioma.
[Validation of miRNA dynamics expression profiles for six miRNAs in different pathological grades]
The expression profiles of miR-107, miR-124, miR-138, miR-149, miR-302b and miR-23a in 6 normal brain tissue and 50 gliomas (6 grade I,20 grade II,9 grade III and 15 grade IV) were analyzed by Real-time PCR. It has been validated that miR-107, miR-124, miR-138, miR-149 are down-regulated in all gliomas and their expression levels are negatively correlated with pathological grades, which are consistent with the results of microarray. The expression level of miR-23a is increased gradually in gliomas and reaches the highest level in grade III, whereas no expression is detected in grade IV. Then we detected the expression level of miR-23a by in situ hybridization in same samples. The results showed that miR-23a is mainly expressed in nucleus of normal neurons and cytoplasma of glials in glioma gradeⅡandⅢ. We speculated that the expression of miR-23a in nucleus of neurons is possibly due to the inability of miR-23a probe to discriminate the pre-miRNA and mature miRNA. We don't exclude the possibility that there has an expression transition of miR-23a during the progression of gliomas. So, further experimental validation is very necessary.
[The tumor suppressor function of miR-124 and miR-149 in glioma cells lines SF126 and U251]
miR-107, miR-124, miR-138 and miR-149 were transfected into glioma cell lines to observe their cellular biological functions by multiple methods. MTT assay showed that miR-107, miR-124, miR-138 and miR-149 can inhibit the growth of SF126 and U251, and the inhibitory effects of miR-124 and miR-149 are the most significant. At the fourth day after transfection with miR-124 and miR-149, the proliferation rate of SF126 has decreased by 40% and 33% respectively, the similar result was observed in U251. Would healing assay indicated that miR-124 and miR-149 can inhibit the migration ability of SF126 and U251 significantly.48 hours after wounding, all cells have repaired their wounds except those transfected with miR-124 and miR-149 mimics. These results implied a possible tumor suppressor role of miR-124 and miR-149 during the tumorigenesis of gliomas.
[Construction of mRNA dynamics expression profiles in gliomas with different pathological grades]
By cDNA microarray, the expression levels of 30968 genes were detected in the same samples that ever used in miRNA microarray, and the same statistical approach used in miRNA data analysis was also applied. We found the expression profiles of significant genes in gliomas with different pathological grades could be classified into 14 categories. BMP1, CDK4, DDX5, DEK, HARS, HNRNPA2B1, IKBKB, LAMA4, p53, PCNA, PLAGL2, TLR3, VCAM1 are up-regulated in all gliomas. The expression levels of HARS, HNRNPA2B1, p53, PLAGL2 showed no apparent correlation with pathological grades while the expression levels of BMP1, DDX5, DEK, IKBKB, LAMA4, PCNA, TLR3 are positively correlated with pathological grades. ANXA1, JAK2, JUN, MMP14, RAP1B, STAT1, VCL, VIM are down-regulated mainly in glioma grade II and III. Additionally, the expression levels of ANK1 and APC are negatively correlated with glioma grades.
[Bioinformatic analysis of differentially expressed mRNAs]
A comprehensive functional annotation software DAVID was used to analyze the Gene Ontology_BP (biological process) of differentially expressed genes. Analysis showed that the up-regulated genes in glioma grade I are mainly involved in transcription, RNA splicing, cell proliferation and so on. While the up-regulated genes in glioma grade III are mainly involved in apoptosis, biological adhesion, RNA splicing, cell cycle, signal transduction, and so on. The down-regulated genes of glioma grade I, II and III are mainly involved in phosphorus metabolic process, intracellular signaling cascade, protein localization, intracellular transport, neuron differentiation, and so on. These results implied that the biological function of down-regulated genes has no significant difference among the glioma grades, whereas the biological function of up-regulated genes has made an obvious transition from low-grade glioma to high-grade glioma.
Then the signaling pathways were analyzed and found several genes that involved in the regulation of MAPK, JAK/STAT, WNT, NF-κB and Toll-like receptor signaling pathways have altered expression in gliomas. The positive factors in regulation of the signaling pathways mentioned above such as RAP1B, JUN, FOS, JAK2, STAT1, TCF7L1, IKBKB, TRL3, MYD88 are up-regulated in gliomas while the negative factors such as DUSP8, APC, CYLD are down-regulated in gliomas. These results implied that MAPK, JAK/STAT, WNT, NF-κB and Toll-like receptor signaling pathways are activated during the tumorigenesis of gliomas.
[Docking study of differentially expressed miRNAs and mRNAs]
Pearson correlation analysis was used for estimating the correlation between differentially expressed miRNAs and their predicted target mRNAs, and the mRNAs negatively correlated in expression level with their corresponding miRNAs are considered as putative target mRNAs. The negative correlation analysis indicates that miR-23a, miR-26a, miR-338-5p, miR-491-3p, miR-107, miR-124, miR-128a/128b, miR-138, miR-149, miR-181a, miR-181b and miR-302b have more putative target mRNAs than others and PLAGL2, HNRNPA2B1, RAP1B, DEK, etc have more corresponding miRNAs. The negative correlation between RAP IB and miR-149 in expression level has been validated by Real-time PCR. These results implied that one miRNA can regulate several mRNAs while one mRNAs can be regulated by several miRNAs. Taken together, the miRNAs and genes mentioned may play an important role in the initiation and progression of gliomas.
[Conclusion]
We have analyzed simultaneously the expression profiles of differentially expressed miRNAs and mRNAs in gliomas with different pathological grades and obtained some preliminary conclusions, which contribute to the further clarification of the molecular mechanisms involved in the tumorigenesis of gliomas and the finding of diagnostic and deteriorated markers in gliomas.
引文
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