环状RNAhsa＿circ＿0082626的特征分析及过表达载体构建
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摘要
目的环状RNA是目前非编码RNA的研究热点,文章旨在研究由抗病毒基因ZC3HAV1基因转录而来的环状RNA hsa_circ_0082626的基本特征以及过表达载体的表达效果。方法利用反向剪切位点的验证、RNA酶消化试验和提取细胞质、细胞核RNA进行细胞内分布定位研究了hsa_circ_0082626的基本特征,RNA酶消化试验中将样品分为RNase R处理组与对照组,RNase R处理组加入20 U(2 U/μg)的RNase R,对照组用等量ddH2O替代,以此检测经RNA酶处理后的表达量变化。转染24、48和72 h后分别收集以转染重组质粒的细胞为过表达组、未经转染的细胞作为阴性对照组,RT-qPCR检测环状RNA表达效果。结果与对照组比较,RNase R处理组ZC3HAV1、GAPDH表达均降低(0.144±0.002 vs 1.000±0.016,0.772±0.058 vs 1.000±0.122,0.077±0.009 vs 1.000±0.164,P<0.05)。hsa_circ_0082626可以抵抗RNase R的处理,主要分布于细胞质。过表达组的hsa_circ_0082626表达量明显较阴性对照组明显增高,而且在转染48 h时的表达量最高(表达量约为对照组的1000倍)。结论成功分析了hsa_circ_0082626的反向剪切位点、RNA酶的抵抗性和在细胞中的表达定位等特征,证明了hsa_circ_0082626确实具有环状结构及成功构建了hsa_circ_0082626的过表达载体,为深入研究环状RNA hsa_circ_0082626的生物功能和机制提供了实验基础。
Objective Circular RNA is a research hotspot of non-coding RNAs. The purpose of this study was to investigate the basic characteristics and expression effect of the overexpression vectors of circular RNA hsa_circ_0082626 transcribed from antiviral gene ZC3 HAV1.Methods The basic characteristics of hsa_circ_0082626 were studied by the verification of reverse cleavage site,RNase digestion assay and intracellular distribution location with extraction of cytoplasmic and nuclear RNA. In the RNase digestion assay,samples were divided into RNase R treatment group and control group. RNase. 20 U(2 U/μg)of RNase R was added to the R treatment group,and the control group was replaced with an equivalent amount of ddH2 O to detect changes in expression levels after RNase treatment. The cells were divided into 2 groups:overexpression group and negative control group. At 24,48 and 72 h after transfection,cells were collected to detect the expression of circular RNA by RT-qPCR.Results Compared with the control group,the expression of ZC3 HAV1,CDR1 as and GAPDH in the RNase R treatment group was increased(0.144±0.002 vs 1.000±0.016,0.772±0.058 vs 1.000±0.122,0.077±0.009 vs 1.000±0.164,P<0.05).Hsa_circ_0082626 could resist the treatment of RNase R and was mainly distributed in cytoplasm. The expression level of hsa_circ_0082626 in the overexpression group was significantly higher than that in the negative control group,and the expression level was the highest at 48 h after transfection.Conclusion The characteristics of hsa_circ_0082626 reverse cleavage site,RNase resistance and expression in cells were successfully analyzed,which proved that hsa_circ_0082626 does have a circular structure. The overexpression vector of hsa_circ_0082626 was successfully constructed to provide an experimental basis for the biological function and mechanism of RNA hsa_circ_0082626.
Objective Circular RNA is a research hotspot of non-coding RNAs. The purpose of this study was to investigate the basic characteristics and expression effect of the overexpression vectors of circular RNA hsa_circ_0082626 transcribed from antiviral gene ZC3 HAV1.Methods The basic characteristics of hsa_circ_0082626 were studied by the verification of reverse cleavage site,RNase digestion assay and intracellular distribution location with extraction of cytoplasmic and nuclear RNA. In the RNase digestion assay,samples were divided into RNase R treatment group and control group. RNase. 20 U(2 U/μg)of RNase R was added to the R treatment group,and the control group was replaced with an equivalent amount of ddH2 O to detect changes in expression levels after RNase treatment. The cells were divided into 2 groups:overexpression group and negative control group. At 24,48 and 72 h after transfection,cells were collected to detect the expression of circular RNA by RT-qPCR.Results Compared with the control group,the expression of ZC3 HAV1,CDR1 as and GAPDH in the RNase R treatment group was increased(0.144±0.002 vs 1.000±0.016,0.772±0.058 vs 1.000±0.122,0.077±0.009 vs 1.000±0.164,P<0.05).Hsa_circ_0082626 could resist the treatment of RNase R and was mainly distributed in cytoplasm. The expression level of hsa_circ_0082626 in the overexpression group was significantly higher than that in the negative control group,and the expression level was the highest at 48 h after transfection.Conclusion The characteristics of hsa_circ_0082626 reverse cleavage site,RNase resistance and expression in cells were successfully analyzed,which proved that hsa_circ_0082626 does have a circular structure. The overexpression vector of hsa_circ_0082626 was successfully constructed to provide an experimental basis for the biological function and mechanism of RNA hsa_circ_0082626.
引文
[1]Salzman J,Chen RE,Olsen MN,et al.Cell-type specific features of circular RNA expression[J].PLoS Genet,2013,9(9):e1003777.
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[4]黄静,李欣晓,吴伟豪,等.人鼻咽癌组织环状RNA差异表达分析[J].医学研究生学报,2018,31(8):807-812.
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[6]Rybak-Wolf A,Stottmeister C,Gla?ar P,et al.Circular RNAs in the Mammalian Brain Are Highly Abundant,Conserved,and Dynamically Expressed[J].Molecular Cell,2015,58(5):870-885.
[7]Conn SJ,Pillman KA,Toubia J,et al.The RNA binding protein quaking regulates formation of circRNAs[J].Cell,2015,160(6):1125-1134.
[8]Yang Y,Gao X,Zhang M,et al.Novel Role of FBXW7 Circular RNA in Repressing Glioma Tumorigenesis[J].J Natl Cancer Inst,2018,110(3):304-315.
[9]何存存,吴会超,成绍敏.环状RNAs在消化系恶性肿瘤中的表达、生物学功能及临床应用研究进展[J].山东医药,2017,57(40):100-103.
[10]顾天伦,包兴洁,毛书奇,等.环状RNA在慢性病发病中作用机制的研究进展[J].生理科学进展,2017(03):167-172.
[11]玄丽佳.环状RNA的研究进展[J].医学研究生学报,2016,29(2):201-205.
[12]Mao R,Nie H,Cai D,et al.Inhibition of hepatitis B virus replication by the host zinc finger antiviral protein[J].PLoS Pathog,2013,9(7):e1003494.
[13]Salzman J,Chen RE,Olsen MN,et al.Cell-type specific features of circular RNA expression[J].PLoS Genet,2013,9(9):e1003777.
[14]Starke S,Jost I,Rossbach O,et al.Exon circularization requires canonical splice signals[J].Cell Rep,2015,10(1):103-111.
[15]Kerns JA,Emerman M,Malik HS.Positive selection and increased antiviral activity associated with the PARP-containing isoform of human zinc-finger antiviral protein[J].PLoS Genet,2008,4(1):e21.
[16]Shi J,Hu N,Li J,et al.Unique expression signatures of circular RNAs in response to DNA tumor virus SV40 infection[J].Oncotarget,2017,8(58):98609-98622.
[17]Hu XL,Zhu M,Zhang X,et al.Identification and characterization of circular RNAs in the silkworm midgut following Bombyx mori cytoplasmic polyhedrosis virus infection[J].RNA Biol,2018,15(2):292-301.
[18]Jost I,Shalamova LA,Gerresheim GK,et al.Functional sequestration of microRNA-122 from Hepatitis C Virus by circular RNA sponges[J].RNA Biol,2018,15(8):1032-1039.
[19]Li X,Liu C,Xue W,et al.Coordinated circRNA Biogenesis and Function with NF90/NF110 in Viral Infection[J].Mol Cell,2017,67(2):214-227.
[20]Zheng Q,Bao C,Guo W,et al.Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs[J].Nat Commun,2016,7:11215.
[21]Globinska A,Pawelczyk M,Kowalski ML.MicroRNAs and the immune response to respiratory virus infections[J].Expert Rev Clin Immunol,2014,10(7):963-971.
[2]Salzman J,Gawad C,Wang PL,et al.Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types[J].PLoS One,2012,7(2):e30733.
[3]Memczak S,Jens M,Elefsinioti A,et al.Circular RNAs are a large class of animal RNAs with regulatory potency[J].Nature,2013,495(7441):333-338.
[4]黄静,李欣晓,吴伟豪,等.人鼻咽癌组织环状RNA差异表达分析[J].医学研究生学报,2018,31(8):807-812.
[5]Hansen TB,Jensen TI,Clausen BH,et al.Natural RNA circles function as efficient microRNA sponges[J].Nature,2013,495(7441):384-388.
[6]Rybak-Wolf A,Stottmeister C,Gla?ar P,et al.Circular RNAs in the Mammalian Brain Are Highly Abundant,Conserved,and Dynamically Expressed[J].Molecular Cell,2015,58(5):870-885.
[7]Conn SJ,Pillman KA,Toubia J,et al.The RNA binding protein quaking regulates formation of circRNAs[J].Cell,2015,160(6):1125-1134.
[8]Yang Y,Gao X,Zhang M,et al.Novel Role of FBXW7 Circular RNA in Repressing Glioma Tumorigenesis[J].J Natl Cancer Inst,2018,110(3):304-315.
[9]何存存,吴会超,成绍敏.环状RNAs在消化系恶性肿瘤中的表达、生物学功能及临床应用研究进展[J].山东医药,2017,57(40):100-103.
[10]顾天伦,包兴洁,毛书奇,等.环状RNA在慢性病发病中作用机制的研究进展[J].生理科学进展,2017(03):167-172.
[11]玄丽佳.环状RNA的研究进展[J].医学研究生学报,2016,29(2):201-205.
[12]Mao R,Nie H,Cai D,et al.Inhibition of hepatitis B virus replication by the host zinc finger antiviral protein[J].PLoS Pathog,2013,9(7):e1003494.
[13]Salzman J,Chen RE,Olsen MN,et al.Cell-type specific features of circular RNA expression[J].PLoS Genet,2013,9(9):e1003777.
[14]Starke S,Jost I,Rossbach O,et al.Exon circularization requires canonical splice signals[J].Cell Rep,2015,10(1):103-111.
[15]Kerns JA,Emerman M,Malik HS.Positive selection and increased antiviral activity associated with the PARP-containing isoform of human zinc-finger antiviral protein[J].PLoS Genet,2008,4(1):e21.
[16]Shi J,Hu N,Li J,et al.Unique expression signatures of circular RNAs in response to DNA tumor virus SV40 infection[J].Oncotarget,2017,8(58):98609-98622.
[17]Hu XL,Zhu M,Zhang X,et al.Identification and characterization of circular RNAs in the silkworm midgut following Bombyx mori cytoplasmic polyhedrosis virus infection[J].RNA Biol,2018,15(2):292-301.
[18]Jost I,Shalamova LA,Gerresheim GK,et al.Functional sequestration of microRNA-122 from Hepatitis C Virus by circular RNA sponges[J].RNA Biol,2018,15(8):1032-1039.
[19]Li X,Liu C,Xue W,et al.Coordinated circRNA Biogenesis and Function with NF90/NF110 in Viral Infection[J].Mol Cell,2017,67(2):214-227.
[20]Zheng Q,Bao C,Guo W,et al.Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs[J].Nat Commun,2016,7:11215.
[21]Globinska A,Pawelczyk M,Kowalski ML.MicroRNAs and the immune response to respiratory virus infections[J].Expert Rev Clin Immunol,2014,10(7):963-971.