Scientia Agricultura Sinica ›› 2018, Vol. 51 ›› Issue (21): 4157-4168.doi: 10.3864/j.issn.0578-1752.2018.21.014

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• SPECIAL FOCUS: SWINE FEVER AND AFRICAN SWINE FEVER • Previous Articles     Next Articles

Screen of MicroRNAs in Classical Swine Fever Virus-Infected PK-15 Cells and the Regulation of Virus Replication by miR-214

ShaoFeng DENG(),ZuoDong YE,ShuangQi FAN,JinDing CHEN,JingYuan ZHANG,MengJiao ZHU,MingQiu ZHAO()   

  1. College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642
  • Received:2018-04-18 Accepted:2018-09-07 Online:2018-11-01 Published:2018-11-01
  • Contact: ShaoFeng DENG,MingQiu ZHAO E-mail:dsf4530@126.com;zmingqiu@scau.edu.cn

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Abstract:

【Objective】 In this study, differential expression of miRNAs in CSFV-infected PK-15 cells were determined by miRNA expression array, and further explore the function of miRNAs in the pathogenic of Classical swine fever virus (CSFV), and provide some new basis for the prevention and control of Classical swine fever (CSF) .【Method】 In order to investigate the changes of the miRNAs expression in CSFV-infected PK-15 cells, we synthesized probes of 326 miRNAs of pig according to the miRBase database version 19.0, and screening of differential expression of miRNAs in CSFV-infected PK-15 cells using miRNA expression array. Then, miR-214, the most obvious difference in expression of CSFV-infected PK-15 cells, was selected as the further study object to investigate the function of miR-214 in the infection process of CSFV. We detected mRNA expression of miR-214 in CSFV-infected PK-15 cells using qRT-PCR. In order to further study the effect of miR-214 of CSFV infection, we synthesized miR-214 analog and inhibitor and transfected into PK-15 cells respectively, follow with CSFV infection at 24 h post-transfection, and then detected CSFV titers and quantity of CSFV genomic copies. In order to further explore the mechanism of miR-214 participate in the regulation of CSFV replication, we predicted the target protein of miR-214 using bioinformatics software and confirmed it by luciferase reporter gene system. Given TRADD can specific interacts with TNFR1 intracellular dead zones and participate in the programmed cell death, we assume that miR-214 influencing apoptosis of PK 15 cells by influencing expression level of target protein TRADD. PK 15 cells transfected with miR-214 and inhibitor respectively, follow with CSFV infection at 24 h post-transfection. At 48 h post-infection, the expression levels of TRADD were detected, and the effect of miR-214 on the apoptosis of CSFV-infected PK-15 cells was detected by flow cytometry. 【Result】 69 miRNAs with different expressions were screened by miRNA expression array in CSFV-infected PK-15 cells. Among which the expression changes of miR-214 were most obvious and up-regulated, and confirmed it by qRT-PCR. After transfected with miR-214 to PK-15 cells, CSFV titers and quantity of CSFV genomic copies decreased significantly, while transfected with miR-214 inhibitor, CSFV titers and quantity of CSFV genomic copies were increased significantly, which suggested that miR-214 promoted the replication of CSFV. In order to further explore the mechanism of miR-214 promoting CSFV replication, we confirmed TRADD is the target protein of miR-214. After transfected miR-214 to PK-15 cells, mRNA and protein expression of TRADD were increased significantly, while transfected with miR-214 inhibitor, that were decreased significantly, suggesting that miR-214.inhibits the expression of TRADD. And then, we verified that CSFV infection inhibits apoptosis of PK-15 cells, and miR-214 inhibits apoptosis of CSFV-infected PK-15 cells. 【Conclusion】 The expression of miR-214 in cells was up-regulated after CSFV infected PK-15. miR-214 inhibits the apoptosis of PK-15 cells and promote the replication of CSFV in cells by targeting inhibits the expression of TRADD protein.

Key words: CSFV, miRNA, replication, TRADD, apoptosis

Table 1

The up-regulated miRNAs in CSFV-infected PK-15 cells"

编号 ID miRNA名称 Name log2比率 log2 ratio P值 P-value
MIMAT0006786 ssc-miR-140-3p 0.48443 0.00026
MIMAT0002147 ssc-miR-214 0.42896 0.00001
MIMAT0013955 ssc-miR-335 0.38587 0.00001
MIMAT0002120 ssc-miR-125b 0.09519 0.00043
MIMAT0025368 ssc-miR-194a 0.31888 0.01203
MIMAT0013944 ssc-miR-342 0.31845 0.03668
MIMAT0010192 ssc-miR-215 0.30804 0.04995
MIMAT0022956 ssc-miR-202-3p 0.29007 0.00009
MIMAT0007759 ssc-miR-185 0.22220 0.00035
MIMAT0013912 ssc-miR-208b 0.21017 0.00771
MIMAT0013876 ssc-miR-191 0.20254 0.03214
MIMAT0025360 ssc-miR-31 0.20020 0.01347
MIMAT0002119 ssc-miR-122 0.19622 0.01318
MIMAT0007758 ssc-miR-130a 0.19402 0.02421
MIMAT0013916 ssc-miR-34c 0.19369 0.01302
MIMAT0017385 ssc-miR-1839-3p 0.19364 0.03377
MIMAT0013906 ssc-miR-664-5p 0.16827 0.00425
MIMAT0013880 ssc-miR-423-5p 0.16298 0.00001
MIMAT0002134 ssc-miR-24-3p 0.16088 0.01132
MIMAT0007757 ssc-miR-34a 0.16044 0.00131
MIMAT0017956 ssc-miR-382 0.15206 0.00146
MIMAT0013954 ssc-miR-1285 0.14589 0.04690
MIMAT0025356 ssc-let-7d-5p 0.14434 0.01134
MIMAT0017376 ssc-miR-365-5p 0.14349 0.03370
MIMAT0028156 ssc-miR-7140-3p 0.13804 0.00007
MIMAT0015300 ssc-miR-30a-3p 0.13721 0.02233
MIMAT0028144 ssc-miR-7134-3p 0.13182 0.00483
MIMAT0013956 ssc-miR-500 0.13164 0.02126
MIMAT0013910 ssc-miR-192 0.12760 0.00862
MIMAT0013922 ssc-miR-130b 0.12228 0.02580
MIMAT0013903 ssc-miR-885-3p 0.11877 0.00246
MIMAT0022921 ssc-miR-139-3p 0.11860 0.00302
MIMAT0028152 ssc-miR-7138-3p 0.11640 0.03755
MIMAT0006018 ssc-miR-99b 0.11097 0.01450
MIMAT0028155 ssc-miR-7140-5p 0.09369 0.00232
MIMAT0013920 ssc-miR-424-5p 0.09366 0.00090
MIMAT0022922 ssc-miR-30c-3p 0.08348 0.01393
MIMAT0013894 ssc-miR-193a-5p 0.08314 0.00302
MIMAT0010188 ssc-miR-450a 0.03115 0.00119

Table 2

The down-regulated miRNAs in CSFV-infected PK-15 cells"

编号 ID miRNA名称 Name log2比率 log2 ratio P值 P-value
MIMAT0025385 ssc-miR-1249 -2.15272 0.00001
MIMAT0013934 ssc-miR-361-3p -1.95208 0.00001
MIMAT0017952 ssc-miR-296-5p -1.05927 0.00001
MIMAT0013937 ssc-miR-1306-5p -0.95394 0.00001
MIMAT0013941 ssc-miR-532-3p -0.68756 0.00001
MIMAT0017980 ssc-miR-4339 -0.6224 0.00001
MIMAT0013871 ssc-miR-30d -0.30603 0.00001
MIMAT0002165 ssc-miR-21 -0.27854 0.00001
MIMAT0025381 ssc-miR-671-5p -0.2569 0.00001
MIMAT0017974 ssc-miR-484 -0.248 0.00001
MIMAT0020586 ssc-miR-129b -0.22805 0.00008
MIMAT0018379 ssc-miR-149 -0.21029 0.00004
MIMAT0015708 ssc-miR-744 -0.20892 0.00386
MIMAT0017965 ssc-miR-1224 -0.18271 0.00033
MIMAT0013900 ssc-miR-345-3p -0.17812 0.00828
MIMAT0025365 ssc-miR-150 -0.15152 0.00775
MIMAT0020596 ssc-miR-1343 -0.14743 0.00461
MIMAT0013935 ssc-miR-1277 -0.13761 0.00482
MIMAT0010186 ssc-miR-133a-3p -0.13388 0.03942
MIMAT0013959 ssc-miR-129a -0.12737 0.01689
MIMAT0013939 ssc-miR-339-5p -0.11957 0.01352
MIMAT0025384 ssc-miR-874 -0.11957 0.00046
MIMAT0028147 ssc-miR-7136-5p -0.11865 0.00367
MIMAT0013909 ssc-miR-92b-3p -0.10387 0.02389
MIMAT0017962 ssc-miR-4332 -0.09669 0.03493
MIMAT0013918 ssc-miR-425-3p -0.09066 0.00617
MIMAT0013869 ssc-miR-133b -0.08309 0.00758
MIMAT0013904 ssc-miR-365-3p -0.06645 0.03768
MIMAT0022958 ssc-miR-296-3p -0.06567 0.04572
MIMAT0013947 ssc-miR-935 -0.05012 0.01506

Fig. 1

Relative expression of miR-214 in CSFV-infected PK-15 cells"

Fig. 2

The expression of miR-214 after transfection with miR-214 mimics or miR-214 inhibitor in PK-15 cells A. The expression of miR-214 in cells after transfected with miR-214; B. The expression of miR-214 in cells after transfected with miR-214 inhibitor"

Fig. 3

The effects of miR-214 on CSFV replication A. The influence of miR-214 on the expression of CSFV NS5B mRNA; B. The effect of miR-214 on the virus titer of CSFV"

Fig. 4

The effects of miR-214 on luciferase activity of pmirGLO-TRADD 3′UTR"

Fig. 5

The potential interaction site between miR-214 and TRADD The red labels in the figure is the complementary base pairing between mir-214 and TRADD 3'-UTR"

Fig. 6

The Effects of miR-214 on the expression of TRADD A. Effect of transfection of mir-214 on TRADD mRNA expression; B. Effect of transfection of mir-214 inhibitor on TRADD mRNA expression"

Fig. 7

The Effects of miR-214 on the expression of TRADD A. Effect of transfection of mir-214 on TRADD protein expression; B. Effect of transfection of mir-214 inhibitor on TRADD protein expression; C and D are the statistical analysis of the intensity band ratio of A and B respectively"

Fig. 8

The effect of CSFV infection on apoptosis in PK-15 cells A. Apoptosis rate of PK-15 cells without infected with CSFV; B. Apoptosis rate of PK -15 cells infected with CSFV; C. Statistical analysis of apoptosis rate of CSFV infection group and control group"

Fig. 9

Effects of miR-214 and miR-214 inhibitors on apoptosis of PK-15 cells A. The effect of miR-214 on apoptosis of PK-15 cells; B. The effect of mir-214 on apoptosis of PK-15 cells infected with CSFV; C. The effect of miR-214 inhibitors on apoptosis of PK-15 cells infected with CSFV"

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