CN114032241B - 检测离体前列腺细胞circKIF4A的m6A甲基化修饰程度的方法 - Google Patents
检测离体前列腺细胞circKIF4A的m6A甲基化修饰程度的方法 Download PDFInfo
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Abstract
环状RNA circKIF4A序列与miR‑1231特定序列产生吸附作用，通过高通量测序鉴定环状RNA circKIF4A靶向miR‑1231特定序列，吸附作用导致前列腺癌细胞癌变的进程，所述的circKIF4A特定序列的m6A甲基化修饰百分比越高，则导致miR‑1231对前列腺癌细胞铁死亡关键基因的表达抑制越强。
Description
技术领域
本申请属于检测前列腺细胞癌变领域，具体分子机制涉及检测环状RNA腺嘌呤核糖核苷酸碱基A的甲基化修饰的表观遗传学领域。
背景技术
已有研究表明环状RNA circKIF4A(hsa_circ_0007255,circBase)通过吸附miRNA，进而调控肝细胞癌和乳头状甲状腺癌的肿瘤细胞铁死亡。通过非编码环状RNA高通量测序发现在前列腺癌组织和正常组织中显著差异表达的环状RNA，生物信息技术可以从小RNA调控层面分析预测发现，该环状RNA抑制表达的miRNAs。从表观遗传学层面分析这种持久的环状RNA高表达进而吸附miRNA，影响其靶基因对肿瘤细胞铁死亡的调控。
但是在相关科学研究领域RIP实验仍然是分析和验证RNA甲基化的标准。环状RNA与miRNA的海绵吸附机制在非编码RNA调控细胞信号通路常见，已有文献报道在甲状腺癌中铁死亡的机制与海绵吸附相关，但是是因何种原因导致吸附的，现有技术并没有给出分子水平的解释，更加没有深入到转录组层面的表观遗传修饰。
发明内容
发明目的：第一通过高通量测序定量分析circKIF4A在癌细胞和正常组织的表达差异；第二通过生物信息分析技术(分析流程)预测与circKIF4A发生吸附作用的miRNAs；第三通过RIP实验技术衡量circKIF4A一段特定序列上腺嘌呤核糖核苷酸A的甲基化处于超甲基化状态(Hypomethylation)。即本权利保护闭合环状RNA circKIF4A的甲基化修饰揭示了前列腺癌恶性进展的一种崭新的分子调控机制。
技术方案：本技术利用高通量测序技术，RIP技术和生物信息分析技术揭示非编码环状RNA circKIF4A的甲基化修饰上调了circKIF4A的表达，进而促进circKIF4A对miRNA1231的吸附作用，而miRNA1231被抑制表达后会上调前列腺癌肿瘤细胞铁死亡中枢因子GPX4抗氧化酶的功能，进而促进前列腺癌恶性进展。
一种环状RNA circKIF4A序列，其特征在于其序列转录本见NM_012310.5。碱基长度4285个碱基，要求权利保护此circRNA的序列3721至4361处，在此范围内我们可以看到这段序列是富含腺嘌呤核糖核苷酸(A)。要求被保护的circRNA在染色体上对应的位置，位于人类X染色体长臂q13.1处，(chrX:69,549,254-69,553,539，参考UCSC GRCh37/hg19基因组)，如图2所示。被甲基化修饰特定碱基序列如下：
3721 CCAGCTTCTT CTCTGGCTGC TCCCCTATCG AAGAAGAGGC CCACTGAAGTTGGAGTCATC
3781 ATCTCTACCC CCAGTCTGGC TTGGGAGATG CTTTCAGGTT GCAGCCAGAAGGGGTTTTTT
3841 AAATGACTTC TCTGGATTTC AGGTTTCTTG CTGTTGAAAA AAGGAACAAAGCGTTACTGA
3901 AAAGAAGGTA ACCTTTGTTG GATGTGGGCC TTAGCCTCCA GGTCCAGACTACTACTCTAT
3961 GTTCTCCAGA AGGGTGCTAA GTCACCTACT GAAGAGAGAA CCAACTGACTTTCCTATTGA
4021 CTCATCAGGA ACCAGTCCTC AGTCTGGTCA AGTTGTTTCT TATTTGTGAGCAGTTCAGGC
4081 TATCTCCTGA TGGGGATGAG GCCAAGGCTT TCTTATCTTT TGGTTGTCTCTGCTTAATGG
4141 AGGAGCCTGG CCTAGGATGG AGGCCTGGCT TAGATCTTTC ATTCCACCTCAGGAATGAGG
4201 TTGTGATCTT TCCTGTCCTG ACCCTCTCTG AATTATGTTT CAATAGTACTCTTGATTGTC
4261 TGCCATGTTG TTGAAGCAAA TGAATTATTT TTAAATGTTA AGTAAGTAAATAAACCTTAG
4321 CCCGTCTACT GTTTGGGAAG ATCCTTCTGT GCTAGAGGGA GAAATAAAATTTCAACCTGT
4381 GTTCCTCA
一种miR-1231特定序列，其特征在于吸附环状RNA circKIF4A特定序列，环状RNAcircKIF4A特定序列与miR-1231特定序列见NO.2。如图3所示。
预测吸附位点举例如下，上方序列为环状RNA circKIF4A序列，下方为miR-1231序列。但是通过生物信息分析，二者通过序列结合的位点不止此一种方式，还可能有其他序列结合位点：
circKIF4A特定序列：UGUUAAUAUUGAUCCCCAGACAG
miR-1231特定序列：CGUCGACAGGCGGGUCUGUG
一种检测离体前列腺细胞circKIF4A的m6A甲基化修饰程度的方法，其特征在于，通过高通量测序检测、生物信息分析环状RNA circKIF4A吸附miR-1231特定序列，所述的circKIF4A序列转录本见NM_012310.5；所述的circKIF4A序列与miR-1231特定序列见NO.2。
所述的circKIF4A的m6A甲基化修饰程度越高，则吸附miR-1231特定序列越多。我们通过高通量测序发现，在前列腺癌患者的癌细胞样本中这段序列的腺嘌呤脱氧核苷酸(A)发生了甲基化，
有益效果：通过高通量测序、分析环状RNA circKIF4A吸附miR-1231特定序列或根据circKIF4A的m6A甲基化修饰程度可以判断细胞癌变程度，进而可以有效地针对性治疗。
附图说明
图1为表观转录组调控层面提示环状RNA抑制表达的miRNAs示意图。
图2为转录本NM_012310.5序列位于染色体位置及碱基序列
图3为：环状RNA circKIF4A特定序列与miR-1231序列NO.2举例
具体实施方式
原理：通过非编码RNA高通量测序(illumina Novaseq平台，双端测序，PE150)，研究circKIF4A在前列腺癌癌细胞铁死亡的调控机制。技术路线如下：
1、通过铁死亡试剂盒，分析circKIF4A和miR-1231对前列腺癌细胞铁死亡细胞程序性调控中枢抑制因子抗氧化酶GPX4的影响。GPX4在癌细胞铁死亡中的表达和活性依赖于谷胱甘肽和硒的存在。哺乳动物细胞和人细胞中已经证明存在以GPX4为核心的抑制细胞铁死亡的信号通路。
2、circKIF4A m6A修饰相关的表观遗传因子METTL16，上调circKIF4A的表达，增加对miR-1231的吸附，进而影响miR-1231的靶基因GPX4，上调铁死亡中枢抑制因子GPX4的表达，增加前列腺癌细胞铁死亡抑制作用，进而使前列腺癌超恶性方向发展。
具体实施例如下：
(1)筛选Circular RNAs
通过RNA-seq高通量测序分析在前列腺癌癌组织中异常表达的circRNAs,在本研究中，根据生物信息分析选择circKIF4A
1.在30对临床前列腺癌组织及其配对癌旁组织中，进行生物学重复的全转录组的RNA-seq分析(包括circRNAs建库)，通过RNA-seq分析筛选出在前列腺癌组织中高表达的circRNAs,通过qPCR在前列腺癌细胞中对筛选到的Circular RNAs进行验证，通过综合分析，选择circKIF4A。
2.在正常前列腺细胞或多种前列腺癌细胞系中，通过qPCR实验分析的circKIF4A。表达，寻找两个circKIF4A表达较高的前列腺癌细胞系，用于后续分析，在此先以PC3 andC4-2细胞为例。
3.分别合成circKIF4A，及其同转录本的mRNA的PCR引物，在PC3 and C4-2细胞中，利用qPCR实验验证及其同转录本的mRNA对RNase R和放线菌铜D的敏感性，以分析circKIF4A的稳定特性。
4.在PC3 and C4-2细胞中，利用qPCR技术，以GAPDH为内参，分析验证circKIF4A在细胞中的表达。
5.利用qPCR实验，在PC3 and C4-2细胞中，从多个shRNAs中筛选出效率最高的shRNA，用于后续实验。
(2)研究circKIF4A对前列腺癌肿瘤细胞铁死亡的影响
通过靶向免疫沉淀、铁死亡试剂盒等细胞生物学、分子生物学和生物化学手段，分析circKIF4A对肿瘤细胞铁死亡的影响。
1.在PC3 and C4-2细胞中，利用circKIF4A shRNAs，通过不同的试剂盒，分析Circular RNA A对iron,lipid ROS,Fe2+,MDA,GSH等铁死亡标志物的影响。
2.在PC3 and C4-2细胞中，利用circKIF4A shRNAs，通过Western blot以及免疫荧光实验，分析circKIF4A对ACSL4、GPX4、xCT、JC-1等重要铁死亡调控蛋白及标志蛋白的影响。
3.在PC3 and C4-2细胞中，利用CCK-8、edu等实验，观察对铁死亡激活剂erastin或GPX4介导的肿瘤细胞增殖抑制的影响。
(3)研究circKIF4A介导的表观遗传修饰影响肿瘤细胞铁死亡的分子机制
通过RNA RIP等细胞生物学、分子生物学和生物化学手段，分析circKIF4A与表观遗传修饰酶(或调控因子)的相互作用，以及对铁死亡相关调控因子对表观遗传修饰和表达影响，分析circKIF4A/表观遗传修饰酶/铁死亡相关调控因子在肿瘤细胞铁死亡中的作用及详细机制。
1.在机制上可以寻找下游的结合蛋白或miRNAs,这里以结合蛋白为例。利用生物素标记circKIF4A,通过RNA RIP实验分析结合蛋白，从中发现甲基转移酶和miRNAs。在PC3and C4-2细胞中，利用RIP实验验证circKIF4A与上述表观遗传调控酶或相关因子的结合，结合文献报道及上述RNA-seq综合分析，筛选出与circKIF4A结合的甲基转移酶METTL16。
2.通过qPCR和Western blot实验，在PC3 and C4-2细胞中，分别利用circKIF4AshRNAs和甲基转移酶METTL16，观察它们对铁死亡调控因子表达的影响。
3.在PC3 and C4-2细胞中，分别利用甲基转移酶METTL16 shRNAs或抑制剂,通过不同的铁死亡试剂盒，分析iron,lipid ROS,Fe2+,MDA,GSH等铁死亡标志物；通过Westernblot以及免疫荧光实验，分析ACSL4、GPX4、xCT、JC-1等重要铁死亡调控蛋白及标志蛋白；利用CCK-8、edu等实验，观察对铁死亡激活剂erastin或RSL3介导的肿瘤细胞增殖的影响。
在PC3 and C4-2细胞中，分别在过表达circKIF4A/过表达circKIF4A+甲基转移酶METTL16 shRNAs/过表达circKIF4A+甲基转移酶METTL16的抑制剂，通过qPCR和Westernblot实验分析铁死亡调控因子GPX4的表达。
5.在PC3 and C4-2细胞中利用铁死亡调控因子GPX4的shRNA,通过不同的试剂盒，分析铁死亡调控因子GPX4；研究GPX4介导的肿瘤细胞增殖的影响。
(4)研究circKIF4A的甲基化修饰，揭示miRNA对前列腺癌铁死亡GPX4的促进机制
通过高通量测序分析circKIF4A序列中腺嘌呤核糖核苷酸A的甲基化修饰。
通过cicrRNA高通量测序分析30对前列腺癌癌组织和癌旁正常配对组织中环状RNA的表达情况，发现前列腺癌癌组织显著性高表达circKIF4A。cicrRNA高通量测序分析表明circKIF4A的表达量在前列腺癌细胞中与正常组织中表达量相比上调54％(测序数据经过分析注释，reads转化为FPKM，然后通过log2(FPKM+1)标准化FPKM数据，计算癌细胞和正常组织circKIF4A的表达量，其中p＝0.0035。测序数据方面，测序质量Q30＝94.41％，测序质量Q20＝97.21％，GC含量比值约等于1。)。并利用荧光定量PCR技术(Q-PCR)在两种前列腺癌细胞系LNCaP和PC-3中，确认circKIF4A显著高表达(设计引物进行荧光定量PCR实验，实验结果表明与house keeping基因GADPH作内参对照，在两种前列腺癌细胞系中，circKIF4A表达量显著上调，p＝0.001)。通过生物信学方法分析，找到与microRNA构建“海绵吸附效应”的miRNAs，并预测circKIF4A吸附miR-1231，促进其靶基因GPX4抗氧化酶的表达，而GPX4是肿瘤细胞铁死亡中枢抑制因子。通过荧光定量PCR实验在前列腺癌细胞系中证实miR-1231在前列腺癌组织中显著低表达(P＝0.001)，通过荧光定量PCR验证GPX4基因高表达(P＝0.001)。由此，揭示环状RNA circKIF4A在前列腺癌细胞中因吸附miR-1231导致其靶基因表达量上升，进而增强对癌细胞铁死亡的抑制作用。这导致了前列腺癌的恶性进展。
利用RIP实验技术，检测circKIF4A m6A(RNA胞嘧啶甲基化)表观遗传修饰，揭示circKIF4A的m6A甲基化修饰(发现37％的circKIF4A序列中的胞嘧啶A发生甲基化修饰，这属于转录组层面的高甲基化修饰)。在两种前列腺癌细胞系LNCaP和PC-3中通过荧光定量PCR发现，甲基化酶METTL家族几个关键的基因表达水平出现异常，其中METTL16表达量上调，作为甲基化转移酶可以增加circRNA的腺嘌呤A甲基化修饰。circRNAs的表观遗传调控因子包括：RNA甲基化转移酶METTL16，RNA去甲基化酶FTO，RNA甲基化阅读蛋白YTHDF1/2/3等。由此，揭示在前列腺癌细胞中表观遗传调控因子METTL16增加circKIF4A m6A甲基化修饰，进而上调circKIF4A表达的机制，增加对miR-1231的吸附。如图1所示。
序列表
<110> 上海裕隆生物科技有限公司
<120> 用环状RNA circKIF4A与miR-1231序列吸附量来检测细胞癌变程度
<141> 2021-11-10
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 668
<212> DNA
<213> Human
<400> 1
ccagcttctt ctctggctgc tcccctatcg aagaagaggc ccactgaagt tggagtcatc 60
atctctaccc ccagtctggc ttgggagatg ctttcaggtt gcagccagaa ggggtttttt 120
aaatgacttc tctggatttc aggtttcttg ctgttgaaaa aaggaacaaa gcgttactga 180
aaagaaggta acctttgttg gatgtgggcc ttagcctcca ggtccagact actactctat 240
gttctccaga agggtgctaa gtcacctact gaagagagaa ccaactgact ttcctattga 300
ctcatcagga accagtcctc agtctggtca agttgtttct tatttgtgag cagttcaggc 360
tatctcctga tggggatgag gccaaggctt tcttatcttt tggttgtctc tgcttaatgg 420
aggagcctgg cctaggatgg aggcctggct tagatctttc attccacctc aggaatgagg 480
ttgtgatctt tcctgtcctg accctctctg aattatgttt caatagtact cttgattgtc 540
tgccatgttg ttgaagcaaa tgaattattt ttaaatgtta agtaagtaaa taaaccttag 600
cccgtctact gtttgggaag atccttctgt gctagaggga gaaataaaat ttcaacctgt 660
gttcctca 668
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cgucgacagg cgggucugug 20
Claims (1)
1.一种检测离体前列腺细胞circKIF4A的m6A甲基化修饰程度的试剂在制备用于前列腺癌细胞癌变程度预测试剂盒中的应用，其特征在于，所述试剂包括miR-1231，所述的miR-1231的序列如SIQ ID NO.3所示，所述的如circKIF4A序列如SIQ ID NO.1所示。
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