生物技术通报 ›› 2022, Vol. 38 ›› Issue (5): 159-168.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1071
李宇航1,2(), 王兴平1,2(), 杨箭1,2, 罗仍卓么1,2, 任倩倩1,2, 魏大为1,2, 马云1,2
收稿日期:
2021-08-20
出版日期:
2022-05-26
发布日期:
2022-06-10
作者简介:
李宇航,女,硕士,研究方向:动物分子遗传育种;E-mail: 基金资助:
LI Yu-hang1,2(), WANG Xing-ping1,2(), YANG Jian1,2, LUORENG Zhuo-ma1,2, REN Qian-qian1,2, WEI Da-wei1,2, MA Yun1,2
Received:
2021-08-20
Published:
2022-05-26
Online:
2022-06-10
摘要:
为探究miR-665在奶牛乳腺上皮细胞炎症中的表达及功能,利用脂多糖(LPS)诱导奶牛乳腺上皮细胞产生炎症反应,在LPS诱导0、3、6和12 h采用qPCR技术检测了miR-665及其潜在靶mRNA的表达水平,并采用生物信息学方法进行了miR-665保守性分析、靶基因预测及其KEGG和GO功能富集分析。结果表明,miR-665在牛、人和小鼠等物种间高度保守;与0 h相比,miR-665在LPS诱导乳腺上皮细胞产生炎症的3、6和12 h的表达量均显著上调,且与促进炎症相关的潜在靶基因(MAPK14、MAP3K2、SMAD2、SP1)的表达量呈相反趋势;miR-665的靶基因预测及其KEGG和GO功能富集结果显示,miR-665可能通过参与MAPK、Th17细胞分化、肿瘤坏死因子等信号通路发挥作用。推测miR-665可能在LPS诱导的乳腺上皮细胞炎症中具有抑制作用。
李宇航, 王兴平, 杨箭, 罗仍卓么, 任倩倩, 魏大为, 马云. miR-665在奶牛乳腺上皮细胞炎症中的表达及功能分析[J]. 生物技术通报, 2022, 38(5): 159-168.
LI Yu-hang, WANG Xing-ping, YANG Jian, LUORENG Zhuo-ma, REN Qian-qian, WEI Da-wei, MA Yun. Expression and Functional Analysis of miR-665 in Bovine Mammary Epithelial Cell Inflammation[J]. Biotechnology Bulletin, 2022, 38(5): 159-168.
基因名称 Gene name | 引物序列 Prime sequence(5'-3') | 产物大小 Product length/bp |
---|---|---|
miR-665 | RT:GTCGTATCCAGTGCAGGGTCCGAGG TATTCGCACTGGATACGACAGGGGCCT | 64 |
F:GTCATGGAACCAGTAGGCCG | ||
R:CAGTGCAGGGTCCGAGGTAT | ||
IL-1β | F:CAACCGTACCTGAACCC | 193 |
R:GACACCACCTGCCTGAA | ||
IL-8 | F:ACACATTCCACACCTTTCCAC | 149 |
R:ACCTTCTGCACCCACTTTTC | ||
GADPH | F:GGCATCGTGGAGGGACTTATG | 186 |
R:GCCAGTGAGCTTCCCGTTGAG | ||
RPS18 | F:GTGGTGTTGAGGAAAGCAGACA | 79 |
R:TGATCACACGTTCCACCTCATC |
表1 miR-665的逆转录及qPCR引物
Table 1 Reverse transcription and qPCR primers of miR-665
基因名称 Gene name | 引物序列 Prime sequence(5'-3') | 产物大小 Product length/bp |
---|---|---|
miR-665 | RT:GTCGTATCCAGTGCAGGGTCCGAGG TATTCGCACTGGATACGACAGGGGCCT | 64 |
F:GTCATGGAACCAGTAGGCCG | ||
R:CAGTGCAGGGTCCGAGGTAT | ||
IL-1β | F:CAACCGTACCTGAACCC | 193 |
R:GACACCACCTGCCTGAA | ||
IL-8 | F:ACACATTCCACACCTTTCCAC | 149 |
R:ACCTTCTGCACCCACTTTTC | ||
GADPH | F:GGCATCGTGGAGGGACTTATG | 186 |
R:GCCAGTGAGCTTCCCGTTGAG | ||
RPS18 | F:GTGGTGTTGAGGAAAGCAGACA | 79 |
R:TGATCACACGTTCCACCTCATC |
基因名称 Gene name | 引物序列 Prime sequence(5'-3') | 产物大小 Product length/bp |
---|---|---|
MAPK14 | F:ATCTTGGATTTCGGACTGGC | 264 |
R:ATGGCTTGGCATCCTGTTA | ||
MAP3K2 | F:CGGAAATACACCCGTCAGA | 324 |
R:AGCCATCGCTTCAAACTCG | ||
SMAD2 | F:GAAGGCAGACGGTAACAAGC | 114 |
R:TTGAGCAACGCACTGAAGG | ||
SP1 | F:CGGTGGGCAGTATGTTGT | 237 |
R:CCTCCACTTCCTCGGTTT |
表2 miR-665潜在关键靶基因的qPCR引物
Table 2 qPCR primers for potential key target genes of miR-665
基因名称 Gene name | 引物序列 Prime sequence(5'-3') | 产物大小 Product length/bp |
---|---|---|
MAPK14 | F:ATCTTGGATTTCGGACTGGC | 264 |
R:ATGGCTTGGCATCCTGTTA | ||
MAP3K2 | F:CGGAAATACACCCGTCAGA | 324 |
R:AGCCATCGCTTCAAACTCG | ||
SMAD2 | F:GAAGGCAGACGGTAACAAGC | 114 |
R:TTGAGCAACGCACTGAAGG | ||
SP1 | F:CGGTGGGCAGTATGTTGT | 237 |
R:CCTCCACTTCCTCGGTTT |
图1 主要炎症因子在LPS诱导的乳腺上皮细胞炎症中的表达 *表示P < 0.05;**表示P < 0.01,下同
Fig.1 Expression of major inflammatory factors in the LPS-induced inflammation of mammary epithelial cells * refers to P < 0.05;** refers to P < 0.01,the same below
基因名称Gene name | 牛Bos taurus | 人Homo sapiens | 小鼠Mus musculus | ||||
---|---|---|---|---|---|---|---|
GenBank ID | GenBank ID | 同源性Homology/% | GenBank ID | 同源性Homology/% | |||
MAPK14 | NM_001102174.1 | NM_001315.3 | 84.34 | NM_011951.3 | 84.88 | ||
MAP3K2 | XM_002685232.6 | NM_006609.5 | 85.91 | NM_011946.3 | 85.14 | ||
SMAD2 | NM_001046218.1 | NM_005901.6 | 93.96 | NM_010754.5 | 88.70 | ||
SP1 | NM_001078027.1 | NM_138473.3 | 91.03 | NM_013672.2 | 88.99 |
表3 牛与人、小鼠相关基因的同源性分析结果
Table 3 Homology analysis of related genes in the bovine,human and mouse
基因名称Gene name | 牛Bos taurus | 人Homo sapiens | 小鼠Mus musculus | ||||
---|---|---|---|---|---|---|---|
GenBank ID | GenBank ID | 同源性Homology/% | GenBank ID | 同源性Homology/% | |||
MAPK14 | NM_001102174.1 | NM_001315.3 | 84.34 | NM_011951.3 | 84.88 | ||
MAP3K2 | XM_002685232.6 | NM_006609.5 | 85.91 | NM_011946.3 | 85.14 | ||
SMAD2 | NM_001046218.1 | NM_005901.6 | 93.96 | NM_010754.5 | 88.70 | ||
SP1 | NM_001078027.1 | NM_138473.3 | 91.03 | NM_013672.2 | 88.99 |
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