生物信息学方法分析前列腺癌组织及细胞系中LMNA基因第七外显子点突变

发布时间：2018-04-14 16:31

本文选题：前列腺癌 + 核纤层蛋白A/C　；参考：《首都医科大学学报》2017年06期

【摘要】：目的探讨前列腺癌中核纤层蛋白A/C(lamin A/C,LMNA)基因第七外显子点突变与LMNA表达差异的相关性。方法采用高通测序分析了永生化正常前列腺上皮细胞(RWPE-1)、低侵袭力前列腺癌上皮细胞(PC-3M-2B4)及高侵袭力前列腺癌上皮细胞(PC-3M-1E8)LMNA基因的12个外显子,识别出在RWPE-1与PC-3M-1E8细胞系中C.1159CCA(p.L387LI)点突变,定位在1号染色体156106006。为了验证点突变在前列腺癌患者中的分布情况,Pub Med GEO数据库下载了3组数据,分别是100例前列腺癌患者石蜡包埋标本的总RNA测序数据、20例前列腺癌和10例配对正常组织的转录组数据及21种前列腺正常及癌细胞株的测序数据。在1号染色体上选取156105950到156106055范围,设置比对序列CTACGCCTG或者NTACGCCTGTCCCCCAGCCC,每次分析比对长度为50 nt。同时,利用GEO数据库分析了突变点周围序列的特点与功能。最后,转染突变质粒,蛋白质电泳分析LMNA点突变对LMNA蛋白质表达的影响。结果在所有分析的样品中,1号染色体LMNA外显子7从156106006到156106011的区域内,存在2种错义突变形式:C/CA(p.L387LI)、C/CG(p.L387LV)和4种同义突变形式:C/CT(p.L387L)、C/CA(p.R388R)、C/CT(p.R388R)、C/CG(p.R388R)。前列腺癌按格里森(Gleason score,GS)评分系统分组,错义突变的总发生率分别占40%(正常)、11%(GS 5-6)、2%(GS 7)和6%(GS 8-10)。在16种前列腺癌细胞系和5种前列腺良性细胞系中,错义突变的总发生率分别占31%和60%。此外,还发现1号染色体上从156106008到156106066是转录因子结合位点,常见转录因子为PAX5、HEN1(NHLH1)、HTF、P53、MIF1、COMP1和NGFIC(NGF4)。通过功能聚类分析,这些转录因子的功能主要集中在染色质重组及调控、RNA代谢过程的正向调节、组蛋白修饰的调控以及程序性细胞死亡的负调节等方面。高表达突变LMNA的细胞系LMNA及磷酸化LMNA蛋白质表达下调。结论 156106006位点突变多发生在前列腺正常组织和前列腺良性细胞株,与LMNA蛋白质表达密切相关,可能是lamin蛋白质异源性表达的机制之一。
[Abstract]:Objective to investigate the relationship between the point mutation of the seventh exon of nuclear laminin A/C(lamin A / C rmna gene and the difference of LMNA expression in prostate cancer.Methods the 12 exons of RWPE-1, PC-3M-2B4 and PC-3M-1E8 LMNA gene in immortalized normal prostatic epithelial cells and low invasive prostate cancer epithelial cells were analyzed by high pass sequencing. The mutation of L387LI gene was identified in RWPE-1 and PC-3M-1E8 cell lines.Located on chromosome 1 156106006.To verify the distribution of point mutations in prostate cancer patients and download three sets of data from the Pub Med GEO database,The total RNA sequencing data were obtained from paraffin-embedded paraffin embedded specimens of 100 prostate cancer patients respectively. The transcriptome data of 20 cases of prostate cancer and 10 cases of matched normal tissues and 21 kinds of prostate normal and cancer cell lines were sequenced.From 156105950 to 156106055 on chromosome 1, the alignment sequence CTACGCCTG or NTACGCCTGTCCCCCAGCCC was set, and the length of each analysis was 50 NT.At the same time, the characteristics and functions of the sequence around the mutation point are analyzed by using GEO database.Finally, the effect of point mutation of LMNA on the expression of LMNA protein was analyzed by protein electrophoresis.Results in all the samples analyzed, there were two missense mutations in the region of exon 7 of chromosome 1 from 156106006 to 156106011. There were two missense mutations: C / CAp.L387LI / L / L ~ (387L) and four synonyms: C / C / C / C / L ~ (387L) / C / C / C / C / R ~ (388R).According to the Gleason scoreGSscoring system, the total incidence of missense mutation in prostate cancer was 40% (normal 11 GS 5-6% GS7) and 6%(GS 8-10%.In 16 prostate cancer cell lines and 5 benign prostate cell lines, the total incidence of missense mutation was 31% and 60%, respectively.It was also found that the transcription factor binding sites on chromosome 1 were from 156106008 to 156106066, and the common transcription factors were PAX5, HEN1, NHLHH1, HTF5, P53, MIF1, COMP1 and NGFICF4, respectively.By functional cluster analysis, these transcription factors mainly focus on the positive regulation of chromatin recombination and regulation of RNA metabolism, the regulation of histone modification and the negative regulation of programmed cell death.The expression of LMNA and phosphorylated LMNA protein was down-regulated in the cell line with high expression of mutant LMNA.Conclusion the 156106006 mutation occurs in normal prostate tissues and benign prostate cell lines, which is closely related to the expression of LMNA protein, which may be one of the mechanisms of heterogenous expression of lamin protein.
【作者单位】：首都医科大学基础医学院生物化学与分子生物学系;首都医科大学生物医学工程学院生物医学信息学系;
【基金】：国家自然科学基金(81272406,81672834) 北京市教育委员会科技计划面上项目(KM201510025009)~~
【分类号】：R737.25

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