MEG8-DMR对人肝癌Huh7细胞DLK1表达水平的影响
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摘要
目的 DLK1-DIO3印记区间位于人14号染色体、小鼠12号染色体上,在两者的表达中高度保守,其间有三个父本控制的蛋白质编码区域,以及多个母本控制的非编码转录本,还有大量的C/D snoRNA和microRNA。其间有三个差异甲基化区,分别为父本甲基化的IG-DMR和MEG3-DMR,以及母本甲基化的MEG8-DMR。先前关于对MEG8-DMR的研究集中于肿瘤方面的较少,因此探究差异甲基化区MEG8-DMR对肿瘤发生的影响以及其调控机制,深入分析印记基因参与肿瘤的发病机理,具有重要的意义。方法本实验通过CRISPR/Cas9基因编辑技术,先依据脱靶位点少的原则选择sgRNA,构建重组载体并通过脂质体法将其转染入Huh7细胞后检测出重组载体在Huh7细胞系中对MEG8-DMR区域进行了有效的敲除。结果敲除MEG8-DMR后细胞形态没有发现明显变化,同时发现敲除MEG8-DMR导致上游基因DLK1表达水平的下调。结论本实验中通过对MEG8-DMR的敲除,导致DLK1表达下调,而DLK1在几种肿瘤包括神经纤维瘤、骨髓增生异常综合征和肝癌患者的血清中呈高表达,且肿瘤的形成是多因素、多基因共同参与的结果,MEG8-DMR上下游区域分布着一系列母系表达非编码转录本,和大量的miRNA和C/DsnoRNA,因此,MEG8-DMR及临近基因在肝细胞性肝癌的发生过程中可能具有表达调控的重要功能。
Objective The imprinted region of DLK1-DIO3 is located on human chromosome 14 and mouse chromosome 12 and is highly conserved in the expression of both. There are three parental control protein coding regions and multiple maternal controlled non-coding transcripts,and a large number of C/D snoRNAs and microRNAs.There are three regions of differential methylation between the parental methylated IG-DMR and MEG3-DMR, and the maternal methylated MEG8-DMR. Previous a few studies on MEG8-DMR focused on tumors.Therefore,it is of great significance to explore the influence of MEG8-DMR on tumorigenesis and its regulation mechanism and to deeply analyze the pathogenesis of the imprinted genes involved in tumors.Methods In this study, CRISPR/Cas9 gene editing techniques were used to select sgRNAs based on the principle of less off-target sites,construct recombinant vectors and transfect them by liposome method.After incorporation into Huh7 cells, it was detected that the recombinant vector effectively knocked out the MEG8-DMR region in the Huh7 cell line.Results No significant changes in cell morphology were observed after knockout of MEG8-DMR.It was also found that the knockdown of MEG8-DMR resulted in the down-regulation of the expression level of the upstream gene DLK1.Conclusion In this experiment,the knockdown of MEG8-DMR resulted in the down-regulation of DLK1 expression.DLK1 was highly expressed in the serum of several tumors,including neurofibroma, myelodysplastic syndrome and liver cancer, and the tumor formation was multifactorial.As a result of multiple genes co-involved,a series of maternal expressive non-coding transcripts and a large number of miRNAs and C/D snoRNAs are distributed in the upstream and downstream regions of MEG8-DMR.Therefore,MEG8-DMR and its adjacent genes may play an important role in the regulation of hepatocellular carcinoma.
Objective The imprinted region of DLK1-DIO3 is located on human chromosome 14 and mouse chromosome 12 and is highly conserved in the expression of both. There are three parental control protein coding regions and multiple maternal controlled non-coding transcripts,and a large number of C/D snoRNAs and microRNAs.There are three regions of differential methylation between the parental methylated IG-DMR and MEG3-DMR, and the maternal methylated MEG8-DMR. Previous a few studies on MEG8-DMR focused on tumors.Therefore,it is of great significance to explore the influence of MEG8-DMR on tumorigenesis and its regulation mechanism and to deeply analyze the pathogenesis of the imprinted genes involved in tumors.Methods In this study, CRISPR/Cas9 gene editing techniques were used to select sgRNAs based on the principle of less off-target sites,construct recombinant vectors and transfect them by liposome method.After incorporation into Huh7 cells, it was detected that the recombinant vector effectively knocked out the MEG8-DMR region in the Huh7 cell line.Results No significant changes in cell morphology were observed after knockout of MEG8-DMR.It was also found that the knockdown of MEG8-DMR resulted in the down-regulation of the expression level of the upstream gene DLK1.Conclusion In this experiment,the knockdown of MEG8-DMR resulted in the down-regulation of DLK1 expression.DLK1 was highly expressed in the serum of several tumors,including neurofibroma, myelodysplastic syndrome and liver cancer, and the tumor formation was multifactorial.As a result of multiple genes co-involved,a series of maternal expressive non-coding transcripts and a large number of miRNAs and C/D snoRNAs are distributed in the upstream and downstream regions of MEG8-DMR.Therefore,MEG8-DMR and its adjacent genes may play an important role in the regulation of hepatocellular carcinoma.
引文
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[6]郑武,谷峰.CRISPR/Cas9的应用及脱靶效应研究进展[J].遗传,2015(10):1003-1010.
[7]Jelinic P,Shaw P.Loss of imprinting and cancer[J].Journal of Pathology,2007,211(3):261.
[2]Court F,Tayama C,Romanelli V,et al.Genome-wide parent-oforigin DNA methylation analysis reveals the intricacies of human imprinting and suggests a germline methylation-independent mechanism of establishment.[J].Genome Res.2014,24(4):554-569.
[3]Donsante A,Miller D G,Li Y,et al.AAV vector integration sites in mouse hepatocellular carcinoma[J].Science,2007,317(5837):477.
[4]Dupuy A J,Rogers L M,Kim J,et al.A modified sleeping beauty transposonsy stemth at can be used to modelawi devariety of human cancer sinmic.[J].Cancer Research,2009,69(20):8150-8156.
[5]Lin S P,Youngson N,Takada S,et al.Asymmetric regulation of imprinting on the maternal and paternal chromosomes at the Dlk1-Gtl2 imprinted cluster on mouse chromosome 12[J].Nature Genetics,2003,35(1):97-102.
[6]郑武,谷峰.CRISPR/Cas9的应用及脱靶效应研究进展[J].遗传,2015(10):1003-1010.
[7]Jelinic P,Shaw P.Loss of imprinting and cancer[J].Journal of Pathology,2007,211(3):261.