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

Bioinformatics analysis about point mutation of exon 7 in LMNA in prostate tissues and cell lines

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.1159C>CA(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。

Objective Our research focuses on investigating the associations between point mutation of exon 7 in LMNA and its expression in prostate cancer.Methods A lamin A/C（LMNA） genomic missense variation C.1159 CCA（p.L387 LI,located chr1：156106006）,identified by DNA sequencing for all 12 exons of LMNA,in HPV-immobilized normal epithelium（RWPE-1） and high-invasive prostate cancer cell line（PC-3 M-1 E8） models was recognized.To verify these point mutations in prostate cancer patients,we downloaded three group data from Pub Med GEO datasets,respectively from global RNA sequencing data,paraffin-embedded（FFPE） prostatectomy samples from 100 patients,from the transcriptome（poly A ＋） data of 20 prostate cancer tumors and 10 matched normal tissues and from sequencing data from 21 prostate cell lines.All reads were selected on the range from 156105950 to 156106055 on chr1 and these reads were aligned with CTACGCCTG or NTACGCCTGTCCCCCAGCCC.The length of reads analyzed is 50 nt for one time.Further,we analyzed thefunction of the sequence around the mutation point using GEO database.At last,we analyzed the effects of LMNA mutation by Western blotting in transfected mutation plasmid cells.Results We found that missense mutation has two forms of C/CA（p.L387 LI） and C/CG（p.L387 LV）,however same-sense mutation has four forms of C/CT（p.L387 L）,C/CA（p.R388 R）,C/CT（p.R388 R） and C/CG（p.R388 R） in the range from 156106006 to 156106011 of exon 7 of chr1 in LMNA from samples or cell lines.However,the incidence of missense mutation accounted for respectively 40%,11%,2% and 6% in normal,Gleason score 5-6,7 and 8-10 of patient samples.In16 prostate cancer cell lines and 5 prostate benign cell lines,the incidence of missense mutation accounted for respectively 31% and 60% in cancer and benign cell lines.In addition,this sequence from 156106008 to 156106066 on chr1 was transcription factors（TFs） binding site;The common TFs in this region were PAX5,HEN1（NHLH1）,HTF,P53,MIF1,COMP1 and NGFIC（NGF4）.By functional cluster analysis,the function of these TFs focuses on chromatin organization,positive regulation of RNA metabolic process,regulation of histone modification,regulation of chromosome organization and negative regulation of programmed cell death.Mutation led to a downregulation of in LMNA and phosphor-LMNA expression levels.Conclusion Missense mutation incidence of 156106006 on chr1 in both prostate cancer tissues and prostate cancer cell lines is lower than that of prostate normal tissue and prostate benign cell lines.It is possible to have relationship between the mutation and expression level or a heterogeneous expression pattern of LMNA.