序列变异对miR-378生物发生以及靶标关系的影响

doi:10.13560/j.cnki.biotech.bull.1985.2021-0137

摘要/Abstract

摘要：

本研究旨在探索序列变异对miR-378的结构、表达水平以及靶标关系的影响。利用PCR测序比对不同猪种miR-378的序列突变,预测突变对miR-378二级结构和自由能的改变;构建miR-378表达载体检测突变对其加工过程中各级产物表达水平的作用;运用TargetScan和TargetRank对突变引起的靶标关系变化进行分析;采用microRNA pull-down 技术验证突变对靶标关系的影响。结果发现,在荣昌猪和内江猪群体内pre-miR-378的+49和+68位均发生A>G的序列变异,且改变了pre-miR-378的二级结构和自由能;这两个位点突变后影响了pri-miR-378到pre-miR-378的加工过程,提升了pre-miR-378和成熟体miR-378的表达水平;同时+49/A>G的突变改变了miR-378的功能及其靶标关系,其中GDF6和 RAB10分别为突变后获得和缺失的靶基因,而Runx1t1、Galnt3为不受突变影响的靶基因。本研究的结果强调了miRNAs序列变异的重要性及其对miRNAs生物发生和功能的显著影响。

关键词: miR-378, 序列变异, 生物发生, 靶基因

Abstract:

The aim of this study is to explore the effects of sequence variation on the structure,expression level and targets relationship of miR-378. PCR-sequencing was employed to align the sequence mutations of miR-378 in different pig breeds and to predict the changes of the secondary structure and free energy of miR-378 caused by the mutation. The miR-378 expression vectors was constructed to detect the effect of mutation on the expression level during the processing. TargetScan and TargetRank were used to analyze the change of target relationship caused by the mutation of miR-378. MicroRNA pull-down technique was adapted to verify the effect of mutation on the target relationship of miR-378. The results showed that there were two A>G mutations on the +49 and +68 position of pre-miR-378 in Rongchang and Neijiang pigs,and the secondary structure and free energy of pre-miR-378 were changed. And these two mutations affected the processing from pri-miR-378 to pre-miR-378,and enhanced the expressions of pre-miR-378 and mature miR-378. The mutation of +49/A>G changed the function and target relationship of miR-378,of which GDF6 and RAB10 were the target genes obtained and deleted after mutation,respectively,while the shared targets（Runx1t1 and Galnt3）were not affected by mutation. These results serve to highlight the importance of miRNA mutations and their impacts on miRNA biogenesis.

Key words: miR-378, sequence variation, biogenesis, targets

张廷焕, 郭宗义, 柴捷, 潘红梅, 张亮, 陈磊, 龙熙. 序列变异对miR-378生物发生以及靶标关系的影响[J]. 生物技术通报, 2022, 38(1): 205-214.

ZHANG Ting-huan, GUO Zong-yi, CHAI Jie, PAN Hong-mei, ZHANG Liang, CHEN Lei, LONG Xi. Effects of Sequence Variation on the Biogenesis and Target Relationship of miR-378[J]. Biotechnology Bulletin, 2022, 38(1): 205-214.

图/表 10

表1 pri-miR-378 PCR扩增引物

Table 1 PCR primers of pri-miR-378

名称Name	序列Sequence	长度 Length/bp
pri-miR-378-F	CGGGATCCAAGGTATTTGGGGCACTGCA	23
pri-miR-378-R	GGGAAGCTTCAGGACAGTTCAGGCAAGGT	20

表2 miR-378 RT-PCR引物

Table 2 RT-PCR primers of miR-378

名称Name	序列Sequence	长度Length/bp
pri-miR-378-F	GAGGGAGGCAGCATGGTAAG	20
pre-miR-378-F	TGAAGGCAGGCAGAACCATT	20
precursor-miR-378-R	CGGGCCTTCTGACTCCAAG	19
miR-378/AA	ACTGGACTTGGAGTCAGAAGGC	22
miR-378/GG	ACTGGGCTTGGAGTCAGAAGGC	22

表3 miR-378靶基因RT-PCR扩增引物

Table 3 RT-PCR primers of miR-378 targets

名称Name	序列Sequence	长度Length/bp
GDF6-F	CTCGAGCTACTAAATGACAG	20
GDF6-R	TCTCCTTCCTCACTGCCTGT	20
Runx1t1-F	ATCGGGAATTCCTTCACAGGC	21
Runx1t1-R	GCTTTTTGCAGCTCCGTCAT	20
Galnt3-F	ACGCAGGTGATTGCTCGTAA	20
Galnt3-R	AGGTCTGGCACATACGCTTC	20
RAB10-F	ATGTACTTGCTCAGCTCAACT	21
RAB10-R	AGGGACTCAAGCACATTATCCA	22

图1 猪miR-378的扩增和测序 A：miR-378 PCR产物的凝胶电泳图;B：不同猪种pre-miR-378序列对比图,红色字母代表miR-378的种子序列。其中,RC代表荣昌猪,NJ代表内江猪,下同

Fig.1 Amplification and sequencing of porcine miR-378 A: Gel electrophoresis of miR-378 PCR products. B: Sequence comparison of pre-miR-378 in different pig breeds and the red letters refer to the seed sequence of miR-378. RC refers to Rongchang pig and NJ refers to Neijiang pig,the same below

图2 miR-378结构和能量预测 A：miR-378的二级结构;B：miR-378的自由能分布。其中,红色方框代表变化最明显的区域

Fig.2 Structure and energy prediction of miR-378 A: Secondary structure of miR-378. B: Energy distribution of miR-378. The red box refers to the areas with the most obvious changes

图3 miR-378过表达载体的构建 A：pri-miR-378 PCR产物的凝胶电泳图;B：过表达载体示意图;C：过表达载体插入片段部分序列图,红色方框代表变异位点

Fig.3 Construction of miR-378 overexpression vector A: Gel electrophoresis of pri-miR-378 PCR products. B: Schematic diagram of overexpression vectors. C: Partial sequence diagram of inserted fragment of overexpression vectors,and the red boxes refer to the mutation sites

图4 miR-378各级产物表达量分析 A：成熟体miR-378的表达量;B：pri-miR-378和pre-miR-378特异性引物的示意图;C：pri-miR-378的表达量;D：pri-miR-378和pre-miR-378表达总量。其中**代表P<0.01,NS代表P>0.05,下同

Fig.4 Analysis of miR-378 products in expressions A: The expression of mature miR-378. B: Schematic diagram of the specific primers designed to amplify pri-miR-378 and pre-miR-378. C: The expression of pri-miR-378. D: The total expression of pre-miR-378 and pri-miR-378. And ** refers to P < 0.01,NS refers to P > 0.05,the same below

图5 miR-378靶标关系和功能预测 A：不同物种间成熟体miR-378序列比对,红色字母代表种子序列;B：靶基因预测以及不同软件之间的多重比对;C：靶基因的功能富集分析

Fig.5 Target relationship and function prediction of miR-378 A: The sequence alignment of mature miR-378 in different species and the red letters refer to the seed sequence. B: Target gene prediction and multiple comparisons among different softwares. C: Functional enrichment analysis of the targets

表4 miR-378的靶基因列表

Table 4 Target list of miR-378

分组名称 Group name	靶基因 Target genes
获得的靶基因 Gained-targets	NFIX、ABAT、ADAM19、ASXL3、ATP1B2、AXUD1、BCL2、BMF、BRUNOL6、C14orf83、C15orf27、C9orf41、CACNA1I、CDH1、CHD5、CHRM1、CLCN6、CRISPLD2、DNAJB12、ENG、ETV1、FAIM2、FBXL11、FBXO33、FNDC3A、FRAS1、GUCY1A2、HEYL、IFI30、INPP5D、ITGA5、KCNA5、KIAA0556、LARP1、MGLL、MMP24、MYLK2、NAV1、NCAM1、NOTCH2、NUAK1、OXSR1、PAF1、PAQR4、PDE11A、PELI3、PLEKHA6、PRNPIP、RBM19、REEP2、RHBG、RORC、SFRS7、SHROOM4、SLAIN2、SLC23A2、SNAP29、SOBP、SP1、SPRN、SYT9、TMC1、TMPRSS4、TPI1、TYRO3、UBE2N、WDR78、ZHX3、ZNF609、BSDC1、COBL、CYP26B1、DDX6、ELAVL3、FLJ14154、GDF6、HECTD3、KCNIP3、KIAA0652、LEF1、MAF、MAPK3、PARD6B、PJA2、SBK1、SMARCD2、SP6、TMEM104、TSC22D3、ZNF292、ARNT2、C12orf4、C9orf105、FBXO41、GAB2、GRHL2、KCTD15、KIAA1853、LRRTM3、MTMR3、PAX2、PHACTR2、POLR2J3、RIMS3、SRRM1、THRB、UNC119B、ZCCHC3
失去的靶基因 Lost-targets	KIAA1522、SDAD1、METTL4、PHC3、SULF1、RBMS3、RRP1B、BMP2、VANGL1、PAPD5、MED12L、PLEKHG2、CBL、GRSF1、SLC38A1、GOLT1A、PLAGL2、SOX7、DCX、DACT1、SLC39A9、KIAA1219、DYRK1A、IPO9、KIAA1576、QSER1、NEK4、TLK2、LBX2、C1orf21、FRMPD4、SFRS3、CALN1、WDR37、DBT、MEF2D、TSPAN17、RAB10
共有的靶基因 Shared-targets	Runx1t1、Galnt3、KPNA6、BCL2L2

图6 miR-378靶基因的富集程度验证 A：GDF6基因富集程度;B：RAB10基因富集程度;C：Galnt3基因富集程度;D：Runx1t1基因富集程度

Fig.6 Verification of enrichment degree of miR-378 targets A: The enrichment of GDF6 gene. B: The enrichment of RAB10 gene. C: The enrichment of Galnt3 gene. D: The enrichment of Runx1t1 gene

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