MicroRNA-1285及其靶标DDX3X对猪塞内卡病毒感染PK-15细胞的调控作用

孙媛; 唐晓钰; 白杨; 陈雨琪; 郑瑶瑶; 吴佼玲; 蓝天; 马静云

doi:10.7671/j.issn.1001-411X.202205038

MicroRNA-1285及其靶标DDX3X对猪塞内卡病毒感染PK-15细胞的调控作用

华南农业大学动物科学学院, 广东广州 510642

基金项目: 广东省基础与应用基础研究基金(2020A1515010295，2022A1515012473)

详细信息

作者简介:
孙媛，讲师，博士，主要从事动物健康养殖与疫病防控研究，E-mail: sunyuan@scau.edu.cn

通讯作者:
马静云，教授，博士，主要从事动物健康养殖与综合防控研究，E-mail: majy2400@scau.edu.cn

中图分类号: S852.42；S852.659.6
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出版历程
- 收稿日期: 2022-05-18
- 网络出版日期: 2023-05-17
- 刊出日期: 2023-05-09

Regulation effects of microRNA-1285 and its target DDX3X on Senecavirus A infected PK-15 cells

College of Animal Science, South China Agricultural University, Guangzhou 510642, China

摘要

摘要:
目的
探究MicroRNA-1285(miR-1285)及其靶标DDX3X在猪塞内卡病毒(Senecavirus A，SVA)感染PK-15细胞中的调控作用。

方法
利用qRT-PCR、双荧光素酶活性及Western blot等方法研究miR-1285和DDX3X对I型干扰素(IFN-β)分泌及RIG-I信号通路的作用，分析miR-1285及DDX3X对SVA 3C蛋白基因表达的影响。

结果
SVA感染PK-15细胞后，miR-1285表达量显著升高，并且miR-1285与DDX3X存在负靶向关系，二者可促进IFN-β转录及蛋白水平的表达。miR-1285通过靶向DDX3X对RIG-I信号通中的MAVS、TRAF3信号分子起调控作用。对于SVA 3C蛋白基因，DDX3X可以显著抑制其转录，并且可以逆转miR-1285所诱导的上调趋势。

结论
SVA感染PK-15细胞后，宿主miR-1285及其靶标DDX3X对IFN-β及病毒3C蛋白的表达具有调控作用，研究结果将为明确miRNAs调控SVA感染的分子机制奠定基础，并为SVA的防控和诊断提供新的科学依据。
- MicroRNA-1285 /
- DDX3X /
- 猪塞内卡病毒 /
- IFN-β /
- RIG-I信号通路
Abstract:
Objective
To explore the regulation roles of microRNA-1285 (miR-1285) and its target DDX3X in Senecavirus A (SVA) infected PK-15 cells.

Method
By qRT-PCR, double luciferase activity and Western blot, the effects of miR-1285 and its target DDX3X on IFN-β secretion and the RIG-I signaling pathway were studied, and their effects on the expression of SVA 3C protein gene were analyzed.

Result
In SVA infected PK-15 cells, the expression of miR-1285 increased significantly, and there was a negative targeting relationship between miR-1285 and DDX3X. Both miR-1285 and DDX3X promoted the transcription and protein expression of IFN-β. MiR-1285 regulated MAVS and TRAF3 signaling molecules in the RIG-I signaling pathway by targeting DDX3X. For SVA 3C protein, DDX3X significantly inhibited the transcription of 3C and reversed the up-regulation trend induced by miR-1285.

Conclusion
After infecting PK-15 cells with SVA, host miR-1285 and its target DDX3X can regulate the expression of IFN-β and the viral 3C protein, which will lay a foundation for clarifying the molecular mechanism of miRNAs regulating SVA infection, and provide a new scientific basis for the prevention, control and diagnosis of SVA.
- MiRNA-1285 /
- DDX3X /
- Senecavirus A /
- IFN-β /
- RIG-I signal pathway

HTML全文

图 1 不同SVA感染时间(A)和感染剂量(B)条件下PK-15细胞中miR-1285的表达量

“*”“**”分别表示处理与对照在P < 0.05和P < 0.01水平差异显著(Duncan’s法)

Figure 1. Expression of miR-1285 in PK-15 cells infected by SVA at different infection time (A) and dosages (B)

“*” and “**” represented statistical difference in comparison with control group at P < 0.05 and P < 0.01 levels respectively (Duncan’s method)

下载: 全尺寸图片幻灯片

图 2 miR-1285与DDX3X的靶向结合位点

Figure 2. Targeted binding site between miR-1285 and DDX3X

下载: 全尺寸图片幻灯片

图 3 转染miR-1285 mimics、inhibitor至PK-15细胞后DDX3X蛋白的表达

Figure 3. DDX3X protein expression after the transfection of miR-1285 mimics, inhibitor into PK-15 cells

下载: 全尺寸图片幻灯片

图 4 转染miR-1285 mimics、inhibitor至PK-15细胞后DDX3X的mRNA相对表达量

“**”表示处理与对照在P < 0.01水平差异显著 (Duncan’s 法)

Figure 4. The relative expression of DDX3X mRNA after transfection of miR-1285 mimics and inhibitor into PK-15 cells

“**” represented statistical difference in comparison with control group at P < 0.01 level (Duncan’s method)

下载: 全尺寸图片幻灯片

图 5 转染不同DDX3X重组载体质粒至PK-15细胞后miR-1285双荧光素酶活性

“*”表示处理与对照在P < 0.05水平差异显著 (Duncan’s 法)

Figure 5. The relative dual-luciferase activity of miR-1285 after transfection of different DDX3X recombinant vector plasmids into PK-15 cells

“*” represented statistical difference in comparison with control group at P < 0.05 level (Duncan’s method)

下载: 全尺寸图片幻灯片

图 6 miR-1285及其靶标DDX3X对IFN-β mRNA表达的调控作用

“*”和“**”分别表示处理与对照在P < 0.05和P < 0.01水平差异显著(Duncan’s法)

Figure 6. Regulation effects of miR-1285 and its target DDX3X on the mRNA expression of IFN-β

“*” and “**” represented statistical difference in comparison with control group at P < 0.05 and P < 0.01 levels respectively (Duncan’s method)

下载: 全尺寸图片幻灯片

图 7 miR-1285及其靶标DDX3X对IFN-β 蛋白表达的调控作用

Figure 7. Regulation effects of miR-1285 and its target DDX3X on the protein expression of IFN-β

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图 8 miR-1285对RIG-I通路信号转导分子的影响

“*”表示处理与对照在P < 0.05水平差异显著(Duncan’s法)

Figure 8. Effects of miR-1285 on signal transduction molecules of the RIG-I pathway

“*” represented statistical difference in comparison with control group at P < 0.05 level respectively (Duncan’s method)

下载: 全尺寸图片幻灯片

图 9 DDX3X沉默对RIG-I通路信号转导分子的影响

“*”和“**”分别表示处理与对照在P < 0.05和P < 0.01水平差异显著(Duncan’s法)

Figure 9. Effects of DDX3X silencing on signal transduction molecules of the RIG-I pathway

“*” and “**” represented statistical difference in comparison with control group at P < 0.05 and P < 0.01 levels respectively (Duncan’s method)

下载: 全尺寸图片幻灯片

图 10 miR-1285及其靶标DDX3X对SVA 3C表达的影响

“**”表示处理与对照在P < 0.01水平差异显著(Duncan’s法)

Figure 10. Effects of miR-1285 and its target DDX3X on the expression of SVA 3C

“**” represented statistical difference in comparison with control group at P < 0.01 level respectively (Duncan’s method)

下载: 全尺寸图片幻灯片

表 1 基因引物序列

Table 1 Primer sequences of genes

基因名称 Gene name	引物/探针序列(5′→3′) Primer/Probe sequence	θ_退火/ ℃ Annealing temperature	产物大小/bp Product size	文献 Reference
RIG-I	F: ATCCCAGCAACGAGAA	60	188	[36]
RIG-I	R: GCCACGTCCAGTCAAT	60	188	[36]
MDA5	F: GAGGAATCAGCACGAGGAA	58	73	[37]
MDA5	R: GTCAGTAATCCACTGGGA	58	73	[37]
MAVS	F: ATAGCCAGCCTTTCTCGG	60	237	[36]
MAVS	R: TAGCCTCAGTCTTGACCTCTTC	60	237	[36]
TRAF3	F: GTGTCAAGAAGGCATCG	60	164	[36]
TRAF3	R: CCTCAAACTGGCAATCA	60	164	[36]
TANK	F: GGACGCCTTGAACTACCTGT	60	119
TANK	R: GCCTGCCGAAAGGCTTCATA	60	119
TBK1	F: GCCTTTCTCGGGGTCTTCAA	60	74
TBK1	R: ACACTTTTCCTGATCCGCCT	60	74
IRF3	F: CCAGTGGTGCCTACACTCCT	61	191	[38]
IRF3	R: AGAGGTGTCTGGCTCAGGAA	61	191	[38]
IRF7	F: CGCCTCCTGGAAAACCAA	60	76	[37]
IRF7	R: CCCTGAGTTGTCCTGCAACA	60	76	[37]
IFN-β	F: GCTAACAAGTGCATCCTCCAAA	60	77	[39]
IFN-β	R:AGCACATCATAGCTCATGGAAAGA	60	77	[39]
GAPDH	F: ACATGGCCTCCAAGGAGTAAGA	60	106	[40]
GAPDH	R: GATCGAGTTGGGGCTGTGACT	60	106	[40]
SVA-3C	F: GAGCTTCAATCTCCTAGA	59	115
SVA-3C	R: GTGTCATCATTCTCGTTAG	59
探针 Probe	CAGACATTCGAGCCAAGCAACAA	69

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